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Asuka Inoue

Section

Graduate School of Pharmaceutical Sciences

Job title

Professor

Degree

博士（薬学）（東京大学）
修士（薬学）（東京大学）

researchmap

https://researchmap.jp/iaska

J-GLOBAL ID

200901057936608094

ORCID

https://orcid.org/0000-0003-0805-4049

Research History 6

2024/04 - Present

Kyoto University　Graduate School of Pharmaceutical Sciences　Professor
2022/04 - Present

Tohoku University　Graduate School of Pharmaceutical Sciences　Professor
2020/04 - 2022/03

Tohoku University　Graduate School of Pharmaceutical Sciences　Associate Professor
2016/09 - 2020/03

Tohoku University　Graduate School of Pharmaceutical Sciences　Associate Professor
2014/07 - 2016/08

Tohoku University　Graduate School of Pharmaceutical Sciences　Assistant Professor
2008/08 - 2014/06

Tohoku University　Graduate School of Pharmaceutical Sciences　Research Associate

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Education 3

The University of Tokyo　Graduate School of Pharmaceutical Sciences

2006/04 - 2008/07
The University of Tokyo　Graduate School of Pharmaceutical Sciences

2004/04 - 2006/03
The University of Tokyo　Faculty of Pharmaceutical Sciences

2000/04 - 2004/03

Professional Memberships 2

日本薬学会
日本生化学会

Research Interests 1

GPCR

Research Areas 3

Life sciences / Pharmacology /
Life sciences / Pharmaceuticals - health and biochemistry /
Life sciences / Functional biochemistry /

Awards 3

Japan Academy Medal

2025/02　The Japan Academy
JSPS PRIZE

2025/02　The Japan Society for the Promotion of Science (JSPS)
The MEXT Young Scientists' Prize.

2021/04　Ministry of Education, Culture, Sports, Science and Technology (MEXT)

Papers 321

Generation of Comprehensive GPCR-Transducer-Deficient Cell Lines to Dissect the Complexity of GPCR Signaling

Ayaki Saito, Ryoji Kise, Asuka Inoue

Pharmacological Reviews　76　(4)　599-619　2024/05/07
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
DOI： 10.1124/pharmrev.124.001186 　

ISSN： 0031-6997

eISSN： 1521-0081
Chemogenetic activation of G12 signaling enhances adipose tissue browning. International-journal

Yuki Ono, Ryo Ito, Kaito Arai, Gurdeep Singh, Tsuyoshi Saitoh, Robert B Russell, Francesco Raimondi, Junken Aoki, Juro Sakai, Asuka Inoue

Signal transduction and targeted therapy　8　(1)　307-307　2023/08/21

DOI： 10.1038/s41392-023-01524-2 　
Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics International-journal

John Janetzko, Ryoji Kise, Benjamin Barsi-Rhyne, Dirk H. Siepe, Franziska M. Heydenreich, Kouki Kawakami, Matthieu Masureel, Shoji Maeda, K. Christopher Garcia, Mark von Zastrow, Asuka Inoue, Brian K. Kobilka

Cell　185　(24)　4560-4573.e19　2022/11/23

DOI： 10.1016/j.cell.2022.10.018 　

ISSN： 0092-8674

eISSN： 1097-4172
Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying β-arrestin transducer bias

Kouki Kawakami, Masataka Yanagawa, Suzune Hiratsuka, Misaki Yoshida, Yuki Ono, Michio Hiroshima, Masahiro Ueda, Junken Aoki, Yasushi Sako, Asuka Inoue

Nature Communications　13　(1)　2022/01/25
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-022-28056-7 　

eISSN： 2041-1723

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Abstract Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and β-arrestins. Although a wealth of structural knowledge about signaling bias at the GPCR level exists (preferential engagement of a specific transducer), little is known about the bias at the transducer level (different functions mediated by a single transducer), partly due to a poor understanding of GPCR kinase (GRK)-mediated GPCR phosphorylation. Here, we reveal a unique role of the Gq heterotrimer as a determinant for GRK-subtype selectivity that regulates subsequent β-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that β-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the β-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and β-arrestin functionality by Ang II is shifted to those of TRV027. Single-molecule imaging identifies relocation of AT1R and GRK5, but not GRK2, to an immobile phase under the Gq-inactive, AT1R-stimulated conditions. These findings uncover a previously unappreciated Gq-regulated mechanism that encodes GRK-subtype selectivity and imparts distinct phosphorylation-barcodes directing downstream β-arrestin functions.
Illuminating G-Protein-Coupling Selectivity of GPCRs. International-journal Peer-reviewed

Asuka Inoue, Francesco Raimondi, Francois Marie Ngako Kadji, Gurdeep Singh, Takayuki Kishi, Akiharu Uwamizu, Yuki Ono, Yuji Shinjo, Satoru Ishida, Nadia Arang, Kouki Kawakami, J Silvio Gutkind, Junken Aoki, Robert B Russell

Cell　177　(7)　1933-1947　2019/06/13

DOI： 10.1016/j.cell.2019.04.044 　

ISSN： 0092-8674

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Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.
TGFα shedding assay: an accurate and versatile method for detecting GPCR activation. International-journal Peer-reviewed

Asuka Inoue, Jun Ishiguro, Hajime Kitamura, Naoaki Arima, Michiyo Okutani, Akira Shuto, Shigeki Higashiyama, Tomohiko Ohwada, Hiroyuki Arai, Kumiko Makide, Junken Aoki

Nature methods　9　(10)　1021-9　2012/10

DOI： 10.1038/nmeth.2172 　

ISSN： 1548-7091
LPA-producing enzyme PA-PLA₁α regulates hair follicle development by modulating EGFR signalling. International-journal Peer-reviewed

Asuka Inoue, Naoaki Arima, Jun Ishiguro, Glenn D Prestwich, Hiroyuki Arai, Junken Aoki

The EMBO journal　30　(20)　4248-60　2011/08/19

DOI： 10.1038/emboj.2011.296 　

ISSN： 0261-4189
Characterization of Gαs and Gαolf activation by catechol and non-catechol dopamine D1 receptor agonists. International-journal

Anh Minh Nguyen, Ana Semeano, Vianna Quach, Asuka Inoue, David E Nichols, Hideaki Yano

iScience　28　(5)　112345-112345　2025/05/16

DOI： 10.1016/j.isci.2025.112345 　

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The dopamine D1 receptor (D1R) couples to Gαs and Gαolf and is crucial in regulating neurological and neuropsychiatric functions. In the brain, Gαolf is predominantly found in the striatum whereas Gαs is expressed elsewhere. Our in vitro assays revealed that the tetracyclic catechol agonists dihydrexidine, methyl-dihydrexidine, doxanthrine, and the non-catechol compounds PF-8294, PF-6142 exerted full agonism for Gαs coupling but only partial agonism for Gαolf coupling. In contrast, the non-catechol agonist tavapadon acted as a full agonist at Gαolf and a partial agonist at Gαs. The effects of these ligands on the thalamocortical and striatonigral electrophysiological events, as well as on the locomotor activity and cognitive function of mice agreed with their selectivity profiles in vitro. These findings suggest the possibility of achieving region-specific pharmacology and open new directions for developing D1R drugs to treat relevant neurological and neuropsychiatric disorders.
An integrated mechanism of Gq regulation of PLCβ enzymes. International-journal

Kanishka Senarath, Isaac J Fisher, Wonjo Jang, Sumin Lu, Asuka Inoue, Evi Kostenis, Angeline M Lyon, Nevin A Lambert

Proceedings of the National Academy of Sciences of the United States of America　122　(16)　e2500318122　2025/04/22

DOI： 10.1073/pnas.2500318122 　

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Phospholipase Cβ (PLCβ) enzymes are the principal effectors activated by Gq heterotrimers. Both Gαq and Gβγ subunits can activate PLCβ, which requires precise positioning of PLCβ at the plasma membrane to relieve structural autoinhibition and give the active site access to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCβ enzymes possess a unique distal C-terminal domain (dCTD) that is critical for activation by Gαq, but the reason for this is unclear. It is also not known how G protein activation affects the subcellular localization of PLCβ enzymes, some of which are found primarily in the cytosol despite needing to act at the plasma membrane. Here, we use bioluminescence spectroscopy, imaging, and gene editing to study the membrane disposition of PLCβ enzymes in living cells and to define the functional roles of the dCTD. We find that PLCβ translocates to the plasma membrane upon Gq activation, primarily by binding to Gαq subunits. This is rapidly counteracted by PIP2 hydrolysis, which promotes PLCβ translocation back into the cytosol. PLCβ translocation and activation require binding of Gαq to the catalytic domain and the dCTD at two distinct interfaces. Gαq binding to the dCTD is required for activation even when PLCβ is artificially tethered to the plasma membrane, suggesting that this domain has functions beyond simply recruiting the enzyme to the PIP2 substrate. We propose that in addition to associating PLCβ with the plasma membrane, the dCTD reorders the αN helix of active Gαq and thus participates directly in the precise positioning of the catalytic domain.
Arrestin-independent internalization of the GLP-1 receptor is facilitated by a GRK, clathrin, and caveolae-dependent mechanism. International-journal

Ee Von Moo, Thor Christian Møller, Frederikke Astrid Sørensen, Asuka Inoue, Hans Bräuner-Osborne

The FEBS journal　292　(7)　1675-1695　2025/04

DOI： 10.1111/febs.17338 　

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The glucagon-like peptide-1 receptor (GLP-1R) plays an important role in regulating insulin secretion and reducing body weight, making it a prominent target in the treatment of type 2 diabetes and obesity. Extensive research on GLP-1R signaling has provided insights into the connection between receptor function and physiological outcomes, such as the correlation between Gs signaling and insulin secretion, yet the exact mechanisms regulating signaling remain unclear. Here, we explore the internalization pathway of GLP-1R, which is crucial for controlling insulin release and maintaining pancreatic beta-cell function. Utilizing a reliable and sensitive time-resolved fluorescence resonance energy transfer (TR-FRET) internalization assay, combined with HEK293-derived knockout cell lines, we were able to directly compare the involvement of different endocytic machinery in GLP-1R internalization. Our findings indicate that the receptor internalizes independently of arrestin and is dependent on Gs and Gi/o activation and G protein-coupled receptor kinase phosphorylation. Mechanistically, we observed that the receptor undergoes distinct clathrin- and caveolae-mediated internalization in HEK293 cells. This study also investigated the role of arrestins in GLP-1R function and regulation. These insights into key endocytic components that are involved in the GLP-1R internalization pathway could enhance the rational design of GLP-1R therapeutics for type 2 diabetes and other GLP-1R-related diseases.
Autocrine/paracrine lysophosphatidylserine signaling suppresses B cell aggregation and tertiary lymphoid structure formation.

Akiharu Uwamizu, Yuji Shinjo, Jumpei Omi, Manae Tatstumi, Kuniyuki Kano, Kumiko Makide, Hajime Kitamura, Michiyo Okudaira, Keita Satoh, Fumiya Fukami, Tasuku Kawano, Tomomitsu Miyasaka, Tomoko Takahashi, Osamu Nakajima, Tomohiko Ohwada, Asuka Inoue, Junken Aoki

iScience　112420-112420　2025/04
Publisher: Elsevier BV
DOI： 10.1016/j.isci.2025.112420 　

ISSN： 2589-0042
Structural insights into lipid chain-length selectivity and allosteric regulation of FFA2. International-journal

Mai Kugawa, Kouki Kawakami, Ryoji Kise, Carl-Mikael Suomivuori, Masaki Tsujimura, Kazuhiro Kobayashi, Asato Kojima, Wakana J Inoue, Masahiro Fukuda, Toshiki E Matsui, Ayami Fukunaga, Junki Koyanagi, Suhyang Kim, Hisako Ikeda, Keitaro Yamashita, Keisuke Saito, Hiroshi Ishikita, Ron O Dror, Asuka Inoue, Hideaki E Kato

Nature communications　16　(1)　2809-2809　2025/03/26

DOI： 10.1038/s41467-025-57983-4 　

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The free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that selectively recognizes short-chain fatty acids to regulate metabolic and immune functions. As a promising therapeutic target, FFA2 has been the focus of intensive development of synthetic ligands. However, the mechanisms by which endogenous and synthetic ligands modulate FFA2 activity remain unclear. Here, we present the structures of the human FFA2-Gi complex activated by the synthetic orthosteric agonist TUG-1375 and the positive allosteric modulator/allosteric agonist 4-CMTB, along with the structure of the inactive FFA2 bound to the antagonist GLPG0974. Structural comparisons with FFA1 and mutational studies reveal how FFA2 selects specific fatty acid chain lengths. Moreover, our structures reveal that GLPG0974 functions as an allosteric antagonist by binding adjacent to the orthosteric pocket to block agonist binding, whereas 4-CMTB binds the outer surface of transmembrane helices 6 and 7 to directly activate the receptor. Supported by computational and functional studies, these insights illuminate diverse mechanisms of ligand action, paving the way for precise GPCR-targeted drug design.
Structure and dynamics determine G protein coupling specificity at a class A GPCR. International-journal

Marina Casiraghi, Haoqing Wang, Patrick C Brennan, Chris Habrian, Harald Hübner, Maximilian F Schmidt, Luis Maul, Biswaranjan Pani, Sherif M F M Bahriz, Bing Xu, Nico Staffen, Tufa E Assafa, Bohan Chen, Elizabeth White, Roger K Sunahara, Asuka Inoue, Yang K Xiang, Robert J Lefkowitz, Ehud Y Isacoff, Nathaniel Nucci, Peter Gmeiner, Michael T Lerch, Brian K Kobilka

Science advances　11　(12)　eadq3971　2025/03/21

DOI： 10.1126/sciadv.adq3971 　

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G protein-coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The β2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.
Discovering Key Activation Hotspots in the M2 Muscarinic Receptor. International-journal

Yuya Sugiura, Tatsuya Ikuta, Yuji Sumii, Hirokazu Tsujimoto, Kohei Suzuki, Ryoji Suno, Putri Nur Arina Binti Mohd Ariff, So Iwata, Norio Shibata, Asuka Inoue, Takuya Kobayashi, Hideki Kandori, Kota Katayama

Journal of the American Chemical Society　147　(14)　11754-11765　2025/03/14

DOI： 10.1021/jacs.4c14385 　

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The M2 muscarinic receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that serves as a model system for understanding ligand recognition and GPCR activation. Here, using vibrational spectroscopy, we identify the mechanisms governing M2R activation by its native agonist, acetylcholine. Combined with mutagenesis, computational chemistry, and organic synthetic chemistry, our analyses found that the precise distance between acetylcholine and Asn404, one of the amino acids constituting the ligand-binding site, is important for M2R activation and that the N404Q mutant undergoes partial active state-like conformational changes. We discovered that a water molecule bridging acetylcholine and Asn404 forms a precise and flexible hydrogen bond network, triggering the outward movement of transmembrane helix 6 in M2R. Consistent with this observation, disruptions in this hydrogen bond network via chemical modification at the α- or β-position of acetylcholine failed to activate M2R. Collectively, our findings pinpoint Asn404 as a critical residue that both senses acetylcholine binding and induces M2R activation.
Endosomal chemokine receptor signalosomes regulate central mechanisms underlying cell migration. International-journal

Hyunggu Hahn, Carole Daly, John Little 4th, Nicole A Perry-Hauser, Emmanuel Flores-Espinoza, Asuka Inoue, Bianca Plouffe, Alex R B Thomsen

eLife　13　2025/02/24

DOI： 10.7554/eLife.99373 　

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Chemokine receptors are GPCRs that regulate the chemotactic migration of a wide variety of cells including immune and cancer cells. Most chemokine receptors contain features associated with the ability to stimulate G protein signaling during β-arrestin-mediated receptor internalization into endosomes. As endosomal signaling of certain non-GPCR receptors plays a major role in cell migration, we chose to investigate the potential role of endosomal chemokine receptor signaling on mechanisms governing this function. Applying a combination of pharmacological and cell biological approaches, we demonstrate that the model chemokine receptor CCR7 recruits G protein and β-arrestin simultaneously upon chemokine stimulation, which enables internalized receptors to activate G protein from endosomes. Furthermore, spatiotemporal-resolved APEX2 proteome profiling shows that endosomal CCR7 uniquely enriches specific Rho GTPase regulators as compared to plasma membrane CCR7, which is directly associated with enhanced activity of the Rho GTPase Rac1 and chemotaxis of immune T cells. As Rac1 drives the formation of membrane protrusions during chemotaxis, our findings suggest an important integrated function of endosomal chemokine receptor signaling in cell migration.
Ligand-Independent Spontaneous Activation of Purinergic P2Y6 Receptor Under Cell Culture Soft Substrate. International-journal

Akiyuki Nishimura, Kazuhiro Nishiyama, Tomoya Ito, Xinya Mi, Yuri Kato, Asuka Inoue, Junken Aoki, Motohiro Nishida

Cells　14　(3)　2025/02/03

DOI： 10.3390/cells14030216 　

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G protein-coupled receptors (GPCRs) exist in the conformational equilibrium between inactive state and active state, where the proportion of active state in the absence of a ligand determines the basal activity of GPCRs. Although many GPCRs have different basal activity, it is still unclear whether physiological stresses such as substrate stiffness affect the basal activity of GPCRs. In this study, we identified that purinergic P2Y6 receptor (P2Y6R) induced spontaneous Ca2+ oscillation without a nucleotide ligand when cells were cultured in a silicon chamber. This P2Y6R-dependent Ca2+ oscillation was absent in cells cultured in glass dishes. Coating substrates, including collagen, laminin, and fibronectin, did not affect the P2Y6R spontaneous activity. Mutation of the extracellular Arg-Gly-Asp (RGD) motif of P2Y6R inhibited spontaneous activity. Additionally, extracellular Ca2+ was required for P2Y6R-dependent spontaneous Ca2+ oscillation. The GPCR screening assay identified cells expressing 10 GPCRs, including purinergic P2Y1R, P2Y2R, and P2Y6R, that exhibited spontaneous Ca2+ oscillation under cell culture soft substrate. Our results suggest that stiffness of the cell adhesion surface modulates spontaneous activities of several GPCRs, including P2Y6R, through a ligand-independent mechanism.
GRK5 regulates endocytosis of FPR2 independent of β-arrestins. International-journal

Christine E Jack, Emily M Cope, Laura Lemel, Meritxell Canals, Julia Drube, Carsten Hoffmann, Asuka Inoue, James N Hislop, Dawn Thompson

The Journal of biological chemistry　301　(2)　108112-108112　2025/02

DOI： 10.1016/j.jbc.2024.108112 　

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The formyl-peptide receptor 2 (FPR2) is a G-protein-coupled receptor that responds to pathogen-derived peptides and regulates both proinflammatory and proresolution cellular processes. While ligand selectivity and G-protein signaling of FPR2 have been well characterized, molecular mechanisms controlling subsequent events such as endocytosis and recycling to the plasma membrane are less understood. Here, we show the key role of the G-protein-coupled receptor kinase 5 (GRK5) in facilitating FPR2 endocytosis and postendocytic trafficking. We found, in response to activation by a synthetic peptide WKYMVm, the recruitment of β-arrestins to the receptor requires both putative phosphorylation sites in the C-terminal region of FPR2 and the presence of GRKs, predominantly GRK5. Furthermore, although GRKs are required for β-arrestin recruitment and endocytosis, the recruitment of β-arrestin is not itself essential for FPR2 endocytosis. Instead, β-arrestin determines postendocytic delivery of FPR2 to subcellular compartments and subsequent plasma membrane delivery and controls the magnitude of downstream signal transduction. Collectively, the newly characterized FPR2 molecular pharmacology will facilitate the design of more efficient therapeutics targeting chronic inflammation.
Role of the G protein-coupled receptor kinase 2/3 N terminus in discriminating the endocytic effects of opioid agonist drugs. International-journal

Joy Li, Asuka Inoue, Aashish Manglik, Mark von Zastrow

Molecular pharmacology　107　(1)　100003-100003　2025/01

DOI： 10.1124/molpharm.124.000951 　

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Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins, and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by G protein-coupled receptor kinases (GRKs) from the GRK2/3 subfamily. While these relatively "downstream" steps in the agonist selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished "upstream" by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs and that agonist selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved Pleckstrin homology domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and uses the NTD as an agonist-selective sensor. SIGNIFICANCE STATEMENT: This study investigates how G protein-coupled receptor kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-type opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N terminus is a key determinant of agonist discrimination.
Optimized 5-HT2b inhibitors for neuropsychiatric syndromes with cognitive dysfunction. International-journal

Saktimayee M Roy, Erica Acquarone, Elentina K Argyrousi, Hong Zhang, Agnieszka Staniszewski, Asuka Inoue, Joshua J Ziarek, Ottavio Arancio, D Martin Watterson

Alzheimer's & dementia (New York, N. Y.)　11　(1)　e70073　2025

DOI： 10.1002/trc2.70073 　

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INTRODUCTION: Neuropsychiatric syndromes such as anxiety and agitation are clinical presentations common to diverse neurodegenerative diseases and brain injury sequelae. They are a concern due to the impact on cognition, social interactions, and non-pharmacological treatments. Cognitive or behavioral disturbances occur at early disease stages and increase with disease progression. Coincident pathologies include the loss of serotonin (5-HT) neurons and appearance of neurofibrillary tangles in the raphe nucleus. Brain 5-HT2b receptor (5-HT2bR) levels are increased in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and post-stroke morbidity. HTR2B gene variants are implicated in psychiatric disorders. 5-HTRs are associated with atypical neurotropic drug mechanisms and behavioral dysfunction in drug abuse. The accumulating body of evidence suggests that selective 5-HT2bR inhibition might mitigate neuropsychiatric syndromes and the associated cognitive dysfunction. Atypical neurotropic drugs interact with a variety of monoamine receptors and outcomes are viewed as a combination of 5-HT and dopamine D2 receptor mediated actions. Clearly, there is a need for insight into precision 5-HT2bR inhibition as a potential pharmacological mechanism for treatment of neuropsychiatric syndromes and cognitive dysfunction associated with dementia. METHODS: Strategic optimization of an atypical neurotropic drug was used to develop MW073, a highly selective and orally bioavailable inhibitor of 5-HT2bR activity and β-arrestin-1 recruitment that is devoid of dopamine receptor recognition and risk of 5-HT2bR agonist activity. RESULTS: MW073 ameliorates amyloid and tau induction of behavioral dysfunction in preventive or disease stage intervention paradigms. Using MW073 as a standard of comparison, risperidone was shown to be a dose-dependent inhibitor 5-HT2bR activity and β-arrestin-1 recruitment. DISCUSSION: Selective inhibition of 5-HT2bR activity is a viable mechanism for the treatment of neuropsychiatric syndromes with synaptic dysfunction as a root cause and is a previously unrealized pharmacodynamic mechanism potentially embedded in current neurotherapeutics. HIGHLIGHTS: A new highly selective 5-HT2bR antagonist, MW073, is described and used as a reference standard.MW073 attenuates synaptic and behavioral dysfunctions an animal models of neuropsychatric syndromes.Risperidone is a dose dependent inhibitor of 5-HT2bR activity and arrestin recruitment.
Structure-Signal Relationships of the δ-Opioid-Receptor (DOR)-Selective Agonist KNT-127-Part I: Impact of the Morphinan Skeleton on the G-Protein-Biased DOR Agonism.

Keita Kajino, Tomoya Sugai, Ryoji Kise, Riko Suzuki, Akihisa Tokuda, Yuki Sekiya, Tomoya Kakumoto, Risako Katamoto, Noriki Kutsumura, Yasuyuki Nagumo, Asuka Inoue, Tsuyoshi Saitoh

Chemical & pharmaceutical bulletin　73　(3)　246-256　2025

DOI： 10.1248/cpb.c25-00012 　

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The δ-opioid receptor (DOR) is a promising target for developing novel analgesics due to its lower risk of causing side effects compared to the μ-opioid receptor (MOR), which is commonly associated with dependence, respiratory depression, and other adverse effects. KNT-127, a DOR-selective agonist with a morphinan skeleton, offers analgesic and antidepressant benefits without inducing convulsions at therapeutic doses, unlike the conventional DOR agonist SNC80. While previous studies have suggested that KNT-127 exhibits reduced β-arrestin recruitment, a signaling pathway implicated in adverse opioid effects, the ligand structural basis for this biased signaling remains unclear. In this study, we explored the structure-signal relationships of KNT-127, focusing on its quinoline moiety, which is known to serve as an address domain responsible for DOR selectivity. Modifying the quinoline moiety by removing the aromatic rings reduced DOR selectivity and potency in relation to G-protein activation while diminishing both the potency and efficacy of β-arrestin recruitment. These results suggest that the morphinan skeleton is critical for reduced β-arrestin recruitment, while the quinoline moiety differentially modulates G-protein activation and β-arrestin recruitment. Together, our study expands the message-address concept, previously limited to receptor selectivity, by providing structural insights into the G-protein-biased agonism of DOR agonists, thereby guiding the design of safer DOR-targeting therapeutics.
Structure-Signal Relationships of the δ-Opioid-Receptor (DOR)-Selective Agonist KNT-127-Part II: Quinoline Ring Modifications for Enhanced G-Protein Signaling and Reduced β-Arrestin Recruitment.

Keita Kajino, Tomoya Sugai, Tomoya Kakumoto, Ryoji Kise, Riko Suzuki, Akihisa Tokuda, Yuki Sekiya, Risako Katamoto, Noriki Kutsumura, Yasuyuki Nagumo, Takatsugu Hirokawa, Asuka Inoue, Tsuyoshi Saitoh

Chemical & pharmaceutical bulletin　73　(4)　336-348　2025

DOI： 10.1248/cpb.c24-00796 　

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The δ-opioid receptor (DOR) continues to attract attention as a therapeutic target for the development of safer analgesics due to its ability to mediate pain relief with a lower risk of adverse effects compared to the μ-opioid receptor (MOR). Building upon our previous findings on KNT-127, a DOR-selective agonist with a morphinan scaffold, this study further explores the structure-signal relationships between quinoline ring modifications and the signaling bias toward Gi-protein activation while minimizing β-arrestin-2 recruitment. Our findings highlight the critical role of the 5'-position in modulating signaling bias. Bulky hydrophobic substituents, such as isopropoxy and cyclohexanoxy groups, effectively reduce β-arrestin-2 recruitment without compromising DOR binding affinity or Gi-protein activation. Molecular-docking and molecular dynamics simulations provided mechanistic insights, showing that these modifications change ligand interactions with the V2816.55-W2846.58-L3007.35 sub-pocket, thus selectively favoring Gi-protein signaling. These insights clarify the key interactions for the signaling bias in DOR agonists, offering a new framework for the design of DOR-targeted therapies with an improved therapeutic profile.
Detection of Human GPCR Activity in Drosophila S2 Cells Using the Tango System. International-journal

Emil Salim, Aki Hori, Kohei Matsubara, Toshiyuki Takano-Shimizu, Andre Rizky Pratomo, Marianne Marianne, Armia Syahputra, Dadang Irfan Husori, Asuka Inoue, Maryam Aisyah Abdullah, Nur Farisya Shamsudin, Kamal Rullah, Takayuki Kuraishi

International journal of molecular sciences　26　(1)　2024/12/29

DOI： 10.3390/ijms26010202 　

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G protein-coupled receptors (GPCRs) are essential cell surface proteins involved in transducing extracellular signals into intracellular responses, regulating various physiological processes. This study validated the use of the Tango assay, a sensitive method for detecting GPCR activation, in Drosophila Schneider 2 (S2) cells, focusing on the human Dopamine Receptor D4 (DRD4). Plasmids encoding the LexA-tagged human DRD4 receptor and a luciferase reporter were co-transfected into Drosophila S2 cells and stimulated with dopamine. Receptor activation was measured by quantifying the luciferase activity. The system showed high specificity for dopamine, with no activation in response to octopamine, a non-ligand for DRD4. Furthermore, the system effectively detects activation by a novel compound. These results demonstrate that Drosophila S2 cells, coupled with the Tango assay, provide a viable model for studying human GPCR function and ligand specificity. This system enables the rapid screening of potential GPCR ligands in a cost-effective cellular model.
Design of allosteric modulators that change GPCR G protein subtype selectivity. International-journal

Madelyn N Moore, Kelsey L Person, Abigail Alwin, Campbell Krusemark, Noah Foster, Caroline Ray, Asuka Inoue, Michael R Jackson, Michael J Sheedlo, Lawrence S Barak, Ezequiel Marron Fernandez de Velasco, Steven H Olson, Lauren M Slosky

Research square　2024/12/11

DOI： 10.21203/rs.3.rs-5538058/v1 　

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G protein-coupled receptors (GPCRs), the largest family of drug targets, can signal through 16 subtypes of Gα proteins. Biased compounds that selectively activate therapy-relevant pathways promise to be safer, more effective medications. The determinants of bias are poorly understood, however, and rationally-designed, G protein-subtype-selective compounds are lacking. Here, using the prototypical class A GPCR neurotensin receptor 1 (NTSR1), we find that small molecules binding the intracellular GPCR-transducer interface change G protein coupling by subtype-specific and predictable mechanisms, enabling rational drug design. We demonstrate that the compound SBI-553 switches NTSR1 G protein preference by acting both as a molecular bumper and a molecular glue. Structurally, SBI-553 occludes G protein binding determinants on NTSR1, promoting association with select G protein subtypes for which an alternative, shallow-binding conformation is energetically favorable. Minor modifications to the SBI-553 scaffold produce allosteric modulators with distinct G protein subtype selectivity profiles. Selectivity profiles are probe-independent, conserved across species, and translate to differences in in vivo activity. These studies demonstrate that G protein selectivity can be tailored with small changes to a single chemical scaffold targeting the receptor-transducer interface and, as this pocket is broadly conserved, present a strategy for pathway-selective drug discovery applicable to the diverse GPCR superfamily.
Conjugated fatty acids drive ferroptosis through chaperone-mediated autophagic degradation of GPX4 by targeting mitochondria. International-journal Peer-reviewed

Yusuke Hirata, Yuto Yamada, Soma Taguchi, Ryota Kojima, Haruka Masumoto, Shinnosuke Kimura, Takuya Niijima, Takashi Toyama, Ryoji Kise, Emiko Sato, Yasunori Uchida, Junya Ito, Kiyotaka Nakagawa, Tomohiko Taguchi, Asuka Inoue, Yoshiro Saito, Takuya Noguchi, Atsushi Matsuzawa

Cell death & disease　15　(12)　884-884　2024/12/06

DOI： 10.1038/s41419-024-07237-w 　

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Conjugated fatty acids (CFAs) have been known for their anti-tumor activity. However, the mechanism of action remains unclear. Here, we identify CFAs as inducers of glutathione peroxidase 4 (GPX4) degradation through chaperone-mediated autophagy (CMA). CFAs, such as (10E,12Z)-octadecadienoic acid and α-eleostearic acid (ESA), induced GPX4 degradation, generation of mitochondrial reactive oxygen species (ROS) and lipid peroxides, and ultimately ferroptosis in cancer cell lines, including HT1080 and A549 cells, which were suppressed by either pharmacological blockade of CMA or genetic deletion of LAMP2A, a crucial molecule for CMA. Mitochondrial ROS were sufficient and necessary for CMA-dependent GPX4 degradation. Oral administration of an ESA-rich oil attenuated xenograft tumor growth of wild-type, but not that of LAMP2A-deficient HT1080 cells, accompanied by increased lipid peroxidation, GPX4 degradation and cell death. Our study establishes mitochondria as the key target of CFAs to trigger lipid peroxidation and GPX4 degradation, providing insight into ferroptosis-based cancer therapy.
A molecular mechanism to diversify Ca²⁺ signaling downstream of Gs protein-coupled receptors International-journal

Julian Brands, Sergi Bravo, Lars Jürgenliemke, Lukas Grätz, Hannes Schihada, Fabian Frechen, Judith Alenfelder, Cy Pfeil, Paul Georg Ohse, Suzune Hiratsuka, Kouki Kawakami, Luna C. Schmacke, Nina Heycke, Asuka Inoue, Gabriele König, Alexander Pfeifer, Dagmar Wachten, Gunnar Schulte, Torsten Steinmetzer, Val J. Watts, Jesús Gomeza, Katharina Simon, Evi Kostenis

Nature Communications　15　(1)　7684-7684　2024/12

DOI： 10.1038/s41467-024-51991-6 　

eISSN： 2041-1723
G12/13-mediated signaling stimulates hepatic glucose production and has a major impact on whole body glucose homeostasis. International-journal

Srinivas Pittala, Dhanush Haspula, Yinghong Cui, Won-Mo Yang, Young-Bum Kim, Roger J Davis, Allison Wing, Yaron Rotman, Owen P McGuinness, Asuka Inoue, Jürgen Wess

Nature communications　15　(1)　9996-9996　2024/11/19

DOI： 10.1038/s41467-024-54299-7 　

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Altered hepatic glucose fluxes are critical during the pathogenesis of type 2 diabetes. G protein-coupled receptors represent important regulators of hepatic glucose production. Recent studies have shown that hepatocytes express GPCRs that can couple to G12/13, a subfamily of heterotrimeric G proteins that has attracted relatively little attention in the past. Here we show, by analyzing several mutant mouse strains, that selective activation of hepatocyte G12/13 signaling leads to pronounced hyperglycemia and that this effect involves the stimulation of the ROCK1-JNK signaling cascade. Using both mouse and human hepatocytes, we also show that activation of endogenous sphingosine-1-phosphate type 1 receptors strongly promotes glucose release in a G12/13-dependent fashion. Studies with human liver samples indicate that hepatic GNA12 (encoding Gα12) expression levels positively correlate with indices of insulin resistance and impaired glucose homeostasis, consistent with a potential pathophysiological role of enhanced hepatic G12/13 signaling.
Chemogenetic activation of hepatic G12 signaling ameliorates hepatic steatosis and obesity. International-journal

Kaito Arai, Yuki Ono, Natsumi Hirai, Yuki Sugiura, Keizo Kaneko, Shigeru Matsuda, Keita Iio, Keita Kajino, Tsuyoshi Saitoh, Fan-Yan Wei, Hideki Katagiri, Asuka Inoue

Biochimica et biophysica acta. Molecular basis of disease　1871　(2)　167566-167566　2024/11/12

DOI： 10.1016/j.bbadis.2024.167566 　

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OBJECTIVE: Hepatic steatosis, the early stage of nonalcoholic fatty liver disease (NAFLD), currently lacks targeted pharmacological treatments. G protein-coupled receptors (GPCRs) in hepatocytes differentially regulate lipid metabolism depending on their coupling profile of G protein subtypes. Unlike Gs, Gi, and Gq signaling, the role of G12 signaling in hepatic steatosis remains elusive. The objective of this study was to investigate the effect of G12 signaling on hepatic steatosis and obesity and its mechanisms. METHODS: We generated mice expressing a G12-coupled designer GPCR in a liver-specific manner. We performed phenotypic analysis in the mice under the condition of fasting (acute hepatic steatosis model) or high-fat diet feeding (chronic hepatic steatosis model). RESULTS: In acute and chronic hepatic steatosis models, chemogenetic activation of hepatic G12 signaling suppressed the progression of hepatic steatosis. The treatment led to an increased triglyceride secretion with little effect on mitochondrial respiratory activity, fatty acid oxidation, de novo lipogenesis, and fatty acid uptake. Furthermore, in a high-fat-diet-induced obesity model, activation of the G12-coupled designer GPCR exerted anti-obesity effects with increased whole-body energy expenditure and fat oxidation. Anti-FGF21 antibody treatment showed that the anti-obesity effects of the hepatic G12D activation relied in part on the hepatokine FGF21. CONCLUSIONS: Our findings indicate that the activation of G12 signaling in the liver has the potential to prevent hepatic steatosis and obesity. This discovery provides a strong rationale for the development of drugs targeting G12-coupled GPCRs expressed in the liver.
Role of the GRK2/3 N-terminus in discriminating the endocytic effects of opioid agonist drugs. International-journal

Joy Li, Asuka Inoue, Aashish Manglik, Mark von Zastrow

Molecular pharmacology　2024/11/06

DOI： 10.1124/molpharm.124.000951 　

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Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by GPCR kinases (GRKs) from the GRK2/3 subfamily. While these relatively 'downstream' steps in the agonist-selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished 'upstream' by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs, and that agonist-selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved PH domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist-selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist-selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and utilizes the NTD as an agonist-selective sensor. Significance Statement This study investigates how GPCR kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist-selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N-terminus is a key determinant of agonist discrimination.
Signal profiles and spatial regulation of β-arrestin recruitment through Gβ5 and GRK3 at the μ-opioid receptor International-journal

Carlo Marion C. Carino, Suzune Hiratsuka, Ryoji Kise, Gaku Nakamura, Kouki Kawakami, Masataka Yanagawa, Asuka Inoue

European Journal of Pharmacology　987　177151-177151　2024/11
Publisher: Elsevier BV
DOI： 10.1016/j.ejphar.2024.177151 　

ISSN： 0014-2999

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The μ-opioid receptor (MOR) is a G-protein-coupled receptor (GPCR) that mediates both analgesic effects and adverse effects of opioid drugs. Despite extensive efforts to develop a signal-biased drug, drugs with sufficiently reduced side effects have not been established, in part owing to lack of comprehensive signal transducer profiles of MOR. In this study, by profiling the activity of signal transducers including G proteins and GPCR kinases (GRKs), we revealed an unprecedented mechanism of selective GRK3 activation by Gβ5, leading to β-arrestin recruitment. By utilizing multiple genome-edited cell lines and functional assays, we found that oliceridine, an FDA-approved G-protein-biased agonist, selectively activates Gαz- and GRK3-mediated signaling. Notably, among the five Gβ subtypes, only Gβ5 distinguishes GRK3 from GRK2. Using single-molecule imaging, we found that GRK3 is recruited to the plasma membrane upon MOR agonist stimulation by Gβ1 and Gβ5, yet their interaction dynamics with GRK3 and mechanisms of action are different. Furthermore, particle diffusion analysis suggests that Gβ5 is enriched in confined membrane domains, through which GRK3 is recruited to the plasma membrane in a freely diffusible state, thereby allowing GRK3 to efficiently interact with MOR. These findings provide a mechanism by which MOR agonists rely on a specific Gα-Gβ-GRK axis to induce β-arrestin recruitment.
The repertoire of G-protein-coupled receptor variations in the Japanese population 54KJPN. International-journal

Tatsuya Ikuta, Riko Suzuki, Asuka Inoue

Genes to cells : devoted to molecular & cellular mechanisms　29　(11)　1026-1036　2024/11

DOI： 10.1111/gtc.13164 　

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G-protein-coupled receptors (GPCRs) are the largest superfamily in the human genome and the major targets for the market drugs. Recent massive genomics studies revealed numerous natural variations in the general population. 54KJPN is the most extensive Japanese population genomics study, curating the whole genome sequences from about 54,000 individuals. Here, by analyzing 390 non-olfactory GPCR genes in the 54KJPN dataset, we annotated 25,443 missense single-nucleotide variations. Among them, we found 120 major variations that appear with an allele frequency greater than 0.5, including variations that occurred on posttranslational modification sites. Structural alignment of GPCRs using the generic numbering system in the GPCRdb reveals enrichment of alterations in the conserved arginine residue within the DRY motif, which contributes to downstream G-protein signaling. A comparison with the worldwide 1000 Genomes Project (1KGP) dataset found 23 variations that were present exclusively in the 54KJPN dataset. This study will be the basis for future pharmacogenomics studies for the Japanese population.
Diverse pathways in GPCR-mediated activation of Ca2+ mobilization in HEK293 cells. International-journal

Francesco De Pascali, Asuka Inoue, Jeffrey L Benovic

The Journal of biological chemistry　300　(11)　107882-107882　2024/10/10

DOI： 10.1016/j.jbc.2024.107882 　

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G protein-coupled receptors transduce extracellular stimuli into intracellular signaling. Ca2+ is a well-known second messenger that can be induced by G protein-coupled receptor activation through the primary canonical pathways involving Gαq- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some Gs-coupled receptors are shown to trigger Ca2+ mobilization, underlying mechanisms remain elusive. Here, we evaluated whether Gs-coupled receptors including the β2-adrenergic receptor (β2AR) and the prostaglandin EP2 and EP4 receptors (EP2R and EP4R) that are endogenously expressed in human embryonic kidney 293 (HEK293) cells utilize common pathways for mediating Ca2+ mobilization. For the β2AR, we found an essential role for Gq in agonist-promoted Ca2+ mobilization while genetic or pharmacological inhibition of Gs or Gi had minimal effect. β-agonist-promoted Ca2+ mobilization was effectively blocked by the Gq-selective inhibitor YM-254890 and was not observed in ΔGαq/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β2AR with Gq. For the EP2R, which couples to Gs, agonist treatment induced Ca2+ mobilization in a pertussis toxin-sensitive but YM-254890-insensitive manner. In contrast, EP4R, which couples to Gs and Gi, exhibited Ca2+ mobilization that was sensitive to both pertussis toxin and YM-254890. Interestingly, both EP2R and EP4R were largely unable to induce Ca2+ mobilization in ΔGαs or ΔPLCβ cells, supporting a strong dependency on Gs signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are used to mediate Ca2+ mobilization in HEK293 cells where the β2AR primarily uses Gq, EP2R uses Gs and Gi, and EP4R uses Gs, Gi, and Gq.
Amylin receptor subunit interactions are modulated by agonists and determine signaling. International-journal

Sandra E Gostynska, Jordan A Karim, Bailee E Ford, Peyton H Gordon, Katie M Babin, Asuka Inoue, Nevin A Lambert, Augen A Pioszak

bioRxiv : the preprint server for biology　2024/10/09

DOI： 10.1101/2024.10.09.617487 　

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Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY1R, AMY2R, and AMY3R are heterodimers consisting of the G protein-coupled calcitonin receptor (CTR) paired with a RAMP1, -2, or -3 accessory subunit, respectively, which increases amylin potency. Little is known about AMYR subunit interactions and their role in signaling. Here, we show that the AMYRs have distinct basal subunit equilibriums that are modulated by peptide agonists and determine the cAMP signaling phenotype. Using a novel biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY1/2R subunit equilibriums favored free CTR and RAMP1/2, and rat amylin and αCGRP agonists promoted subunit association. A stronger CTR-RAMP3 transmembrane domain interface yielded a more stable AMY3R, and human and salmon calcitonin agonists promoted AMY3R dissociation. Similar changes in subunit association-dissociation were observed in live cell membranes, and G protein coupling and cAMP signaling assays showed how these altered signaling. Our findings reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.
Cell swelling enhances ligand-driven β-adrenergic signaling. International-journal

Alexei Sirbu, Marc Bathe-Peters, Jothi L M Kumar, Asuka Inoue, Martin J Lohse, Paolo Annibale

Nature communications　15　(1)　7822-7822　2024/09/07

DOI： 10.1038/s41467-024-52191-y 　

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G protein-coupled receptors' conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the β2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors' function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the β2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.
Insights into lysophosphatidylserine recognition and Gα12/13-coupling specificity of P2Y10. International-journal

Han Yin, Nozomi Kamakura, Yu Qian, Manae Tatsumi, Tatsuya Ikuta, Jiale Liang, Zhenmei Xu, Ruixue Xia, Anqi Zhang, Changyou Guo, Asuka Inoue, Yuanzheng He

Cell chemical biology　31　(11)　1899-1908　2024/08/29

DOI： 10.1016/j.chembiol.2024.08.005 　

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The lysophosphatidylserine (LysoPS) receptor P2Y10, also known as LPS2, plays crucial roles in the regulation of immune responses and holds promise for the treatment of autoimmune diseases. Here, we report the cryoelectron microscopy (cryo-EM) structure of LysoPS-bound P2Y10 in complex with an engineered G13 heterotrimeric protein. The structure and a mutagenesis study highlight the predominant role of a comprehensive polar network in facilitating the binding and activation of the receptor by LysoPS. This interaction pattern is preserved in GPR174, but not in GPR34. Moreover, our structural study unveils the essential interactions that underlie the Gα13 engagement of P2Y10 and identifies key determinants for Gα12-vs.-Gα13-coupling selectivity, whose mutations selectively disrupt Gα12 engagement while preserving the intact coupling of Gα13. The combined structural and functional studies provide insights into the molecular mechanisms of LysoPS recognition and Gα12/13 coupling specificity.
Intracellular <scp>TAS2Rs</scp> act as a gatekeeper for the excretion of harmful substances via <scp>ABCB1</scp> in keratinocytes International-journal

Sazanami Mori, Natsuki Nakamura, Ayane Fuchigami, Satoshi Yoshimoto, Moe Sakakibara, Toshiyuki Ozawa, Junken Aoki, Asuka Inoue, Hayakazu Sumida, Hideya Ando, Motonao Nakamura

FASEB BioAdvances　6　(10)　424-441　2024/08/27
Publisher: Wiley
DOI： 10.1096/fba.2024-00074 　

ISSN： 2573-9832

eISSN： 2573-9832

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Abstract Bitter taste receptors (TAS2Rs) are not only expressed in the oral cavity but also in skin. Extraoral TAS2Rs are thought to be involved in non‐taste perception and tissue‐specific functions. Keratinocytes that express TAS2Rs in the skin provide a first‐line defense against external threats. However, the functional roles of these receptors in host defense remain unclear. Here, we demonstrated the sensory role of intracellularly located TAS2Rs against toxic substances in keratinocytes. Although many G protein‐coupled receptors elicit signals from the surface, TAS2Rs were found to localize intracellularly, possibly to the ER, in human keratinocytes and HaCaT cells. TAS2R38, one of the TAS2R members, activated the G_α12/13/RhoA/ROCK/p38 MAP kinase/NF‐κB pathway upon stimulation by phenylthiocarbamide (PTC), an agonist for this receptor, leading to the production of ABC transporters, such as ABCB1, in these cells. Notably, treatment with bitter compounds, such as PTC and saccharin, induced the upregulation of ABCB1 in HaCaT cells. Mechanistically, intracellular TAS2R38 and its downstream signaling G_α12/13/RhoA/ROCK/p38 MAP kinase/NF‐κB pathway were identified to be responsible for the above effect. Pretreatment with PTC prevented the accumulation of rhodamine 123 because of its excretion via ABCB1. Furthermore, pretreatment with PTC or saccharin counteracted the effect of the toxic compound, diphenhydramine, and pretreated HaCaT cells were found to proliferate faster than untreated cells. This anti‐toxic effect was suppressed by treatment with verapamil, an ABCB1 inhibitor, indicating that enhanced ABCB1 helps clear toxic substances. Altogether, harmless activators of TAS2Rs may be promising drugs that enhance the excretion of toxic substances from the human skin.
Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor. International-journal

Shirsha Saha, Basavraj Khanppnavar, Jagannath Maharana, Heeryung Kim, Carlo Marion C Carino, Carole Daly, Shane Houston, Saloni Sharma, Nashrah Zaidi, Annu Dalal, Sudha Mishra, Manisankar Ganguly, Divyanshu Tiwari, Poonam Kumari, Gagan Deep Jhingan, Prem N Yadav, Bianca Plouffe, Asuka Inoue, Ka Young Chung, Ramanuj Banerjee, Volodymyr M Korkhov, Arun K Shukla

Cell　187　(17)　4751-4769　2024/08/22

DOI： 10.1016/j.cell.2024.07.005 　

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The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.
Molecular mechanism of the endothelin receptor type B interactions with Gs, Gi, and Gq. International-journal

Donghee Ham, Wataru Shihoya, Osamu Nureki, Asuka Inoue, Ka Young Chung

Structure (London, England : 1993)　32　(10)　1632-1639　2024/07/15

DOI： 10.1016/j.str.2024.06.020 　

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The endothelin receptor type B (ETB) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ETB activation. ETB coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ETB in ETB-Gi1 or ETB-Gq coupling. Although The C terminus of Gα is essential for ETB-Gi1 and ETB-Gq coupling, ETB ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ETB with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ETB-induced activation.
Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling. International-journal

Yusman Manchanda, Liliane ElEid, Affiong I Oqua, Zenouska Ramchunder, Jiyoon Choi, Maria M Shchepinova, Guy A Rutter, Asuka Inoue, Edward W Tate, Ben Jones, Alejandra Tomas

Science signaling　17　(843)　eabq7038　2024/07/02

DOI： 10.1126/scisignal.abq7038 　

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Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked β-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.
G protein-coupled receptor endocytosis generates spatiotemporal bias in β-arrestin signaling. International-journal

András D Tóth, Bence Szalai, Orsolya T Kovács, Dániel Garger, Susanne Prokop, Eszter Soltész-Katona, András Balla, Asuka Inoue, Péter Várnai, Gábor Turu, László Hunyady

Science signaling　17　(842)　eadi0934　2024/06/25

DOI： 10.1126/scisignal.adi0934 　

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The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of β-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the β-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine β-arrestin recruitment. The ligand-dependent variance in β-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the β-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-β-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-β-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained β-arrestin binding: the V2 vasopressin receptor and a mutant β2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in β-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.
Structure and dynamics of the pyroglutamylated RF-amide peptide QRFP receptor GPR103. International-journal

Aika Iwama, Ryoji Kise, Hiroaki Akasaka, Fumiya K Sano, Hidetaka S Oshima, Asuka Inoue, Wataru Shihoya, Osamu Nureki

Nature communications　15　(1)　4769-4769　2024/06/19

DOI： 10.1038/s41467-024-49030-5 　

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Pyroglutamylated RF-amide peptide (QRFP) is a peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates a class A G-protein-coupled receptor (GPCR) GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP26-GPR103-Gq complex at 3.19 Å resolution. QRFP26 adopts an extended structure bearing no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains of GPR103 respectively. This movement, reminiscent of class B1 GPCRs except for orientation and structure of the ligand, is critical for the high-affinity binding and receptor specificity of QRFP26. Mutagenesis experiments validate the functional importance of the binding mode of QRFP26 by GPR103. Structural comparisons with closely related receptors, including RY-amide peptide-recognizing GPCRs, revealed conserved and diversified peptide recognition mechanisms, providing profound insights into the biological significance of RF-amide peptides. Collectively, this study not only advances our understanding of GPCR-ligand interactions, but also paves the way for the development of novel therapeutics targeting metabolic and appetite disorders and emergency medical care.
ArreSTick motif controls β-arrestin-binding stability and extends phosphorylation-dependent β-arrestin interactions to non-receptor proteins. International-journal

András Dávid Tóth, Eszter Soltész-Katona, Katalin Kis, Viktor Guti, Sharon Gilzer, Susanne Prokop, Roxána Boros, Ádám Misák, András Balla, Péter Várnai, Lilla Turiák, András Ács, László Drahos, Asuka Inoue, László Hunyady, Gábor Turu

Cell reports　43　(5)　114241-114241　2024/05/16

DOI： 10.1016/j.celrep.2024.114241 　

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The binding and function of β-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established β-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with β-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and β-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by β-arrestin2. Our findings unveil a broader role for β-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.
Identification of Gα12-vs-Gα13-coupling determinants and development of a Gα12/13-coupled designer GPCR. International-journal

Manae Tatsumi, Christian Cruz, Nozomi Kamakura, Riku Kuwabara, Gaku Nakamura, Tatsuya Ikuta, Ravinder Abrol, Asuka Inoue

Scientific reports　14　(1)　11119-11119　2024/05/15

DOI： 10.1038/s41598-024-61506-4 　

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G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα12 and Gα13 belong to the G12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα12 or Gα13, highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα12-coupled designer receptor exclusively activated by designer drugs (DREADD; G12D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα12-vs-Gα13 selectivity. Residue-swapping experiments showed that G12D distinguishes differences between Gα12 and Gα13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378G.H5.23 in Gα12 interacts with N1032.39, S1693.53 and Y17634.53 in G12D, while H364G.H5.09 in Gα12 interact with Q2645.71 in G12D. Screening of mutations at these positions in G12D identified G12D mutants that enhanced coupling with Gα12 and to an even greater extent with Gα13. Combined mutations, most notably the dual Y17634.53H and Q2645.71R mutant, further enhanced Gα12/13 coupling, thereby serving as a potential Gα12/13-DREADD. Such novel Gα12/13-DREADD may be useful in future efforts to develop drugs that target Gα12/13 signaling as well as to identify their therapeutic indications.
Development and basic performance verification of a rapid homogeneous bioassay for agonistic antibodies against the thyroid-stimulating hormone receptor. International-journal

Motoki Hoshina, Shiomi Ojima, Atsushi Kawasaki, Kosuke Doi, Satoshi Ohta, Asuka Inoue, Hiroshi Murayama

Journal of immunological methods　528　113655-113655　2024/05

DOI： 10.1016/j.jim.2024.113655 　

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Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).1 The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor. The measurement consists of three steps: thawing frozen cells, blood sample addition, and luminescence detection. The procedures can be conducted within 1 h. The World Health Organization International Standard TSAb (NIBSC 08/204) stimulated the cells co-expressing TSHR and cAMP biosensor. The intra- and inter-assay coefficients of variance were < 10%. Stimulation activity using wild-type TSHR and chimeric TSHR (Mc4) almost completely correlated with the tested Graves' disease and normal samples. In the rapid-TSAb assay, the evaluation of 39 samples, including TSHR antibody-positive sera, yielded a sensitivity of 100.0% and a specificity of 90.9%, compared to the TSAb-EIA control. The rapid-TSAb assay enables simple and rapid measurement of TSAb and is promising for improving the diagnosis of autoimmune thyroid diseases.
A cAMP-biosensor-based assay for measuring plasma arginine-vasopressin levels. International-journal

Kosuke Doi, Kouki Kawakami, Tatsuya Ikuta, Asuka Inoue

Scientific reports　14　(1)　9453-9453　2024/04/24

DOI： 10.1038/s41598-024-60035-4 　

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Arginine-vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.
The role of G protein-coupled receptor kinases in GLP-1R β-arrestin recruitment and internalisation

Samantha M. McNeill, Jessica Lu, Carlo Marion C. Carino, Asuka Inoue, Peishen Zhao, Patrick M. Sexton, Denise Wootten

Biochemical Pharmacology　222　116119-116119　2024/04
Publisher: Elsevier BV
DOI： 10.1016/j.bcp.2024.116119 　

ISSN： 0006-2952
Structural and Dynamic Insights into the Biased Signaling Mechanism of the Human Kappa Opioid Receptor

Suno-Ikeda C, Nishikawa R, Suzuki R, Iwata S, Takai T, Ogura T, Hirose M, Inoue A, Asai E, Kise R, Sugita Y, Kato T, Nagase H, Saitoh T, Katayama K, Suno R

2024/04

DOI： 10.1101/2024.04.11.588819 　
GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics. International-journal

Davide Zecchin, Nicole Knöpfel, Anna K Gluck, Mark Stevenson, Aimie Sauvadet, Satyamaanasa Polubothu, Sara Barberan-Martin, Fanourios Michailidis, Dale Bryant, Asuka Inoue, Kate E Lines, Fadil M Hannan, Robert K Semple, Rajesh V Thakker, Veronica A Kinsler

The Journal of investigative dermatology　144　(4)　811-819　2024/04

DOI： 10.1016/j.jid.2023.08.028 　

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Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.
Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq. International-journal

Donghee Ham, Asuka Inoue, Jun Xu, Yang Du, Ka Young Chung

Communications biology　7　(1)　362-362　2024/03/23

DOI： 10.1038/s42003-024-06056-1 　

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Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. We propose a detailed analysis of M3-Gq coupling through examination of previously well-defined binding interfaces and neglected regions. Our findings suggest potential binding interfaces between M3 and Gq in pre-assembled and functionally active complexes. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling.
Suppression of Mast Cell Activation by GPR35: GPR35 Is a Primary Target of Disodium Cromoglycate. International-journal

Masumi Oka, Sohta Akaki, Osamu Ohno, Maho Terasaki, Yuho Hamaoka-Tamura, Michiko Saito, Shinichi Kato, Asuka Inoue, Junken Aoki, Kenji Matsuno, Kazuyuki Furuta, Satoshi Tanaka

The Journal of pharmacology and experimental therapeutics　389　(1)　76-86　2024/03/15

DOI： 10.1124/jpet.123.002024 　

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Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.
Identification of oleic acid as an endogenous ligand of GPR3. International-journal

Yangjie Xiong, Zhenmei Xu, Xinzhi Li, Yuqin Wang, Jing Zhao, Na Wang, Yaning Duan, Ruixue Xia, Zhengbin Han, Yu Qian, Jiale Liang, Anqi Zhang, Changyou Guo, Asuka Inoue, Yu Xia, Zheng Chen, Yuanzheng He

Cell research　34　(3)　232-244　2024/03

DOI： 10.1038/s41422-024-00932-5 　

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Although GPR3 plays pivotal roles in both the nervous system and metabolic processes, such as cold-induced thermogenesis, its endogenous ligand remains elusive. Here, by combining structural approach (including cryo-electron microscopy), mass spectrometry analysis, and functional studies, we identify oleic acid (OA) as an endogenous ligand of GPR3. Our study reveals a hydrophobic tunnel within GPR3 that connects the extracellular side of the receptor to the middle of plasma membrane, enabling fatty acids to readily engage the receptor. Functional studies demonstrate that OA triggers downstream Gs signaling, whereas lysophospholipids fail to activate the receptor. Moreover, our research reveals that cold stimulation induces the secretion of OA in mice, subsequently activating Gs/cAMP/PKA signaling in brown adipose tissue. Notably, brown adipose tissues from Gpr3 knockout mice do not respond to OA during cold stimulation, reinforcing the significance of GPR3 in this process. Finally, we propose a "born to be activated and cold to enhance" model for GPR3 activation. Our study provides a starting framework for the understanding of GPR3 signaling in cold-stimulated thermogenesis.
Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation. International-journal

Yueh-Chien Lin, Steven Swendeman, Irina S Moreira, Avishek Ghosh, Andrew Kuo, Nícia Rosário-Ferreira, Shihui Guo, Alan Culbertson, Michel V Levesque, Andreane Cartier, Takahiro Seno, Alec Schmaier, Sylvain Galvani, Asuka Inoue, Samir M Parikh, Garret A FitzGerald, David Zurakowski, Maofu Liao, Robert Flaumenhaft, Zeynep H Gümüş, Timothy Hla

Science signaling　17　(824)　eadg9256　2024/02/20

DOI： 10.1126/scisignal.adg9256 　

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High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.
Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor. International-journal

Tharindunee Jayakody, Asuka Inoue, Srinivasaraghavan Kannan, Gaku Nakamura, Kouki Kawakami, Krishan Mendis, Thanh-Binh Nguyen, Jianguo Li, Deron R Herr, Chandra S Verma, Gavin S Dawe

Science signaling　17　(823)　eabl5880　2024/02/13

DOI： 10.1126/scisignal.abl5880 　

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The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and β-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of β-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of β-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.
GPCR signaling bias: an emerging framework for opioid drug development International-journal

Ryoji Kise, Asuka Inoue

The Journal of Biochemistry　175　(4)　367-376　2024/02/02
Publisher: Oxford University Press (OUP)
DOI： 10.1093/jb/mvae013 　

ISSN： 0021-924X

eISSN： 1756-2651

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Abstract Biased signaling, also known as functional selectivity, has emerged as an important concept in drug development targeting G-protein-coupled receptors (GPCRs). Drugs that provoke biased signaling are expected to offer an opportunity for enhanced therapeutic effectiveness with minimized side effects. Opioid analgesics, whilst exerting potent pain-relieving effects, have become a social problem owing to their serious side effects. For the development of safer pain medications, there has been extensive exploration of agonists with a distinct balance of G-protein and β-arrestin (βarr) signaling. Recently, several approaches based on protein–protein interactions have been developed to precisely evaluate individual signal pathways, paving the way for the comprehensive analysis of biased signals. In this review, we describe an overview of bias signaling in opioid receptors, especially the μ-opioid receptor (MOR), and how to evaluate signaling bias in the GPCR field. We also discuss future directions for rational drug development through the integration of diverse signal datasets.
Basal interaction of the orphan receptor GPR101 with arrestins leads to constitutive internalization International-journal

Dayana Abboud, Clauda Abboud, Asuka Inoue, Jean-Claude Twizere, Julien Hanson

Biochemical Pharmacology　220　116013-116013　2024/02
Publisher: Elsevier BV
DOI： 10.1016/j.bcp.2023.116013 　

ISSN： 0006-2952

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GPR101 is an orphan G protein-coupled receptor that promotes growth hormone secretion in the pituitary. The microduplication of the GPR101 gene has been linked with the X-linked acrogigantism, or X-LAG, syndrome. This disease is characterized by excessive growth hormone secretion and abnormal rapid growth beginning early in life. Mechanistically, GPR101 induces growth hormone secretion through constitutive activation of multiple heterotrimeric G proteins. However, the full scope of GPR101 signaling remains largely elusive. Herein, we investigated the association of GPR101 to multiple transducers and uncovered an important basal interaction with Arrestin 2 (β-arrestin 1) and Arrestin 3 (β-arrestin 2). By using a GPR101 mutant lacking the C-terminus and cell lines with an Arrestin 2/3 null background, we show that the arrestin association leads to constitutive clathrin- and dynamin-mediated GPR101 internalization. To further highlight GPR101 intracellular fate, we assessed the colocalization of GPR101 with Rab protein markers. Internalized GPR101 was mainly colocalized with the early endosome markers, Rab5 and EEA-1, and to a lesser degree with the late endosome marker Rab7. However, GPR101 was not colocalized with the recycling endosome marker Rab11. These findings show that the basal arrestin recruitment by GPR101 C-terminal tail drives the receptor constitutive clathrin-mediated internalization. Intracellularly, GPR101 concentrates in the endosomal compartment and is degraded through the lysosomal pathway. In conclusion, we uncovered a constitutive intracellular trafficking of GPR101 that potentially represents an important layer of regulation of its signaling and function.
Structural and functional characterization of the endogenous agonist for orphan receptor GPR3 International-journal

Geng Chen, Nico Staffen, Zhangsong Wu, Xinyu Xu, Jinheng Pan, Asuka Inoue, Tingyi Shi, Peter Gmeiner, Yang Du, Jun Xu

Cell Research　34　(3)　262-265　2024/01/30
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41422-023-00919-8 　

eISSN： 1748-7838
Four-color single-molecule imaging system for tracking GPCR dynamics with fluorescent HiBiT peptide

Toshiki Yoda, Yasushi Sako, Asuka Inoue, Masataka Yanagawa

Biophysics and Physicobiology　21　(3)　e210020　2024
Publisher: Biophysical Society of Japan
DOI： 10.2142/biophysico.bppb-v21.0020 　

eISSN： 2189-4779

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Single-molecule imaging provides information on diffusion dynamics, oligomerization, and protein-protein interactions in living cells. To simultaneously monitor different types of proteins at the single-molecule level, orthogonal fluorescent labeling methods with different photostable dyes are required. G-protein-coupled receptors (GPCRs), a major class of drug targets, are prototypical membrane receptors that have been studied using single-molecule imaging techniques. Here we developed a method for labeling cell-surface GPCRs inspired by the HiBiT system, which utilizes the high affinity complementation between LgBiT and HiBiT fragments of the NanoLuc luciferase. We synthesized four fluorescence-labeled HiBiT peptides (F-FiBiTs) with a different color dye (Setau-488, TMR, SaraFluor 650 and SaraFluor 720). We constructed a multicolor total internal reflection fluorescence microscopy system that allows us to track four color dyes simultaneously. As a proof-of-concept experiment, we labeled an N-terminally LgBiT-fused GPCR (Lg-GPCR) with a mixture of the four F-FiBiTs and successfully tracked each dye within a cell at the single-molecule level. The F-FiBiT-labeled Lg-GPCRs showed agonist-dependent changes in the diffusion dynamics and accumulation into the clathrin-coated pits as observed with a conventional method using a C-terminally HaloTag-fused GPCR. Taking advantage of luciferase complementation by the F-FiBiT and Lg-GPCRs, the F-FiBiT was also applicable to bioluminescence plate-reader-based assays. By combining existing labeling methods such as HaloTag, SNAP-tag, and fluorescent proteins, the F-FiBiT method will be useful for multicolor single-molecule imaging and will enhance our understanding of GPCR signaling at the single-molecule level.
Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants. International-journal

Ritsuki Kuramoto, Ryoji Kise, Mayu Kanno, Kouki Kawakami, Tatsuya Ikuta, Noriko Makita, Asuka Inoue

PloS one　19　(5)　e0303507　2024

DOI： 10.1371/journal.pone.0303507 　

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Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting β-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.
A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling. International-journal

Pauline Raynaud, Vinesh Jugnarain, Océane Vaugrente, Amandine Vallet, Thomas Boulo, Camille Gauthier, Asuka Inoue, Nathalie Sibille, Christophe Gauthier, Frédéric Jean-Alphonse, Eric Reiter, Pascale Crépieux, Gilles Bruneau

FEBS letters　598　(2)　220-232　2024/01

DOI： 10.1002/1873-3468.14765 　

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Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.
Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins International-journal

Thor C. Møller, Ee Von Moo, Asuka Inoue, Mie F. Pedersen, Hans Bräuner-Osborne

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research　1871　(1)　119584-119584　2024/01
Publisher: Elsevier BV
DOI： 10.1016/j.bbamcr.2023.119584 　

ISSN： 0167-4889

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G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.
Interaction modes of human orexin 2 receptor with selective and nonselective antagonists studied by NMR spectroscopy International-journal

Kayo Imamura, Ken-Ichi Akagi, Yohei Miyanoiri, Hirokazu Tsujimoto, Takatsugu Hirokawa, Hideo Ashida, Kaori Murakami, Asuka Inoue, Ryoji Suno, Takahisa Ikegami, Naotaka Sekiyama, So Iwata, Takuya Kobayashi, Hidehito Tochio

Structure　32　(3)　352-361　2024/01
Publisher: Elsevier BV
DOI： 10.1016/j.str.2023.12.008 　

ISSN： 0969-2126

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Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.
Reply to: How carvedilol does not activate β2-adrenoceptors. International-journal

Evi Kostenis, Jesus Gomeza, Elke Miess-Tanneberg, Nina Kathleen Blum, Tobias Benkel, Andy Chevigné, Carsten Hoffmann, Peter Kolb, Viacheslav Nikolaev, Maria Waldhoer, Martyna Szpakowska, Asuka Inoue, Stefan Schulz

Nature communications　14　(1)　7867-7867　2023/11/30

DOI： 10.1038/s41467-023-42849-4 　
Structural basis for ligand recognition and signaling of hydroxy-carboxylic acid receptor 2. International-journal

Jae-Hyun Park, Kouki Kawakami, Naito Ishimoto, Tatsuya Ikuta, Mio Ohki, Toru Ekimoto, Mitsunori Ikeguchi, Dong-Sun Lee, Young-Ho Lee, Jeremy R H Tame, Asuka Inoue, Sam-Yong Park

Nature communications　14　(1)　7150-7150　2023/11/06

DOI： 10.1038/s41467-023-42764-8 　

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Hydroxycarboxylic acid receptors (HCAR1, HCAR2, and HCAR3) transduce Gi/o signaling upon biding to molecules such as lactic acid, butyric acid and 3-hydroxyoctanoic acid, which are associated with lipolytic and atherogenic activity, and neuroinflammation. Although many reports have elucidated the function of HCAR2 and its potential as a therapeutic target for treating not only dyslipidemia but also neuroimmune disorders such as multiple sclerosis and Parkinson's disease, the structural basis of ligand recognition and ligand-induced Gi-coupling remains unclear. Here we report three cryo-EM structures of the human HCAR2-Gi signaling complex, each bound with different ligands: niacin, acipimox or GSK256073. All three agonists are held in a deep pocket lined by residues that are not conserved in HCAR1 and HCAR3. A distinct hairpin loop at the HCAR2 N-terminus and extra-cellular loop 2 (ECL2) completely enclose the ligand. These structures also reveal the agonist-induced conformational changes propagated to the G-protein-coupling interface during activation. Collectively, the structures presented here are expected to help in the design of ligands specific for HCAR2, leading to new drugs for the treatment of various diseases such as dyslipidemia and inflammation.
Stepwise phosphorylation of BLT1 defines complex assemblies with β-arrestin serving distinct functions. International-journal

Riko Tatsumi, Saki Aihara, Seiya Matsune, Junken Aoki, Asuka Inoue, Takao Shimizu, Motonao Nakamura

FASEB journal : official publication of the Federation of American Societies for Experimental Biology　37　(11)　e23213　2023/11

DOI： 10.1096/fj.202301440R 　

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G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4 ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310 . Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308 , leading to a shift in ligand affinity from a high-to-low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1-engaged conformations of β-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2-GRK pathway, resulting in a β-arrestin-bound low-affinity state. This configuration, referred to as the "low-LTB4 -induced complex," necessitates the finger loop region and the phosphoinositide-binding motif of β-arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low-affinity BLT1 again binds to the ligand and triggers the generation of the low-LTB4 -induced complex into a different form termed "high-LTB4 -induced complex." This change is propelled by The308 -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB4 -induced complex, β-arrestin adapts a unique configuration that involves additional N domain interaction to the low-affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein β-arrestins perform distinct functions.
Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor International-journal

Valeria Burghi, Justine S. Paradis, Adam Officer, Sendi Rafael Adame-Garcia, Xingyu Wu, Edda S.F. Matthees, Benjamin Barsi-Rhyne, Dana J. Ramms, Lauren Clubb, Monica Acosta, Pablo Tamayo, Michel Bouvier, Asuka Inoue, Mark von Zastrow, Carsten Hoffmann, J. Silvio Gutkind

Journal of Biological Chemistry　299　(11)　105293-105293　2023/11
Publisher: Elsevier BV
DOI： 10.1016/j.jbc.2023.105293 　

ISSN： 0021-9258

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β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.
GPCRとβアレスチンの結合多様性と機能的役割の解析

中村楽, 桑原莉来, 川上耕季, 井上飛鳥

日本生化学会大会プログラム・講演要旨集　96回　[1T07a-346)]　2023/10
Publisher: (公社)日本生化学会
遊離脂肪酸受容体(FFAR)の脂肪酸受容における鎖長選択性の解明

九川真衣, 木瀬亮次, 福田昌弘, 川上耕季, 松井俊貴, 小林和弘, 井上飛鳥, 加藤英明

日本生化学会大会プログラム・講演要旨集　96回　[2P-080]　2023/10
Publisher: (公社)日本生化学会
レポーターアッセイを用いたGPCRシグナルネットワークの解明

齋藤郁貴, 木瀬亮次, 川上耕季, 井上飛鳥

日本生化学会大会プログラム・講演要旨集　96回　[2P-381]　2023/10
Publisher: (公社)日本生化学会
G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for depression. International-journal

Naoto Kajitani, Mami Okada-Tsuchioka, Asuka Inoue, Kanako Miyano, Takeshi Masuda, Shuken Boku, Kazuya Iwamoto, Sumio Ohtsuki, Yasuhito Uezono, Junken Aoki, Minoru Takebayashi

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology　49　(3)　561-572　2023/09/06

DOI： 10.1038/s41386-023-01727-9 　

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Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors, in treating severe depression. However, the clinical contribution of non-monoaminergic effects of TCAs remains elusive. In this study, we discovered that amitriptyline, a typical TCA, directly binds to the lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, and activates downstream G protein signaling, while exerting a little effect on β-arrestin recruitment. This suggests that amitriptyline acts as a G protein-biased agonist of LPAR1. This biased agonism was specific to TCAs and was not observed with other antidepressants. LPAR1 was found to be involved in the behavioral effects of amitriptyline. Notably, long-term infusion of mouse hippocampus with the potent G protein-biased LPAR agonist OMPT, but not the non-biased agonist LPA, induced antidepressant-like behavior, indicating that G protein-biased agonism might be necessary for the antidepressant-like effects. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in the hippocampus. Pathway analysis indicated that long-term treatment with OMPT activated LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. Our findings provide insights into the mechanisms underlying the non-monoaminergic antidepressant effects of TCAs and identify the G protein-biased agonism of LPAR1 as a promising target for the development of novel antidepressants.
Eriodictyol and thymonin act as GPR35 agonists International-journal

Fumie Nakashima, Wei Qi Loh, Mayuka Wakabayashi, Sayako Shimomura, Hiroyuki Hattori, Masaki Kita, Asuka Inoue, Koji Uchida, Takahiro Shibata

Bioscience, Biotechnology, and Biochemistry　87　(12)　1514-1522　2023/09/04
Publisher: Oxford University Press (OUP)
DOI： 10.1093/bbb/zbad125 　

eISSN： 1347-6947

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ABSTRACT Although herbs and spices have been used in traditional medicine for more than a century owing to their health benefits, the associated underlying mechanism is still not clear. Since the G protein-coupled receptor 35 (GPR35) has been linked to exert various antioxidant and anti-inflammatory effects, we screened 19 different herbs and spices for possible GPR35 agonist(s) to understand the GPR35-dependent functions of herbs and spices. Among the screened extracts, the ethyl acetate extract of thyme exhibited a remarkable GPR35 agonistic activity. Activity-guided separations allowed us to identify 2 polyphenolic phytochemicals, eriodictyol and thymonin, acting as GPR35 agonists. Both eriodictyol and thymonin showed a potent and specific agonist activity toward GPR35 with half maximal effective concentration values of 5.48 and 8.41 µm, respectively. These findings indicate that these phytochemicals may have beneficial health effects upon GPR35 activation.
Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system. International-journal

Parishmita Sarma, Carlo Marion C Carino, Deeksha Seetharama, Shubhi Pandey, Hemlata Dwivedi-Agnihotri, Xue Rui, Yubo Cao, Kouki Kawakami, Poonam Kumari, Yu-Chih Chen, Kathryn E Luker, Prem N Yadav, Gary D Luker, Stéphane A Laporte, Xin Chen, Asuka Inoue, Arun K Shukla

Nature communications　14　(1)　4808-4808　2023/08/09

DOI： 10.1038/s41467-023-40482-9 　

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Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust βarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced βarr-coupling compared to CXCR7. These two receptors induce distinct βarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for βarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.
A2B adenosine receptor activation and modulation by protein kinase C. International-journal

Zhan-Guo Gao, Ian M Levitan, Asuka Inoue, Qiang Wei, Kenneth A Jacobson

iScience　26　(7)　107178-107178　2023/07/21

DOI： 10.1016/j.isci.2023.107178 　

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Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A2B adenosine receptor (AR)-mediated, but not β2-adrenergic receptor-mediated, cAMP accumulation, in H9C2 cardiomyocyte-like and HEK293 cells. In addition to enhancement, PKC (PMA-treatment) also activated A2BAR with low Emax (H9C2 and NIH3T3 cells endogenously expressing A2BAR), or with high Emax (A2BAR-overexpressing HEK293 cells) to induce cAMP accumulation. A2BAR activation induced by PKC was inhibited by A2BAR and PKC inhibitors but enhanced by A2BAR overexpression. Gαi isoforms and PKCγ isoform were found to be involved in both enhancement of A2BAR function and A2BAR activation. Thus, we establish PKC as an endogenous modulator and activator of A2BAR, involving Giα and PKCγ. Depending on signaling pathway, PKC could activate and enhance, or alternatively inhibit A2BAR activity. These findings are relevant to common functions of A2BAR and PKC, e.g. cardioprotection and cancer progression/treatment.
Cannabinoid receptor type 1 (CB1R) inhibits hypothalamic leptin signaling via β-arrestin1 in complex with TC-PTP and STAT3. International-journal

Gergő Szanda, Tony Jourdan, Éva Wisniewski, Resat Cinar, Grzegorz Godlewski, Anikó Rajki, Jie Liu, Lee Chedester, Bence Szalai, András Dávid Tóth, Eszter Soltész-Katona, László Hunyady, Asuka Inoue, Viktória Bea Horváth, András Spät, Joseph Tam, George Kunos

iScience　26　(7)　107207-107207　2023/07/21

DOI： 10.1016/j.isci.2023.107207 　

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Molecular interactions between anorexigenic leptin and orexigenic endocannabinoids, although of great metabolic significance, are not well understood. We report here that hypothalamic STAT3 signaling in mice, initiated by physiological elevations of leptin, is diminished by agonists of the cannabinoid receptor 1 (CB1R). Measurement of STAT3 activation by semi-automated confocal microscopy in cultured neurons revealed that this CB1R-mediated inhibition requires both T cell protein tyrosine phosphatase (TC-PTP) and β-arrestin1 but is independent of changes in cAMP. Moreover, β-arrestin1 translocates to the nucleus upon CB1R activation and binds both STAT3 and TC-PTP. Consistently, CB1R activation failed to suppress leptin signaling in β-arrestin1 knockout mice in vivo, and in neural cells deficient in CB1R, β-arrestin1 or TC-PTP. Altogether, CB1R activation engages β-arrestin1 to coordinate the TC-PTP-mediated inhibition of the leptin-evoked neuronal STAT3 response. This mechanism may restrict the anorexigenic effects of leptin when hypothalamic endocannabinoid levels rise, as during fasting or in diet-induced obesity.
GPCRome-wide analysis of G-protein-coupling diversity using a computational biology approach International-journal

Marin Matic, Pasquale Miglionico, Manae Tatsumi, Asuka Inoue, Francesco Raimondi

Nature Communications　14　(1)　4361-4361　2023/07/19
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-023-40045-y 　

eISSN： 2041-1723

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Abstract GPCRs are master regulators of cell signaling by transducing extracellular stimuli into the cell via selective coupling to intracellular G-proteins. Here we present a computational analysis of the structural determinants of G-protein-coupling repertoire of experimental and predicted 3D GPCR-G-protein complexes. Interface contact analysis recapitulates structural hallmarks associated with G-protein-coupling specificity, including TM5, TM6 and ICLs. We employ interface contacts as fingerprints to cluster G_s vs G_i complexes in an unsupervised fashion, suggesting that interface residues contribute to selective coupling. We experimentally confirm on a promiscuous receptor (CCKAR) that mutations of some of these specificity-determining positions bias the coupling selectivity. Interestingly, G_s-GPCR complexes have more conserved interfaces, while G_i/o proteins adopt a wider number of alternative docking poses, as assessed via structural alignments of representative 3D complexes. Binding energy calculations demonstrate that distinct structural properties of the complexes are associated to higher stability of G_s than G_i/o complexes. AlphaFold2 predictions of experimental binary complexes confirm several of these structural features and allow us to augment the structural coverage of poorly characterized complexes such as G_12/13.
Structural basis of CXC chemokine receptor 1 ligand binding and activation. International-journal

Naito Ishimoto, Jae-Hyun Park, Kouki Kawakami, Michiko Tajiri, Kenji Mizutani, Satoko Akashi, Jeremy R H Tame, Asuka Inoue, Sam-Yong Park

Nature communications　14　(1)　4107-4107　2023/07/11

DOI： 10.1038/s41467-023-39799-2 　

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Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8 (also known as interleukin-8, abbreviated IL-8) and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes.
Transcriptome Profiling of Anhidrotic Eccrine Sweat Glands Reveals that Olfactory Receptors on Eccrine Sweat Glands Regulate Perspiration in a Ligand-Dependent Manner International-journal

Naoya Murayama, Takafumi Miyaki, Daisuke Okuzaki, Yasuaki Shibata, Takehiko Koji, Asuka Inoue, Junken Aoki, Hideki Hayashi, Yoshimasa Tanaka, Hiroyuki Murota

JID Innovations　3　(4)　100196-100196　2023/07
Publisher: Elsevier BV
DOI： 10.1016/j.xjidi.2023.100196 　

ISSN： 2667-0267

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Sweat maintains systemic homeostasis in humans. Although sweating disorders may cause multifaceted health problems, therapeutic options for sweat disorders have not yet been established. To gain new insight into the mechanism underlying the regulation of perspiration, we compared eccrine sweat gland transcriptomes from hidrotic and anhidrotic lesions from patients with anhidrosis and found out that olfactory receptors were expressed differentially in anhidrotic and hidrotic eccrine sweat glands. We then confirmed OR51A7 and OR51E2 expression in human eccrine sweat glands by in situ hybridization and immunohistochemistry. An alkaline phosphatase-TGFα shedding assay revealed that β-ionone activates G-proteins through OR51A7 or OR51E2. The effect of topically applied β-ionone on sweating was examined with the quantitative sudomotor axon reflex test, which showed that responses to β-ionone differed between sexes. Topical β-ionone attenuated female sweating and augmented male sweating. Taken together, this study suggests that olfactory receptors expressed in eccrine sweat glands may regulate sweating in response to odorous ligands on the basis of sex. These unexpected results indicate that olfactory receptors may modulate sweating and that olfactory receptor modulators may contribute to the management of sweat disorders.
Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314 International-journal

Fabian Bumbak, James B. Bower, Skylar C. Zemmer, Asuka Inoue, Miquel Pons, Juan Carlos Paniagua, Fei Yan, James Ford, Hongwei Wu, Scott A. Robson, Ross A. D. Bathgate, Daniel J. Scott, Paul R. Gooley, Joshua J. Ziarek

Nature Communications　14　(1)　3328-3328　2023/06/07
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-023-38894-8 　

eISSN： 2041-1723

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Abstract The neurotensin receptor 1 (NTS₁) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS₁ structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used ¹³C^εH₃-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS₁:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.
Structural basis of omega-3 fatty acid receptor FFAR4 activation and G protein coupling selectivity International-journal

Han Yin, Asuka Inoue, Zhengxiong Ma, Xinyan Zhu, Ruixue Xia, Zhenmei Xu, Na Wang, Yaning Duan, Anqi Zhang, Changyou Guo, Yuanzheng He

Cell Research　33　(8)　644-647　2023/06/07
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41422-023-00835-x 　

eISSN： 1748-7838
Class B1 GPCR activation by an intracellular agonist. International-journal

Kazuhiro Kobayashi, Kouki Kawakami, Tsukasa Kusakizako, Atsuhiro Tomita, Michihiro Nishimura, Kazuhiro Sawada, Hiroyuki H Okamoto, Suzune Hiratsuka, Gaku Nakamura, Riku Kuwabara, Hiroshi Noda, Hiroyasu Muramatsu, Masaru Shimizu, Tomohiko Taguchi, Asuka Inoue, Takeshi Murata, Osamu Nureki

Nature　618　(7967)　1085-1093　2023/06/07

DOI： 10.1038/s41586-023-06169-3 　

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G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and β-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than β-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.
Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms. International-journal

Yufang Deng, Ee Von Moo, Asuka Inoue, Hans Bräuner-Osborne

FEBS letters　597　(11)　1528-1540　2023/06

DOI： 10.1002/1873-3468.14622 　

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GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into β-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.
Molecular mechanism of fatty acid activation of FFAR1 International-journal

Punita Kumari, Asuka Inoue, Karen Chapman, Peng Lian, Daniel M. Rosenbaum

Proceedings of the National Academy of Sciences　120　(22)　e2219569120　2023/05/22
Publisher: Proceedings of the National Academy of Sciences
DOI： 10.1073/pnas.2219569120 　

ISSN： 0027-8424

eISSN： 1091-6490

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FFAR1 is a G-protein-coupled receptor (GPCR) that responds to circulating free fatty acids to enhance glucose-stimulated insulin secretion and release of incretin hormones. Due to the glucose-lowering effect of FFAR1 activation, potent agonists for this receptor have been developed for the treatment of diabetes. Previous structural and biochemical studies of FFAR1 showed multiple sites of ligand binding to the inactive state but left the mechanism of fatty acid interaction and receptor activation unknown. We used cryo–electron microscopy to elucidate structures of activated FFAR1 bound to a G _q mimetic, which were induced either by the endogenous FFA ligand docosahexaenoic acid or γ-linolenic acid and the agonist drug TAK-875. Our data identify the orthosteric pocket for fatty acids and show how both endogenous hormones and synthetic agonists induce changes in helical packing along the outside of the receptor that propagate to exposure of the G-protein-coupling site. These structures show how FFAR1 functions without the highly conserved “DRY” and “NPXXY” motifs of class A GPCRs and also illustrate how the orthosteric site of a receptor can be bypassed by membrane-embedded drugs to confer full activation of G protein signaling.
Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction. International-journal

Akiko Ogawa, Seiya Ohira, Yuri Kato, Tatsuya Ikuta, Shota Yanagida, Xinya Mi, Yukina Ishii, Yasunari Kanda, Motohiro Nishida, Asuka Inoue, Fan-Yan Wei

Communications biology　6　(1)　511-511　2023/05/12

DOI： 10.1038/s42003-023-04888-x 　

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Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future.
Structural basis for activation of CB1 by an endocannabinoid analog. International-journal

Kaavya Krishna Kumar, Michael J Robertson, Elina Thadhani, Haoqing Wang, Carl-Mikael Suomivuori, Alexander S Powers, Lipin Ji, Spyros P Nikas, Ron O Dror, Asuka Inoue, Alexandros Makriyannis, Georgios Skiniotis, Brian Kobilka

Nature communications　14　(1)　2672-2672　2023/05/09

DOI： 10.1038/s41467-023-37864-4 　

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Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex. Compared to previous structures, the ligand binding pocket shows some differences. Using docking, molecular dynamics simulations, and signaling assays we investigated the functional consequences of ligand interactions with the "toggle switch" residues F2003.36 and W3566.48. Further, we show that ligand-TM2 interactions drive changes to residues on the intracellular side of TM2 and are a determinant of efficacy in activating G protein. These intracellular TM2 rearrangements are unique to CB1 and are exploited by a CB1-specific allosteric modulator.
Antagonist of sphingosine 1-phosphate receptor 3 reduces cold injury of rat donor hearts for transplantation International-journal

Eisho Kanemitsu, Xiangdong Zhao, Keiko Iwaisako, Asuka Inoue, Akihide Takeuchi, Shintaro Yagi, Hidetoshi Masumoto, Hiroaki Ohara, Motoyasu Hosokawa, Tomonari Awaya, Junken Aoki, Etsuro Hatano, Shinji Uemoto, Masatoshi Hagiwara

Translational Research　255　26-36　2023/05
Publisher: Elsevier BV
DOI： 10.1016/j.trsl.2022.11.003 　

ISSN： 1931-5244

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Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-β-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.
Design and Synthesis of Orexin 1 Receptor-Selective Agonists International-journal

Keita Iio, Kao Hashimoto, Yasuyuki Nagumo, Mao Amezawa, Taisei Hasegawa, Naoshi Yamamoto, Noriki Kutsumura, Katsuhiko Takeuchi, Yukiko Ishikawa, Hikari Yamamoto, Akihisa Tokuda, Tetsu Sato, Yasuo Uchida, Asuka Inoue, Ryuji Tanimura, Masashi Yanagisawa, Hiroshi Nagase, Tsuyoshi Saitoh

Journal of Medicinal Chemistry　66　(8)　5453-5464　2023/04/12
Publisher: American Chemical Society (ACS)
DOI： 10.1021/acs.jmedchem.2c01773 　

ISSN： 0022-2623

eISSN： 1520-4804

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Orexins are a family of neuropeptides that regulate various physiological events, such as sleep/wakefulness as well as emotional and feeding behavior, and that act on two G-protein-coupled receptors, i.e., orexin 1 (OX1R) and orexin 2 receptors (OX2R). Since the discovery that dysfunction of the orexin/OX2R system causes the sleep disorder narcolepsy, several OX2R-selective and OX1/2R dual agonists have been disclosed. However, an OX1R-selective agonist has not yet been reported, despite the importance of the biological function of OX1R. Herein, we report the discovery of a potent OX1R-selective agonist, (R,E)-3-(4-methoxy-3-(N-(8-(2-(3-methoxyphenyl)-N-methylacetamido)-5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)-N-(pyridin-4-yl)acrylamide [(R)-YNT-3708; EC50 = 7.48 nM for OX1R; OX2R/OX1R EC50 ratio = 22.5]. The OX1R-selective agonist (R)-YNT-3708 exhibited antinociceptive and reinforcing effects through the activation of OX1R in mice.
Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor. International-journal

Jie Heng, Yunfei Hu, Guillermo Pérez-Hernández, Asuka Inoue, Jiawei Zhao, Xiuyan Ma, Xiaoou Sun, Kouki Kawakami, Tatsuya Ikuta, Jienv Ding, Yujie Yang, Lujia Zhang, Sijia Peng, Xiaogang Niu, Hongwei Li, Ramon Guixà-González, Changwen Jin, Peter W Hildebrand, Chunlai Chen, Brian K Kobilka

Nature communications　14　(1)　2005-2005　2023/04/10

DOI： 10.1038/s41467-023-37233-1 　

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Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The β2 adrenergic receptor's (β2AR) 71 amino acid CT is a substrate for GPCR kinases and binds β-arrestins to regulate signaling. Here we show that the β2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking β-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged β2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.
Exploration of LPS2 agonist binding modes using the combination of a new hydrophobic scaffold and homology modeling. International-journal

Luying Chen, Akiharu Uwamizu, Misa Sayama, Kuniyuki Kano, Yuko Otani, Sho Kondo, Asuka Inoue, Junken Aoki, Tomohiko Ohwada

European journal of medicinal chemistry　252　115271-115271　2023/04/05

DOI： 10.1016/j.ejmech.2023.115271 　

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Lysophosphatidylserine (LysoPS) is an endogenous pan-agonist of three G-protein coupled receptors (GPCRs): LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, and we previously reported a series of LysoPS-based agonists of these receptors. Interestingly, we found that LPS1 agonist activity was very sensitive to structural change at the hydrophobic fatty acid moiety, whereas LPS2 agonist activity was not. Here, to probe the molecular basis of LPS2 agonist binding, we developed a new class of hydrophobic fatty acid surrogates having a biphenyl-ether scaffold. The LPS2 agonist activity of these compounds proved sensitive to molecular modification of the hydrophobic skeleton. Thus, we next constructed an LPS2 model by homology modeling and docking/molecular dynamics (MD) simulation, and validated it by means of SAR studies together with point mutations of selected receptor amino-acid residues. The putative ligand-binding site of LPS2 is Γ-shaped, with a hydrophilic site horizontally embedded in the receptor transmembrane helix bundles and a perpendicular hydrophobic groove adjoining transmembrane domains 4 and 5 that is open to the membrane bilayer. The binding poses of LPS2 agonists to this site are consistent with easy incorporation of various kinds of fatty acid surrogates. Structural development based on this model afforded a series of potent and selective LPS2 full agonists, which showed enhanced in vitro actin stress fiber formation effect.
Migration mediated by the oxysterol receptor GPR183 depends on arrestin coupling but not receptor internalization International-journal

Viktoria M. S. Kjær, Viktorija Daugvilaite, Tomasz M. Stepniewski, Christian M. Madsen, Astrid S. Jørgensen, Kaustubh R. Bhuskute, Asuka Inoue, Trond Ulven, Tau Benned-Jensen, Siv A. Hjorth, Gertrud M. Hjortø, Ee Von Moo, Jana Selent, Mette M. Rosenkilde

Science Signaling　16　(779)　eabl4283　2023/04/04
Publisher: American Association for the Advancement of Science (AAAS)
DOI： 10.1126/scisignal.abl4283 　

ISSN： 1945-0877

eISSN： 1937-9145

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The chemotactic G protein–coupled receptor GPR183 and its most potent endogenous oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) are important for immune cell positioning in secondary lymphoid tissues. This receptor-ligand pair is associated with various diseases, in some cases contributing favorably and in other cases adversely, making GPR183 an attractive target for therapeutic intervention. We investigated the mechanisms underlying GPR183 internalization and the role of internalization in the main biological function of the receptor, chemotaxis. We found that the C terminus of the receptor was important for ligand-induced internalization but less so for constitutive (ligand-independent) internalization. β-arrestin potentiated ligand-induced internalization but was not required for ligand-induced or constitutive internalization. Caveolin and dynamin were the main mediators of both constitutive and ligand-induced receptor internalization in a mechanism independent of G protein activation. Clathrin-mediated endocytosis also contributed to constitutive GPR183 internalization in a β-arrestin–independent manner, suggesting the existence of different pools of surface-localized GPR183. Chemotaxis mediated by GPR183 depended on receptor desensitization by β-arrestins but could be uncoupled from internalization, highlighting an important biological role for the recruitment of β-arrestin to GPR183. The role of distinct pathways in internalization and chemotaxis may aid in the development of GPR183-targeting drugs for specific disease contexts.
Phosphorylation barcodes direct biased chemokine signaling at CXCR3. International-journal

Dylan S Eiger, Jeffrey S Smith, Tujin Shi, Tomasz Maciej Stepniewski, Chia-Feng Tsai, Christopher Honeycutt, Noelia Boldizsar, Julia Gardner, Carrie D Nicora, Ahmed M Moghieb, Kouki Kawakami, Issac Choi, Chloe Hicks, Kevin Zheng, Anmol Warman, Priya Alagesan, Nicole M Knape, Ouwen Huang, Justin D Silverman, Richard D Smith, Asuka Inoue, Jana Selent, Jon M Jacobs, Sudarshan Rajagopal

Cell chemical biology　30　(4)　362-382　2023/04/03

DOI： 10.1016/j.chembiol.2023.03.006 　

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G protein-coupled receptor (GPCR)-biased agonism, selective activation of certain signaling pathways relative to others, is thought to be directed by differential GPCR phosphorylation "barcodes." At chemokine receptors, endogenous chemokines can act as "biased agonists", which may contribute to the limited success when pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomics studies. Mutation of CXCR3 phosphosites altered β-arrestin 2 conformation in cellular assays and was consistent with conformational changes observed in molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes, leading to distinct physiological processes.
Insights into distinct signaling profiles of the µOR activated by diverse agonists. International-journal

Qianhui Qu, Weijiao Huang, Deniz Aydin, Joseph M Paggi, Alpay B Seven, Haoqing Wang, Soumen Chakraborty, Tao Che, Jeffrey F DiBerto, Michael J Robertson, Asuka Inoue, Carl-Mikael Suomivuori, Bryan L Roth, Susruta Majumdar, Ron O Dror, Brian K Kobilka, Georgios Skiniotis

Nature chemical biology　19　(4)　423-430　2023/04

DOI： 10.1038/s41589-022-01208-y 　

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Drugs targeting the μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and β-arrestin recruitment. Cryo-EM structures of μOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and β-arrestins bind. These observations highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes.
Structure-affinity and structure-residence time relationships of macrocyclic Gαq protein inhibitors International-journal

Jan H. Voss, Max Crüsemann, Christian R.O. Bartling, Stefan Kehraus, Asuka Inoue, Gabriele M. König, Kristian Strømgaard, Christa E. Müller

iScience　26　(4)　106492-106492　2023/04
Publisher: Elsevier BV
DOI： 10.1016/j.isci.2023.106492 　

ISSN： 2589-0042

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The macrocyclic depsipeptides YM-254890 (YM) and FR900359 (FR) are potent inhibitors of Gαq/11 proteins. They are important pharmacological tools and have potential as therapeutic drugs. The hydrogenated, tritium-labeled YM and FR derivatives display largely different residence times despite similar structures. In the present study we established a competition-association binding assay to determine the dissociation kinetics of unlabeled Gq protein inhibitors. Structure-affinity and structure-residence time relationships were analyzed. Small structural modifications had a large impact on residence time. YM and FR exhibited 4- to 10-fold higher residence times than their hydrogenated derivatives. While FR showed pseudo-irreversible binding, YM displayed much faster dissociation from its target. The isopropyl anchor present in FR and some derivatives was essential for slow dissociation. These data provide a basis for future drug design toward modulating residence times of macrocyclic Gq protein inhibitors, which has been recognized as a crucial determinant for therapeutic outcome.
Phosphorylation barcodes direct biased chemokine signaling at CXCR3. International-journal

Dylan S Eiger, Jeffrey S Smith, Tujin Shi, Tomasz Maciej Stepniewski, Chia-Feng Tsai, Christopher Honeycutt, Noelia Boldizsar, Julia Gardner, Carrie D Nicora, Ahmed M Moghieb, Kouki Kawakami, Issac Choi, Kevin Zheng, Anmol Warman, Priya Alagesan, Nicole M Knape, Ouwen Huang, Justin D Silverman, Richard D Smith, Asuka Inoue, Jana Selent, Jon M Jacobs, Sudarshan Rajagopal

bioRxiv : the preprint server for biology　2023/03/14

DOI： 10.1101/2023.03.14.532634 　

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G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or "phosphorylation barcodes." At chemokine receptors, ligands act as "biased agonists" with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered β-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes.
Identification and Functional Characterization of Fungal Chalcone Synthase and Chalcone Isomerase. International-journal

Sho Furumura, Taro Ozaki, Akihiro Sugawara, Yohei Morishita, Kento Tsukada, Tatsuya Ikuta, Asuka Inoue, Teigo Asai

Journal of natural products　86　(2)　398-405　2023/02/24

DOI： 10.1021/acs.jnatprod.2c01027 　

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By mining fungal genomic information, a noncanonical iterative type I PKS fused with an N-terminal adenylation-thiolation didomain, which catalyzes the formation of naringenin chalcone, was found. Structural prediction and molecular docking analysis indicated that a C-terminal thioesterase domain was involved in the Claisen-type cyclization. An enzyme responsible for formation of (2S)-flavanone in the biosynthesis of fungal flavonoids was also identified. Collectively, these findings demonstrate unprecedented fungal biosynthetic machinery leading to plant-like metabolites.
Structural basis of lysophosphatidylserine receptor GPR174 ligand recognition and activation. International-journal

Jiale Liang, Asuka Inoue, Tatsuya Ikuta, Ruixue Xia, Na Wang, Kouki Kawakami, Zhenmei Xu, Yu Qian, Xinyan Zhu, Anqi Zhang, Changyou Guo, Zhiwei Huang, Yuanzheng He

Nature communications　14　(1)　1012-1012　2023/02/23

DOI： 10.1038/s41467-023-36575-0 　

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Lysophosphatidylserine (LysoPS) is a lipid mediator that induces multiple cellular responses through binding to GPR174. Here, we present the cryo-electron microscopy (cryo-EM) structure of LysoPS-bound human GPR174 in complex with Gs protein. The structure reveals a ligand recognition mode, including the negatively charged head group of LysoPS forms extensive polar interactions with surrounding key residues of the ligand binding pocket, and the L-serine moiety buries deeply into a positive charged cavity in the pocket. In addition, the structure unveils a partially open pocket on transmembrane domain helix (TM) 4 and 5 for a lateral entry of ligand. Finally, the structure reveals a Gs engaging mode featured by a deep insertion of a helix 5 (αH5) and extensive polar interactions between receptor and αH5. Taken together, the information revealed by our structural study provides a framework for understanding LysoPS signaling and a rational basis for designing LysoPS receptor-targeting drugs.
Correction to: Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via β-arrestin/ERK signaling. International-journal

Vinay Kumar Sharma, Xuyu Yang, Soo-Kyung Kim, Amirhossein Mafi, Daniel Saiz-Sanchez, Patricia Villanueva-Anguita, Lan Xiao, Asuka Inoue, William A Goddard 3rd, Y Peng Loh, Leila Toulabi

Cellular and molecular life sciences : CMLS　80　(3)　65-65　2023/02/20

DOI： 10.1007/s00018-023-04711-0 　
Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1). International-journal

Fabian Bumbak, Miquel Pons, Asuka Inoue, Juan Carlos Paniagua, Fei Yan, Hongwei Wu, Scott A Robson, Ross A D Bathgate, Daniel J Scott, Paul R Gooley, Joshua J Ziarek

Cell reports　42　(1)　112015-112015　2023/01/31

DOI： 10.1016/j.celrep.2023.112015 　

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Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination.
Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs. International-journal

Yuki Ono, Kouki Kawakami, Gaku Nakamura, Satoru Ishida, Junken Aoki, Asuka Inoue

Communications biology　6　(1)　112-112　2023/01/28

DOI： 10.1038/s42003-023-04465-2 　

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G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein α subunit i (Gαi) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the Gαi subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all Gαi subunits, which enables the measurement of GPCR-Gαi coupling upon transient re-expression of a specific Gαi subunit. We profile Gαi-coupling selectivity across 11 GPCRs by measuring ligand-induced inhibitory activity for cAMP accumulation. The coupling profiles are then classified into three clusters, representing those preferentially coupled to Gαz, those to Gαo, and those with unapparent selectivity. These results indicate that individual Gαi-coupled GPCRs fine-tune Gαi signaling by exerting coupling preference at the Gαi-subunit level.
Medium-chain fatty acids suppress lipotoxicity-induced hepatic fibrosis via the immunomodulating receptor GPR84 International-journal

Ryuji Ohue-Kitano, Hazuki Nonaka, Akari Nishida, Yuki Masujima, Daisuke Takahashi, Takako Ikeda, Akiharu Uwamizu, Miyako Tanaka, Motoyuki Kohjima, Miki Igarashi, Hironori Katoh, Tomohiro Tanaka, Asuka Inoue, Takayoshi Suganami, Koji Hase, Yoshihiro Ogawa, Junken Aoki, Ikuo Kimura

JCI Insight　8　(2)　2023/01/24
Publisher: American Society for Clinical Investigation
DOI： 10.1172/jci.insight.165469 　

eISSN： 2379-3708

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Medium-chain triglycerides (MCTs), which consist of medium-chain fatty acids (MCFAs), are unique forms of dietary fat with various health benefits. G protein-coupled 84 (GPR84) acts as a receptor for MCFAs (especially C10:0 and C12:0); however, GPR84 is still considered an orphan receptor, and the nutritional signaling of endogenous and dietary MCFAs via GPR84 remains unclear. Here, we showed that endogenous MCFA-mediated GPR84 signaling protected hepatic functions from diet-induced lipotoxicity. Under high-fat diet (HFD) conditions, GPR84-deficient mice exhibited nonalcoholic steatohepatitis (NASH) and the progression of hepatic fibrosis but not steatosis. With markedly increased hepatic MCFA levels under HFD, GPR84 suppressed lipotoxicity-induced macrophage overactivation. Thus, GPR84 is an immunomodulating receptor that suppresses excessive dietary fat intake-induced toxicity by sensing increases in MCFAs. Additionally, administering MCTs, MCFAs (C10:0 or C12:0, but not C8:0), or GPR84 agonists effectively improved NASH in mouse models. Therefore, exogenous GPR84 stimulation is a potential strategy for treating NASH.
Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor. International-journal

Jun Xu, Qinggong Wang, Harald Hübner, Yunfei Hu, Xiaogang Niu, Haoqing Wang, Shoji Maeda, Asuka Inoue, Yuyong Tao, Peter Gmeiner, Yang Du, Changwen Jin, Brian K Kobilka

Nature communications　14　(1)　376-376　2023/01/23

DOI： 10.1038/s41467-022-35726-z 　

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The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.
Isosteric Replacement of Ester Linkage of Lysophospholipids with Heteroaromatic Rings Retains Potency and Subtype Selectivity.

Masaya Ikubo, Akiharu Uwamizu, Luying Chen, Sho Nakamura, Misa Sayama, Hiroki Kawana, Yuko Otani, Kuniyuki Kano, Asuka Inoue, Junken Aoki, Tomohiko Ohwada

Chemical & pharmaceutical bulletin　71　(7)　584-615　2023

DOI： 10.1248/cpb.c23-00250 　

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Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.
Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels. International-journal

Manae Tatsumi, Takayuki Kishi, Satoru Ishida, Hiroki Kawana, Akiharu Uwamizu, Yuki Ono, Kouki Kawakami, Junken Aoki, Asuka Inoue

PloS one　18　(1)　e0280448　2023

DOI： 10.1371/journal.pone.0280448 　

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Transient receptor potential (TRP) channels are activated by various extracellular and intracellular stimuli and are involved in many physiological events. Because compounds that act on TRP channels are potential candidates for therapeutic agents, a simple method for evaluating TRP channel activation is needed. In this study, we demonstrated that a transforming growth factor alpha (TGFα) shedding assay, previously developed for detecting G-protein-coupled receptor (GPCR) activation, can also detect TRP channel activation. This assay is a low-cost, easily accessible method that requires only an absorbance microplate reader. Mechanistically, TRP-channel-triggered TGFα shedding is achieved by both of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and 17 (ADAM17), whereas the GPCR-induced TGFα shedding response depends solely on ADAM17. This difference may be the result of qualitative or quantitative differences in intracellular Ca2+ kinetics between TRP channels and GPCRs. Use of epidermal growth factor (EGF) and betacellulin (BTC), substrates of ADAM10, improved the specificity of the shedding assay by reducing background responses mediated by endogenously expressed GPCRs. This assay for TRP channel measurement will not only facilitate the high-throughput screening of TRP channel ligands but also contribute to understanding the roles played by TRP channels as regulators of membrane protein ectodomain shedding.
Constitutively active GPR43 is crucial for proper leukocyte differentiation. International-journal

Sosuke Miyasato, Kurumi Iwata, Reika Mura, Shou Nakamura, Keisuke Yanagida, Hideo Shindou, Yosuke Nagata, Masahiro Kawahara, Satoshi Yamaguchi, Junken Aoki, Asuka Inoue, Teruyuki Nagamune, Takao Shimizu, Motonao Nakamura

FASEB journal : official publication of the Federation of American Societies for Experimental Biology　37　(1)　e22676　2023/01

DOI： 10.1096/fj.202201591R 　

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The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.
Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody International-journal

Mithu Baidya, Madhu Chaturvedi, Hemlata Dwivedi-Agnihotri, Ashutosh Ranjan, Dominic Devost, Yoon Namkung, Tomasz Maciej Stepniewski, Shubhi Pandey, Minakshi Baruah, Bhanupriya Panigrahi, Parishmita Sarma, Manish K. Yadav, Jagannath Maharana, Ramanuj Banerjee, Kouki Kawakami, Asuka Inoue, Jana Selent, Stéphane A. Laporte, Terence E. Hébert, Arun K. Shukla

Nature Communications　13　(1)　4634-4634　2022/12

DOI： 10.1038/s41467-022-32386-x 　

eISSN： 2041-1723
Control of Gαq signaling dynamics and GPCR cross-talk by GRKs. International-journal

Guoqing Xiang, Amanda Acosta-Ruiz, Arthur Radoux-Mergault, Melanie Kristt, Jihye Kim, Jared D Moon, Johannes Broichhagen, Asuka Inoue, Francis S Lee, Miriam Stoeber, Jeremy S Dittman, Joshua Levitz

Science advances　8　(47)　eabq3363　2022/11/25

DOI： 10.1126/sciadv.abq3363 　

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Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.
How Carvedilol activates β2-adrenoceptors. International-journal

Tobias Benkel, Mirjam Zimmermann, Julian Zeiner, Sergi Bravo, Nicole Merten, Victor Jun Yu Lim, Edda Sofie Fabienne Matthees, Julia Drube, Elke Miess-Tanneberg, Daniela Malan, Martyna Szpakowska, Stefania Monteleone, Jak Grimes, Zsombor Koszegi, Yann Lanoiselée, Shannon O'Brien, Nikoleta Pavlaki, Nadine Dobberstein, Asuka Inoue, Viacheslav Nikolaev, Davide Calebiro, Andy Chevigné, Philipp Sasse, Stefan Schulz, Carsten Hoffmann, Peter Kolb, Maria Waldhoer, Katharina Simon, Jesus Gomeza, Evi Kostenis

Nature communications　13　(1)　7109-7109　2022/11/19

DOI： 10.1038/s41467-022-34765-w 　

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Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β1-adrenoceptors, arrestin-biased signalling via β2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system.
Enhanced membrane binding of oncogenic G protein αqQ209L confers resistance to inhibitor YM-254890. International-journal

Clinita E Randolph, Morgan B Dwyer, Jenna L Aumiller, Alethia J Dixon, Asuka Inoue, Patrick Osei-Owusu, Philip B Wedegaertner

The Journal of biological chemistry　298　(11)　102538-102538　2022/11

DOI： 10.1016/j.jbc.2022.102538 　

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Heterotrimeric G proteins couple activated G protein-coupled receptors (GPCRs) to intracellular signaling pathways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the αqQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT αq and GPCR-independent αqQ209L. Although YM inhibits WT αq by binding to αq-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular αqQ209L remains to be fully understood. Here, we show that YM promotes a subcellular redistribution of αqQ209L from the plasma membrane (PM) to the cytoplasm. To test if this loss of PM localization could contribute to the mechanism of inhibition of αqQ209L by YM, we developed and examined N-terminal mutants of αqQ209L, termed PM-restricted αqQ209L, in which the addition of membrane-binding motifs enhanced PM localization and prevented YM-promoted redistribution. Treatment of cells with YM failed to inhibit signaling by these PM-restricted αqQ209L. Additionally, pull-down experiments demonstrated that YM promotes similar conformational changes in both αqQ209L and PM-restricted αqQ209L, resulting in increased binding to βγ and decreased binding to regulator RGS2, and effectors p63RhoGEF-DH/PH and phospholipase C-β. GPCR-dependent signaling by PM-restricted WT αq is strongly inhibited by YM, demonstrating that resistance to YM inhibition by membrane-binding mutants is specific to constitutively active αqQ209L. Together, these results indicate that changes in membrane binding impact the ability of YM to inhibit αqQ209L and suggest that YM contributes to inhibition of αqQ209L by promoting its relocalization.
マルチレイヤー解析技術によるシグナル伝達-生命現象の解読オピオイド受容体のシグナル解読とバイアス型リガンドによる自在制御

川上耕季, 生田達也, 井上飛鳥

日本生化学会大会プログラム・講演要旨集　95回　1S15e-03　2022/11
Publisher: (公社)日本生化学会
PTH1受容体における内在性リガンドの認識機構と構造ダイナミクス

小林和弘, 川上耕季, 草木迫司, 郷野弘剛, 富田敦弘, 小林幹, 志甫谷渉, 山下恵太郎, 西澤知宏, 加藤英明, 井上飛鳥, 濡木理

日本生化学会大会プログラム・講演要旨集　95回　2T12a-07　2022/11
Publisher: (公社)日本生化学会
β-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells. International-journal

Raphael S Haider, Edda S F Matthees, Julia Drube, Mona Reichel, Ulrike Zabel, Asuka Inoue, Andy Chevigné, Cornelius Krasel, Xavier Deupi, Carsten Hoffmann

Nature communications　13　(1)　5638-5638　2022/09/26

DOI： 10.1038/s41467-022-33307-8 　

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β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.
Functional rewiring of G protein-coupled receptor signaling in human labor. International-journal

Abigail R Walker, Camilla B Larsen, Samit Kundu, Christina Stavrinidis, Sung Hye Kim, Asuka Inoue, David F Woodward, Yun S Lee, Roberta Migale, David A MacIntyre, Vasso Terzidou, Francesca Fanelli, Shirin Khanjani, Phillip R Bennett, Aylin C Hanyaloglu

Cell reports　40　(10)　111318-111318　2022/09/06

DOI： 10.1016/j.celrep.2022.111318 　

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Current strategies to manage preterm labor center around inhibition of uterine myometrial contractions, yet do not improve neonatal outcomes as they do not address activation of inflammation. Here, we identify that during human labor, activated oxytocin receptor (OTR) reprograms the prostaglandin E2 receptor, EP2, in the pregnant myometrium to suppress relaxatory/Gαs-cAMP signaling and promote pro-labor/inflammatory responses via altered coupling of EP2 from Gαq/11 to Gαi/o. The ability of EP2 to signal via Gαi/o is recapitulated with in vitro OT and only following OTR activation, suggesting direct EP2-OTR crosstalk. Super-resolution imaging with computational modeling reveals OT-dependent reorganization of EP2-OTR complexes to favor conformations for Gαi over Gαs activation. A selective EP2 ligand, PGN9856i, activates the relaxatory/Gαs-cAMP pathway but not the pro-labor/inflammatory responses in term-pregnant myometrium, even following OT. Our study reveals a mechanism, and provides a potential therapeutic solution, whereby EP2-OTR functional associations could be exploited to delay preterm labor.
Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor. International-journal

Franziska Marie Heydenreich, Bianca Plouffe, Aurélien Rizk, Dalibor Milić, Joris Zhou, Billy Breton, Christian Le Gouill, Asuka Inoue, Michel Bouvier, Dmitry B Veprintsev

Molecular pharmacology　102　(3)　139-149　2022/09

DOI： 10.1124/molpharm.122.000497 　

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Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that the V2R is promiscuous in its ability to engage several G proteins and that its' signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT: By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.
Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex International-journal

Ryoji Suno, Yukihiko Sugita, Kazushi Morimoto, Hiroko Takazaki, Hirokazu Tsujimoto, Mika Hirose, Chiyo Suno-Ikeda, Norimichi Nomura, Tomoya Hino, Asuka Inoue, Kenji Iwasaki, Takayuki Kato, So Iwata, Takuya Kobayashi

Cell Reports　40　(11)　111323-111323　2022/09
Publisher: Elsevier BV
DOI： 10.1016/j.celrep.2022.111323 　

ISSN： 2211-1247

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Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.
Endogenous ligand recognition and structural transition of a human PTH receptor. International-journal

Kazuhiro Kobayashi, Kouki Kawakami, Tsukasa Kusakizako, Hirotake Miyauchi, Atsuhiro Tomita, Kan Kobayashi, Wataru Shihoya, Keitaro Yamashita, Tomohiro Nishizawa, Hideaki E Kato, Asuka Inoue, Osamu Nureki

Molecular cell　82　(18)　3468-3483　2022/08/04

DOI： 10.1016/j.molcel.2022.07.003 　

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Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.
クライオ電子顕微鏡によるヒト由来メラトニン受容体シグナル伝達複合体の立体構造解析(Cryo-EM structure of the human MT1-Gi signaling complex)

Okamoto Hiroyuki, Miyauchi Hirotake, Inoue Asuka, Raimondi Francesco, Tsujimoto Hirokazu, Kusakizako Tsukasa, Shihoya Wataru, Yamashita Keitaro, Suno Ryoji, Nomura Norimichi, Kobayashi Takuya, Iwata So, Nishizawa Tomohiro, Nureki Osamu

生物物理　62　(Suppl.1-2)　S347-S347　2022/08
Publisher: (一社)日本生物物理学会
ISSN： 0582-4052

eISSN： 1347-4219
Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface. International-journal

Yunseok Heo, Naito Ishimoto, Ye-Eun Jeon, Ji-Hye Yun, Mio Ohki, Yuki Anraku, Mina Sasaki, Shunsuke Kita, Hideo Fukuhara, Tatsuya Ikuta, Kouki Kawakami, Asuka Inoue, Katsumi Maenaka, Jeremy R H Tame, Weontae Lee, Sam-Yong Park

PLoS biology　20　(8)　e3001714　2022/08

DOI： 10.1371/journal.pbio.3001714 　

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Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hβ2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.
Cryo-EM structures of the β3 adrenergic receptor bound to solabegron and isoproterenol. International-journal

Ikko Nureki, Kazuhiro Kobayashi, Tatsuki Tanaka, Kanae Demura, Asuka Inoue, Wataru Shihoya, Osamu Nureki

Biochemical and biophysical research communications　611　158-164　2022/06/30

DOI： 10.1016/j.bbrc.2022.04.065 　

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The β3-adrenergic receptor (β3AR) is the most essential drug target for overactive bladder and has therapeutic potentials for the treatments of type 2 diabetes and obesity. Here, we report the cryo-electron microscopy structures of the β3AR-Gs signaling complexes with the selective agonist, solabegron and the nonselective agonist, isoproterenol. Comparison of the isoproterenol-, mirabegron-, and solabegron-bound β3AR structures revealed that the extracellular loop 2 changes its conformation depending on the bound agonist and plays an essential role in solabegron binding. Moreover, β3AR has an intrinsically narrow exosite, regardless of the agonist type. This structural feature clearly explains why β3AR prefers mirabegron and solabegron, as the narrow exosite is suitable for binding with agonists with elongated shapes. Our study deepens the understanding of the binding characteristics of β3AR agonists and may pave the way for developing β3AR-selective drugs.
Effect of Ligands and Transducers on the Neurotensin Receptor 1 Conformational Ensemble. International-journal

Austin D Dixon, Asuka Inoue, Scott A Robson, Kelly J Culhane, Jonathan C Trinidad, Sivaraj Sivaramakrishnan, Fabian Bumbak, Joshua J Ziarek

Journal of the American Chemical Society　144　(23)　10241-10250　2022/06/15

DOI： 10.1021/jacs.2c00828 　

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Using a discrete, intracellular 19F nuclear magnetic resonance (NMR) probe on transmembrane helix 6 of the neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptor's structural ensemble in a solution. For apo NTS1, 19F NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the millisecond to second timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other G protein-coupled receptors (GPCRs), the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to β-arrestin-1 (βArr1) or the C-terminal helix of Gαq, which comprises ≳60% of the GPCR/G protein interface surface area. Whereas βArr1 exclusively selects for pre-existing active-like substates, the Gαq peptide induces a new substate. Both transducer molecules promote substantial line broadening of active-like states, suggesting contributions from additional microsecond to millisecond exchange processes. Together, our study suggests that (i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and (ii) the available static structures do not represent the entire conformational ensemble observed in a solution.
PRECOGx: exploring GPCR signaling mechanisms with deep protein representations. International-journal

Marin Matic, Gurdeep Singh, Francesco Carli, Natalia De Oliveira Rosa, Pasquale Miglionico, Lorenzo Magni, J Silvio Gutkind, Robert B Russell, Asuka Inoue, Francesco Raimondi

Nucleic acids research　50　(W1)　W598-610　2022/05/26

DOI： 10.1093/nar/gkac426 　

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In this study we show that protein language models can encode structural and functional information of GPCR sequences that can be used to predict their signaling and functional repertoire. We used the ESM1b protein embeddings as features and the binding information known from publicly available studies to develop PRECOGx, a machine learning predictor to explore GPCR interactions with G protein and β-arrestin, which we made available through a new webserver (https://precogx.bioinfolab.sns.it/). PRECOGx outperformed its predecessor (e.g. PRECOG) in predicting GPCR-transducer couplings, being also able to consider all GPCR classes. The webserver also provides new functionalities, such as the projection of input sequences on a low-dimensional space describing essential features of the human GPCRome, which is used as a reference to track GPCR variants. Additionally, it allows inspection of the sequence and structural determinants responsible for coupling via the analysis of the most important attention maps used by the models as well as through predicted intramolecular contacts. We demonstrate applications of PRECOGx by predicting the impact of disease variants (ClinVar) and alternative splice forms from healthy tissues (GTEX) of human GPCRs, revealing the power to dissect system biasing mechanisms in both health and disease.
Serodolin, a β-arrestin-biased ligand of 5-HT7 receptor, attenuates pain-related behaviors. International-journal

Chayma El Khamlichi, Flora Reverchon, Nadège Hervouet-Coste, Elodie Robin, Nicolas Chopin, Emmanuel Deau, Fahima Madouri, Cyril Guimpied, Cyril Colas, Arnaud Menuet, Asuka Inoue, Andrzej J Bojarski, Gérald Guillaumet, Franck Suzenet, Eric Reiter, Séverine Morisset-Lopez

Proceedings of the National Academy of Sciences of the United States of America　119　(21)　e2118847119　2022/05/24

DOI： 10.1073/pnas.2118847119 　

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G protein–coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT7 receptor (5-HT7R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a β-arrestin–dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT7R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT7R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT7R stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. In this study, using a β-arrestin–biased agonist, we provided insight into the molecular mechanism triggered by 5-HT7R and revealed its therapeutic potential in the modulation of pain response.
Agonist-Dependent Coupling of the Promiscuous Adenosine A2B Receptor to Gα Protein Subunits. International-journal

Jan Hendrik Voss, Andhika B Mahardhika, Asuka Inoue, Christa E Müller

ACS pharmacology & translational science　5　(5)　373-386　2022/05/13

DOI： 10.1021/acsptsci.2c00020 　

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The adenosine A2B receptor (A2BAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family. It is upregulated under hypoxic conditions, in inflammation and cancer. Previous studies indicated the coupling of the A2BAR to different G proteins, mainly Gs, but in some cases Gq/11 or Gi, depending on the cell type. We have now utilized novel technologies, (i) heterologous expression of individual members of the Gαq/11 protein family (Gαq, Gα11, Gα14, and Gα15) in Gαq/11 knockout cells, and (ii) the TRUPATH platform, allowing the direct observation of Gα protein activation for each of the Gα subunits by bioluminescence resonance energy transfer (BRET) measurements. Three structurally diverse A2BAR agonists were studied: the cognate agonist adenosine, its metabolically stable analog NECA, and the non-nucleosidic partial agonist BAY 60-6583. Adenosine and NECA activated most members of all four Gα protein families (Gαs, Gαq/11, Gαi, and Gα12/13). Significant differences in potencies and efficacies were observed; the highest efficacies were determined at the Gα15, Gαs, and Gα12 proteins, and for NECA additionally at the Gαi2 protein. In contrast, the partial agonist BAY 60-6583 only activated Gα15, Gαs, and Gα12 proteins. Adenosine deaminase, an allosteric modulator of ARs, selectively increased the potency and efficacy of NECA and BAY 60-6583 at the Gα15 protein, while it had no effect or decreased efficacy at the other Gα proteins. We conclude that the A2BAR is preferably coupled to the Gα15, Gαs, and Gα12 proteins. Upon upregulation of receptor or Gα protein expression, coupling to further Gα proteins likely occurs. Importantly, different agonists can display different activation profiles.
GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision. International-journal

Wesley B Asher, Daniel S Terry, G Glenn A Gregorio, Alem W Kahsai, Alessandro Borgia, Bing Xie, Arnab Modak, Ying Zhu, Wonjo Jang, Alekhya Govindaraju, Li-Yin Huang, Asuka Inoue, Nevin A Lambert, Vsevolod V Gurevich, Lei Shi, Robert J Lefkowitz, Scott C Blanchard, Jonathan A Javitch

Cell　185　(10)　1661-1675　2022/05/12

DOI： 10.1016/j.cell.2022.03.042 　

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β-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that β-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the β-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that β-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for β-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of β-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream β-arrestin-mediated events are directed.
Abnormal male reproduction and embryonic development induced by downregulation of a phospholipid fatty acid-introducing enzyme Lpgat1 in zebrafish. International-journal

Takeaki Shibata, Hiroki Kawana, Yuri Nishino, Yoshiko Ito, Hiroyasu Sato, Hirofumi Onishi, Kuniyuki Kano, Asuka Inoue, Yoshitaka Taketomi, Makoto Murakami, Satoshi Kofuji, Hiroshi Nishina, Atsuo Miyazawa, Nozomu Kono, Junken Aoki

Scientific reports　12　(1)　7312-7312　2022/05/04

DOI： 10.1038/s41598-022-11002-4 　

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Phospholipids in the membrane consist of diverse pairs of fatty acids bound to a glycerol backbone. The biological significance of the diversity, however, remains mostly unclear. Part of this diversity is due to lysophospholipid acyltransferases (LPLATs), which introduce a fatty acid into lysophospholipids. The human genome has 14 LPLATs and most of them are highly conserved in vertebrates. Here, we analyzed the function of one of these enzymes, lysophosphatidylglycerol acyltransferase 1 (Lpgat1), in zebrafish. We found that the reproduction of heterozygous (lpgat1+/-) male mutants was abnormal. Crosses between heterozygous males and wild-type females produced many eggs with no obvious cleavage, whereas eggs produced by crosses between heterozygous females and wild-type males cleaved normally. Consistent with this, spermatozoa from heterozygous males had reduced motility and abnormal morphology. We also found that the occurrence of lpgat1 homozygous (lpgat1-/-) mutants was far lower than expected. In addition, downregulation of lpgat1 by morpholino antisense oligonucleotides resulted in severe developmental defects. Lipidomic analysis revealed that selective phospholipid species with stearic acid and docosahexaenoic acid were reduced in homozygous larvae and spermatozoa from heterozygotes. These results suggest that the specific phospholipid molecular species produced by Lpgat1 have an essential role in sperm fertilization and in embryonic development.
Activation and allosteric regulation of the orphan GPR88-Gi1 signaling complex. International-journal

Geng Chen, Jun Xu, Asuka Inoue, Maximilian F Schmidt, Chen Bai, Qiuyuan Lu, Peter Gmeiner, Zheng Liu, Yang Du

Nature communications　13　(1)　2375-2375　2022/05/02

DOI： 10.1038/s41467-022-30081-5 　

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GPR88 is an orphan class A G-protein-coupled receptor that is highly expressed in the striatum and regulates diverse brain and behavioral functions. Here we present cryo-EM structures of the human GPR88-Gi1 signaling complex with or without a synthetic agonist (1R, 2R)-2-PCCA. We show that (1R, 2R)-2-PCCA is an allosteric modulator binding to a herein identified pocket formed by the cytoplasmic ends of transmembrane segments 5, 6, and the extreme C terminus of the α5 helix of Gi1. We also identify an electron density in the extracellular orthosteric site that may represent a putative endogenous ligand of GPR88. These structures, together with mutagenesis studies and an inactive state model obtained from metadynamics simulations, reveal a unique activation mechanism for GPR88 with a set of distinctive structure features and a water-mediated polar network. Overall, our results provide a structural framework for understanding the ligand binding, activation and signaling mechanism of GPR88, and will facilitate the innovative drug discovery for neuropsychiatric disorders and for deorphanization of this receptor.
Phosphorylation barcode ensembles encoded by biased CXCR3 agonists direct non-redundant chemokine signaling

Dylan S. Eiger, Jeffrey Smith, Tujin Shi, Tomasz M. Stepniewski, Christopher Honeycutt, Noelia Boldizsar, Julia Gardner, Chia Feng Tsai, Carrie Nicora, Ahmed Moghieb, Kouki Kawakami, Issac Choi, Richard Smith, Asuka Inoue, Jana Selent, Jon Jacobs, Sudarshan Rajagopal

FASEB journal : official publication of the Federation of American Societies for Experimental Biology　36　2022/05/01

DOI： 10.1096/fasebj.2022.36.S1.0R486 　

eISSN： 1530-6860
Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae International-journal

Takeaki Shibata, Kouki Kawakami, Hiroki Kawana, Junken Aoki, Asuka Inoue

Biochemical and Biophysical Research Communications　602　70-76　2022/04

DOI： 10.1016/j.bbrc.2022.02.098 　

ISSN： 0006-291X

eISSN： 1090-2104
OZITX, a pertussis toxin-like protein for occluding inhibitory G protein signalling including Gαz. International-journal

Alastair C Keen, Maria Hauge Pedersen, Laura Lemel, Daniel J Scott, Meritxell Canals, Dene R Littler, Travis Beddoe, Yuki Ono, Lei Shi, Asuka Inoue, Jonathan A Javitch, J Robert Lane

Communications biology　5　(1)　256-256　2022/03/23

DOI： 10.1038/s42003-022-03191-5 　

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Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB5 toxin, inhibits Gαi/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gαz. The role of Gαz signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gαi/o and Gαz G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein.
Biased agonists of the chemokine receptor CXCR3 differentially signal through Gαi:β-arrestin complexes. International-journal

Kevin Zheng, Jeffrey S Smith, Dylan S Eiger, Anmol Warman, Issac Choi, Christopher C Honeycutt, Noelia Boldizsar, Jaimee N Gundry, Thomas F Pack, Asuka Inoue, Marc G Caron, Sudarshan Rajagopal

Science signaling　15　(726)　eabg5203　2022/03/22

DOI： 10.1126/scisignal.abg5203 　

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G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and signal through the proximal effectors, G proteins and β-arrestins, to influence nearly every biological process. The G protein and β-arrestin signaling pathways have largely been considered separable; however, direct interactions between Gα proteins and β-arrestins have been described that appear to be part of a distinct GPCR signaling pathway. Within these complexes, Gαi/o, but not other Gα protein subtypes, directly interacts with β-arrestin, regardless of the canonical Gα protein that is coupled to the GPCR. Here, we report that the endogenous biased chemokine agonists of CXCR3 (CXCL9, CXCL10, and CXCL11), together with two small-molecule biased agonists, differentially formed Gαi:β-arrestin complexes. Formation of the Gαi:β-arrestin complexes did not correlate well with either G protein activation or β-arrestin recruitment. β-arrestin biosensors demonstrated that ligands that promoted Gαi:β-arrestin complex formation generated similar β-arrestin conformations. We also found that Gαi:β-arrestin complexes did not couple to the mitogen-activated protein kinase ERK, as is observed with other receptors such as the V2 vasopressin receptor, but did couple with the clathrin adaptor protein AP-2, which suggests context-dependent signaling by these complexes. These findings reinforce the notion that Gαi:β-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of the spectrum of biased agonism.
Common coupling map advances GPCR-G protein selectivity. International-journal

Alexander S Hauser, Charlotte Avet, Claire Normand, Arturo Mancini, Asuka Inoue, Michel Bouvier, David E Gloriam

eLife　11　2022/03/18

DOI： 10.7554/eLife.74107 　

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Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein 'couplome'. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on G protein-coupled receptor (GPCR)-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling toward the exploitation of G protein signaling bias in design of safer drugs.
Secreted phospholipase A2 modifies extracellular vesicles and accelerates B cell lymphoma International-journal

Kai Kudo, Yoshimi Miki, Joaquim Carreras, Shunya Nakayama, Yasushi Nakamoto, Masatoshi Ito, Etsuko Nagashima, Kei Yamamoto, Hiroshi Higuchi, Shin-ya Morita, Asuka Inoue, Junken Aoki, Kiyoshi Ando, Naoya Nakamura, Makoto Murakami, Ai Kotani

Cell Metabolism　34　(4)　615-633.e8　2022/03
Publisher: Elsevier BV
DOI： 10.1016/j.cmet.2022.02.011 　

ISSN： 1550-4131

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Extracellular vesicles (EVs) including exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of EV lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived EVs is augmented via secreted phospholipase A2 (sPLA2)-driven lipid metabolism. Hydrolysis of EV phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids, and their metabolites. sPLA2-treated EVs were smaller and self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor-associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice, while treatment with sPLA2-modified EVs reversed this phenotype. Furthermore, sPLA2 expression in human large B cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated EV modification promotes tumor development, highlighting a non-canonical mechanistic action of EVs as an extracellular hydrolytic platform of sPLA2.
βアレスチンの結合様式と機能の包括的理解

倉本律輝, 川上耕季, 生田達也, 桑原莉来, 吉田美沙紀, 井上飛鳥

日本薬学会年会要旨集　142年会　26N-am07S　2022/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Author Correction: Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism (Nature Chemical Biology, (2022), 18, 3, (281-288), 10.1038/s41589-021-00930-3) International-journal

Zhenmei Xu, Tatsuya Ikuta, Kouki Kawakami, Ryoji Kise, Yu Qian, Ruixue Xia, Ming Xia Sun, Anqi Zhang, Changyou Guo, Xue Hui Cai, Zhiwei Huang, Asuka Inoue, Yuanzheng He

Nature Chemical Biology　18　(3)　352-352　2022/03

DOI： 10.1038/s41589-022-00968-x 　

ISSN： 1552-4450

eISSN： 1552-4469
Phenylalanine 193 in Extracellular Loop 2 of the β 2-Adrenergic Receptor Coordinates β-Arrestin Interaction. International-journal

Michael Ippolito, Francesco De Pascali, Asuka Inoue, Jeffrey L Benovic

Molecular pharmacology　101　(2)　87-94　2022/02

DOI： 10.1124/molpharm.121.000332 　

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G protein-coupled receptors (GPCRs) transduce a diverse variety of extracellular stimuli into intracellular signaling. These receptors are the most clinically productive drug targets at present. Despite decades of research on the signaling consequences of molecule-receptor interactions, conformational components of receptor-effector interactions remain incompletely described. The β 2-adrenergic receptor (β 2AR) is a prototypical and extensively studied GPCR that can provide insight into this aspect of GPCR signaling thanks to robust structural data and rich pharmacopeia. Using bioluminescence resonance energy transfer -based biosensors, second messenger assays, and biochemical techniques, we characterize the properties of β 2AR-F193A. This single point mutation in extracellular loop 2 of the β 2AR is sufficient to intrinsically bias the β 2AR away from β-arrestin interaction and demonstrates altered regulatory outcomes downstream of this functional selectivity. This study highlights the importance of extracellular control of intracellular response to stimuli and suggests a previously undescribed role for the extracellular loops of the receptor and the extracellular pocket formed by transmembrane domains 2, 3, and 7 in GPCR regulation that may contribute to biased signaling at GPCRs. SIGNIFICANCE STATEMENT: The role of extracellular G protein-coupled receptor (GPCR) domains in mediating intracellular interactions is poorly understood. We characterized the effects of extracellular loop mutations on agonist-promoted interactions of GPCRs with G protein and β-arrestin. Our studies reveal that F193 in extracellular loop 2 in the β2-adrenergic receptor mediates interactions with G protein and β-arrestin with a biased loss of β-arrestin binding. These results provide new insights on the role of the extracellular domain in differentially modulating intracellular interactions with GPCRs.
Intestinal microbe-dependent ω3 lipid metabolite αKetoA prevents inflammatory diseases in mice and cynomolgus macaques. International-journal

Takahiro Nagatake, Shigenobu Kishino, Emiko Urano, Haruka Murakami, Nahoko Kitamura, Kana Konishi, Harumi Ohno, Prabha Tiwari, Sakiko Morimoto, Eri Node, Jun Adachi, Yuichi Abe, Junko Isoyama, Kento Sawane, Tetsuya Honda, Asuka Inoue, Akiharu Uwamizu, Takashi Matsuzaka, Yoichi Miyamoto, So-Ichiro Hirata, Azusa Saika, Yuki Shibata, Koji Hosomi, Ayu Matsunaga, Hitoshi Shimano, Makoto Arita, Junken Aoki, Masahiro Oka, Akira Matsutani, Takeshi Tomonaga, Kenji Kabashima, Motohiko Miyachi, Yasuhiro Yasutomi, Jun Ogawa, Jun Kunisawa

Mucosal immunology　15　(2)　289-300　2022/02

DOI： 10.1038/s41385-021-00477-5 　

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Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body's use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.
Synthesis of unnatural morphinan compounds to induce itch-like behaviors in mice: Towards the development of MRGPRX2 selective ligands. International-journal Peer-reviewed

Keita Iio, Noriki Kutsumura, Yasuyuki Nagumo, Tsuyoshi Saitoh, Akihisa Tokuda, Kao Hashimoto, Naoshi Yamamoto, Ryoji Kise, Asuka Inoue, Hirokazu Mizoguchi, Hiroshi Nagase

Bioorganic & Medicinal Chemistry Letters　56　128485-128485　2022/01/15

DOI： 10.1016/j.bmcl.2021.128485 　

ISSN： 0960-894X

eISSN： 1464-3405
Lateral access mechanism of LPA receptor probed by molecular dynamics simulation. International-journal

Rieko Suenaga, Mizuki Takemoto, Asuka Inoue, Ryuichiro Ishitani, Osamu Nureki

PloS one　17　(2)　e0263296　2022

DOI： 10.1371/journal.pone.0263296 　

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G-protein-coupled receptors (GPCR) are a family of membrane receptors that play important roles in the regulation of various physiological phenomena. LPA receptors (LPA1-6) are members of the class A GPCRs, which transduce a lysophosphatidic acid (LPA) signal across the cell membrane and evoke various responses, including cellular survival, proliferation, differentiation, and migration. The crystal structure of LPA6 revealed a gap between its transmembrane helices (TMs), which is opened toward the membrane side. This led to the proposal of the "lateral access model," in which its lipophilic ligand directly enters the binding pocket through the gap structure at the membrane. In this study, we performed molecular dynamics (MD) simulations and Markov state model (MSM) analyses of LPA6 and LPA, to elucidate the long timescale dynamics of the ligand binding process. The results from the 71.4-μs MD simulation suggested that the flexibility of the TMs constituting the gap structure enables the lateral entrance of the ligand, and the key interactions between the receptor and ligand facilitate the transition state of the ligand binding process.
Structural transition of human PTH1R revealed distinct molecular recognition and molecular switch for sustainable activation

Kazuhiro Kobayashi, Kawakami Kouki, Kusakizako Tshukasa, Gono Hirotake, Tomita Atsuhiro, Kobayashi Kan, Shihoya Wataru, Yamashita Keitaro, Nishizawa Tomohiro, Kato Hideaki, Inoue Asuka, Nureki Osamu

Proceedings for Annual Meeting of The Japanese Pharmacological Society　95　2-YIA-66　2022
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.95.0_2-yia-66 　

eISSN： 2435-4953

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Parathyroid hormone receptor 1 (PTH1R) is a class B G-protein-coupled receptor (GPCR), consisting of extracellular domain (ECD) and transmembrane domain (TMD). PTH1R is activated by two endogenous peptide hormones called PTH and PTHrP. These hormones share similar sequences and activate the stimulatory G-protein (Gs) signaling pathway but show different physiological functions by the differences in the ligand dissociation kinetics. However, the structural basis for ligand recognition and ligand kinetics remains elusive. We revealed the activated PTH1R structure binding the two endogenous hormones, respectively. The structures and mutagenesis revealed distinct molecular recognition for each ligand and conserved active mechanism of PTH1R. Moreover, these structures elucidate molecular switch toggling signaling periods, responsible for the different pharmacological effects. Furthermore, we revealed five distinct structures PTH-PTH1R-Gs toward inactive transition. These sequential structures and molecular dynamics simulations revealed that an unwinding middle region of PTH induces PTH dissociating from PTH1R. This is the first GPCR structure that suggests the ECD allosterically modulates the activation of the receptor. Our structure provides structural insight for a different signal duration and another strategy for drag development for fine-tuning a duration of the receptor activation.
An intrabody sensor to monitor conformational activation of β-arrestins International-journal

Hemlata Dwivedi-Agnihotri, Parishmita Sarma, S. Deeksha, Kouki Kawakami, Asuka Inoue, Arun K. Shukla

Methods in Cell Biology　169　267-278　2022

DOI： 10.1016/bs.mcb.2021.12.023 　

ISSN： 0091-679X
Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via β-arrestin/ERK signaling. International-journal

Vinay Kumar Sharma, Xuyu Yang, Soo-Kyung Kim, Amirhossein Mafi, Daniel Saiz-Sanchez, Patricia Villanueva-Anguita, Lan Xiao, Asuka Inoue, William A Goddard 3rd, Y Peng Loh

Cellular and molecular life sciences : CMLS　79　(1)　24-24　2021/12/29

DOI： 10.1007/s00018-021-04021-3 　

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Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in β-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was β-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited β-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of β-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the β-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.
Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism International-journal

Zhenmei Xu, Tatsuya Ikuta, Kouki Kawakami, Ryoji Kise, Yu Qian, Ruixue Xia, Ming-Xia Sun, Anqi Zhang, Changyou Guo, Xue-Hui Cai, Zhiwei Huang, Asuka Inoue, Yuanzheng He

Nature Chemical Biology　18　(3)　281-288　2021/12/22
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41589-021-00930-3 　

ISSN： 1552-4450

eISSN： 1552-4469

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Sphingosine-1-phosphate receptor 1 (S1PR1) is a master regulator of lymphocyte egress from the lymph node and an established drug target for multiple sclerosis (MS). Mechanistically, therapeutic S1PR1 modulators activate the receptor yet induce sustained internalization through a potent association with β-arrestin. However, a structural basis of biased agonism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of Gi-bound S1PR1 in complex with S1P, fingolimod-phosphate (FTY720-P) and siponimod (BAF312). In combination with functional assays and molecular dynamics (MD) studies, we reveal that the β-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxxY motifs. Specifically, the intermediate flipping of W2696.48 and the retained interaction between F2656.44 and N3077.49 are the key features of the β-arrestin bias. We further identify ligand-receptor interactions accounting for the S1PR subtype specificity of BAF312. These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.
Vibrational spectroscopy analysis of ligand efficacy in human M2 muscarinic acetylcholine receptor (M2R). International-journal

Kota Katayama, Kohei Suzuki, Ryoji Suno, Ryoji Kise, Hirokazu Tsujimoto, So Iwata, Asuka Inoue, Takuya Kobayashi, Hideki Kandori

Communications biology　4　(1)　1321-1321　2021/11/23

DOI： 10.1038/s42003-021-02836-1 　

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The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.
An ATX-LPA6-Gα13-ROCK axis shapes and maintains caudal vein plexus in zebrafish. International-journal

Ryohei Okasato, Kuniyuki Kano, Ryoji Kise, Asuka Inoue, Shigetomo Fukuhara, Junken Aoki

iScience　24　(11)　103254-103254　2021/11/19

DOI： 10.1016/j.isci.2021.103254 　

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Lysophosphatidic acid (LPA) is a potential regulator of vascular formation derived from blood. In this study, we utilized zebrafish as a model organism to monitor the blood vessel formation in detail. Zebrafish mutant of ATX, an LPA-producing enzyme, had a defect in the caudal vein plexus (CVP). Pharmacological inhibition of ATX resulted in a fusion of the delicate vessels in the CVP to form large sac-like vessels. Mutant embryos of LPA6 receptor and downstream Gα13 showed the same phenotype. Administration of OMPT, a stable LPA-analog, induced rapid CVP constriction, which was attenuated significantly in the LPA6 mutant. We also found that blood flow-induced CVP formation was dependent on ATX. The present study demonstrated that the ATX-LPA6 axis acts cooperatively with blood flow and contributes to the formation and maintenance of the CVP by generating contractive force in endothelial cells.
スフィンゴシン-1-リン酸受容体におけるバイアスドアゴニズム機構

前田信太郎, 椎村祐樹, 浅田秀基, 平田邦生, Luo Fangjia, 南後恵理子, 井上飛鳥, 青木淳賢, 岩田想, 萩原正敏

日本生化学会大会プログラム・講演要旨集　94回　[1T15m-207)]　2021/11
Publisher: (公社)日本生化学会
Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling. International-journal

Emma Rose McGlone, Yusman Manchanda, Ben Jones, Phil Pickford, Asuka Inoue, David Carling, Stephen R Bloom, Tricia Tan, Alejandra Tomas

Molecular metabolism　53　101296-101296　2021/11

DOI： 10.1016/j.molmet.2021.101296 　

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OBJECTIVES: Receptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon (GCG) is important for carbohydrate and lipid metabolism. METHODS: Subcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking the Wiskott-Aldrich Syndrome protein and scar homologue (WASH) complex and the trafficking inhibitor monensin were used to investigate the effect of halted recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 upregulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. RESULTS: GCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular accumulation of GCG-stimulated GCGR in cells lacking the WASH complex or in the presence of monensin indicates that activated GCGR undergoes continuous cycles of internalisation and recycling, despite apparent GCGR plasma membrane localisation up to 40 min post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from β-arrestin-2 recruitment coupled with increased activation of Gαs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only a minor disturbance of carbohydrate metabolism was observed in mice with upregulated hepatic RAMP2. CONCLUSIONS: By retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted.
Unraveling binding mechanism and kinetics of macrocyclic Gαq protein inhibitors. International-journal

Jan H Voss, Jessica Nagel, Muhammad Rafehi, Ramon Guixà-González, Davide Malfacini, Julian Patt, Stefan Kehraus, Asuka Inoue, Gabriele M König, Evi Kostenis, Xavier Deupi, Vigneshwaran Namasivayam, Christa E Müller

Pharmacological research　173　105880-105880　2021/11

DOI： 10.1016/j.phrs.2021.105880 　

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G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gαq protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the "lipophilic anchors" of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development.
Loss of β-Arrestins or six Gα proteins in HEK293 cells caused Warburg effect and prevented progesterone-induced rapid proteasomal degradation of progesterone receptor membrane component 1. International-journal

Mohammad Golam Sabbir, Asuka Inoue, Carla G Taylor, Peter Zahradka

The Journal of steroid biochemistry and molecular biology　214　105995-105995　2021/11

DOI： 10.1016/j.jsbmb.2021.105995 　

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Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.
選択的薬剤が結合したbeta3アドレナリン受容体の構造

志甫谷渉, 名切千彩恵, 井上飛鳥, 濡木理

日本生化学会大会プログラム・講演要旨集　94回　[P-994]　2021/11
Publisher: (公社)日本生化学会
Membrane phosphoinositides stabilize GPCR-arrestin complexes and offer temporal control of complex assembly and dynamics

John Janetzko, Ryoji Kise, Benjamin Barsi-Ryne, Dirk H. Siepe, Franziska M. Heydenreich, Matthieu Masureel, Kouki Kawakami, K. Christopher Garcia, Mark von Zastrow, Asuka Inoue, Brian K. Kobilka

2021/10/10
Publisher: Cold Spring Harbor Laboratory
DOI： 10.1101/2021.10.09.463790 　

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<title>Summary</title>Arrestins recognize activated and phosphorylated G protein-coupled receptors (GPCRs) and are responsible for promoting acute desensitization of receptors as well as their endocytosis. As phosphatidylinositols have been shown to bind to components of the endocytic machinery, including arrestins, we examined the role of phosphoinositide (PIP) binding in GPCR-arrestin complexes. Using a PIP-binding-deficient mutant of arrestin we find that GPCRs stratify into two groups based on whether arrestin-PIP-interactions are required for arrestin recruitment to activated receptors. This requirement for arrestin-PIP-interactions depends on receptor phosphorylation, with receptors having more limited phosphorylation requiring arrestin-PIP-binding capacity. In vitro, this arrestin lipid binding functions to stabilize receptor-arrestin complexes and is crucial for promoting a core-engaged state of the complex. In the absence of a bound GPCR, PIP2, but not endosome resident PI(3)P, promotes conformational changes in arrestin that parallel activation, including movement of the finger and gate loops, but without release of the arrestin C-terminus. These results suggest a model for arrestin recruitment that depends on three components that each function to potentiate the conformation of arrestin: the GPCR core, phosphorylated GPCR C-terminus and membrane phosphoinositides. Integration of a phosphoinositide-dependence into arrestin-GPCR complex assembly provides a mechanism for release of arrestin from GPCRs with insufficient phosphorylation, allowing for their rapid recycling, while explaining how GPCRs that form stable complexes with arrestin can remain associated yet switch from desensitized to allowing G protein coupling in endosomes.
Corrigendum to "BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes" [Biochem. Pharmacol. 158 (2108) 232-242]. International-journal

Shannon L O'Brien, Elizabeth K M Johnstone, Dominic Devost, Jacinta Conroy, Melissa E Reichelt, Brooke W Purdue, Mohammed A Ayoub, Tatsuo Kawai, Asuka Inoue, Satoru Eguchi, Terence E Hébert, Kevin D G Pfleger, Walter G Thomas

Biochemical pharmacology　192　114756-114756　2021/10

DOI： 10.1016/j.bcp.2021.114756 　
Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor. International-journal

Francesco De Pascali, Mohammed Akli Ayoub, Riccardo Benevelli, Silvia Sposini, Jordan Lehoux, Nathalie Gallay, Pauline Raynaud, Flavie Landomiel, Frédéric Jean-Alphonse, Christophe Gauthier, Lucie P Pellissier, Pascale Crépieux, Anne Poupon, Asuka Inoue, Nicolas Joubert, Marie-Claude Viaud-Massuard, Livio Casarini, Manuela Simoni, Aylin C Hanyaloglu, Selva G Nataraja, Henry N Yu, Stephen S Palmer, Romain Yvinec, Eric Reiter

International journal of molecular sciences　22　(18)　2021/09/12

DOI： 10.3390/ijms22189850 　

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Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, β-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or β-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for β-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or β-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.
Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors. International-journal

Ulla Seibel-Ehlert, Nicole Plank, Asuka Inoue, Guenther Bernhardt, Andrea Strasser

International journal of molecular sciences　22　(18)　2021/09/09

DOI： 10.3390/ijms22189739 　

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G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such "invasive" techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1-4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1-4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1-4Rs.
Cryo-EM structure of the β3-adrenergic receptor reveals the molecular basis of subtype selectivity. International-journal

Chisae Nagiri, Kazuhiro Kobayashi, Atsuhiro Tomita, Masahiko Kato, Kan Kobayashi, Keitaro Yamashita, Tomohiro Nishizawa, Asuka Inoue, Wataru Shihoya, Osamu Nureki

Molecular cell　81　(15)　3205-3215　2021/08/05

DOI： 10.1016/j.molcel.2021.06.024 　

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The β3-adrenergic receptor (β3AR) is predominantly expressed in adipose tissue and urinary bladder and has emerged as an attractive drug target for the treatment of type 2 diabetes, obesity, and overactive bladder (OAB). Here, we report the cryogenic electron microscopy structure of the β3AR-Gs signaling complex with the selective agonist mirabegron, a first-in-class drug for OAB. Comparison of this structure with the previously reported β1AR and β2AR structures reveals a receptor activation mechanism upon mirabegron binding to the orthosteric site. Notably, the narrower exosite in β3AR creates a perpendicular pocket for mirabegron. Mutational analyses suggest that a combination of both the exosite shape and the amino-acid-residue substitutions defines the drug selectivity of the βAR agonists. Our findings provide a molecular basis for βAR subtype selectivity, allowing the design of more-selective agents with fewer adverse effects.
Cryo-EM structure of the human MT1–Gi signaling complex International-journal

Hiroyuki H. Okamoto, Hirotake Miyauchi, Asuka Inoue, Francesco Raimondi, Hirokazu Tsujimoto, Tsukasa Kusakizako, Wataru Shihoya, Keitaro Yamashita, Ryoji Suno, Norimichi Nomura, Takuya Kobayashi, So Iwata, Tomohiro Nishizawa, Osamu Nureki

Nature Structural & Molecular Biology　28　(8)　694-701　2021/08/05
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41594-021-00634-1 　

ISSN： 1545-9993

eISSN： 1545-9985

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Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1-Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.
Switching Lysophosphatidylserine G Protein-Coupled Receptor Agonists to Antagonists by Acylation of the Hydrophilic Serine Amine. International-journal

Misa Sayama, Akiharu Uwamizu, Masaya Ikubo, Luying Chen, Ge Yan, Yuko Otani, Asuka Inoue, Junken Aoki, Tomohiko Ohwada

Journal of medicinal chemistry　64　(14)　10059-10101　2021/07/22

DOI： 10.1021/acs.jmedchem.1c00347 　

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Three human G protein-coupled receptors (GPCRs)-GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3-are activated specifically by lysophosphatidylserine (LysoPS), an endogenous hydrolysis product of a cell membrane component, phosphatidylserine (PS). LysoPS consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages. We previously generated potent and selective GPCR agonists by modification of the three modules and the ester linkage. Here, we show that a novel modification of the hydrophilic serine moiety, that is, N-acylations of the serine amine, converted a GPR174 agonist to potent GPR174 antagonists. Structural exploration of the amide functionality provided access to a range of activities from agonist to partial agonist to antagonist. The present study would provide a new strategy for the development of lysophospholipid receptor antagonists.
Heterogeneity of G protein activation by the calcium-sensing receptor. International-journal

Hasnat Ali Abid, Asuka Inoue, Caroline M Gorvin

Journal of molecular endocrinology　67　(2)　41-53　2021/06/21

DOI： 10.1530/JME-21-0058 　

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The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messengers or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G protein activation directly, we took advantage of a recently described NanoBiT G protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G proteins were similarly expressed, suggesting CaSR may activate multiple G proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.
Extracellular succinate hyperpolarizes M2 macrophages through SUCNR1/GPR91-mediated Gq signaling. International-journal

Mette Trauelsen, Thomas K Hiron, Da Lin, Jacob E Petersen, Billy Breton, Anna Sofie Husted, Siv A Hjorth, Asuka Inoue, Thomas M Frimurer, Michel Bouvier, Chris A O'Callaghan, Thue W Schwartz

Cell reports　35　(11)　109246-109246　2021/06/15

DOI： 10.1016/j.celrep.2021.109246 　

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Succinate functions both as a classical TCA cycle metabolite and an extracellular metabolic stress signal sensed by the mainly Gi-coupled succinate receptor SUCNR1. In the present study, we characterize and compare effects and signaling pathways activated by succinate and both classes of non-metabolite SUCNR1 agonists. By use of specific receptor and pathway inhibitors, rescue in G-protein-depleted cells and monitoring of receptor G protein activation by BRET, we identify Gq rather than Gi signaling to be responsible for SUCNR1-mediated effects on basic transcriptional regulation. Importantly, in primary human M2 macrophages, in which SUCNR1 is highly expressed, we demonstrate that physiological concentrations of extracellular succinate act through SUCNR1-activated Gq signaling to efficiently regulate transcription of immune function genes in a manner that hyperpolarizes their M2 versus M1 phenotype. Thus, sensing of stress-induced extracellular succinate by SUCNR1 is an important transcriptional regulator in human M2 macrophages through Gq signaling.
Endogenous agonist-bound S1PR3 structure reveals determinants of G protein-subtype bias. International-journal

Shintaro Maeda, Yuki Shiimura, Hidetsugu Asada, Kunio Hirata, Fangjia Luo, Eriko Nango, Nobuo Tanaka, Masayasu Toyomoto, Asuka Inoue, Junken Aoki, So Iwata, Masatoshi Hagiwara

Science advances　7　(24)　2021/06

DOI： 10.1126/sciadv.abf5325 　

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Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the "quartet core") exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein-coupled receptors and will help the design of biased ligands for optimized therapeutics.
Smoothened transduces hedgehog signals via activity-dependent sequestration of PKA catalytic subunits International-journal

Corvin D. Arveseth, John T. Happ, Danielle S. Hedeen, Ju Fen Zhu, Jacob L. Capener, Dana Klatt Shaw, Ishan Deshpande, Jiahao Liang, Jiewei Xu, Sara L. Stubben, Isaac B. Nelson, Madison F. Walker, Kouki Kawakami, Asuka Inoue, Nevan J. Krogan, David J. Grunwald, Ruth Hüttenhain, Aashish Manglik, Benjamin R. Myers

PLoS Biology　19　(4)　e3001191　2021/04

DOI： 10.1371/journal.pbio.3001191 　

ISSN： 1544-9173

eISSN： 1545-7885
Noncanonical scaffolding of Gαi and β-arrestin by G protein-coupled receptors. International-journal

Jeffrey S Smith, Thomas F Pack, Asuka Inoue, Claudia Lee, Kevin Zheng, Issac Choi, Dylan S Eiger, Anmol Warman, Xinyu Xiong, Zhiyuan Ma, Gayathri Viswanathan, Ian M Levitan, Lauren K Rochelle, Dean P Staus, Joshua C Snyder, Alem W Kahsai, Marc G Caron, Sudarshan Rajagopal

Science (New York, N.Y.)　371　(6534)　2021/03/12

DOI： 10.1126/science.aay1833 　

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Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and β-arrestin adaptor proteins. G protein-mediated signaling and β-arrestin-mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between Gαi protein subtype family members and β-arrestins regardless of their canonical Gα protein subtype coupling. Gαi:β-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with β-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:β-arrestin signaling complexes.
Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2. International-journal

Natalia V Ortiz Zacarías, Kirti K Chahal, Tereza Šimková, Cas van der Horst, Yi Zheng, Asuka Inoue, Emy Theunissen, Lloyd Mallee, Daan van der Es, Julien Louvel, Adriaan P IJzerman, Tracy M Handel, Irina Kufareva, Laura H Heitman

Journal of medicinal chemistry　64　(5)　2608-2621　2021/03/11

DOI： 10.1021/acs.jmedchem.0c01137 　

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Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound 14 as an intracellular covalent ligand for CCR2. In silico modeling followed by site-directed mutagenesis confirmed that 14 forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound 14 represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.
N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand. International-journal

Akiko Ogawa, Chisae Nagiri, Wataru Shihoya, Asuka Inoue, Kouki Kawakami, Suzune Hiratsuka, Junken Aoki, Yasuhiro Ito, Takeo Suzuki, Tsutomu Suzuki, Toshihiro Inoue, Osamu Nureki, Hidenobu Tanihara, Kazuhito Tomizawa, Fan-Yan Wei

Molecular cell　81　(4)　659-674　2021/02/18

DOI： 10.1016/j.molcel.2020.12.038 　

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About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.
Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors International-journal

Shubhi Pandey, Punita Kumari, Mithu Baidya, Ryoji Kise, Yubo Cao, Hemlata Dwivedi-Agnihotri, Ramanuj Banerjee, Xaria X. Li, Cedric S. Cui, John D. Lee, Kouki Kawakami, Madhu Chaturvedi, Ashutosh Ranjan, Stéphane A. Laporte, Trent M. Woodruff, Asuka Inoue, Arun K. Shukla

Molecular cell　81　(22)　4605-4621　2021/02/02
Publisher: Cold Spring Harbor Laboratory
DOI： 10.1101/2021.02.02.429298 　

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<title>Abstract</title>G protein-coupled receptors (GPCRs) are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and β-arrestins (βarrs). Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. This raises the question if some naturally-occurring 7TMRs may selectively engage one of these two signal-transducers, even in response to their endogenous agonists. Although there are scattered hints in the literature that some 7TMRs lack G-protein coupling but interact with βarrs, an in-depth understanding of their transducer-coupling preference, GRK-engagement, downstream signaling and structural mechanism remains elusive. Here, we use an array of cellular, biochemical and structural approaches to comprehensively characterize two non-canonical 7TMRs namely, the human decoy D6 receptor (D6R) and the human complement C5a receptor (C5aR2), in parallel with their canonical GPCR counterparts, CCR2 and C5aR1, respectively. We discover that D6R and C5aR2 couple exclusively to βarrs, exhibit distinct GRK-preference, and activate non-canonical downstream signaling partners. We also observe that βarrs, in complex with these receptors, adopt distinct conformations compared to their canonical GPCR counterparts despite being activated by a common natural agonist. Our study therefore establishes D6R and C5aR2 as bona-fide arrestin-coupled receptors (ACRs), and provides important insights into their regulation by GRKs and downstream signaling with direct implications for biased agonism.
S1PR3-G12-biased agonist ALESIA targets cancer metabolism and promotes glucose starvation. International-journal

Masayasu Toyomoto, Asuka Inoue, Kei Iida, Masatsugu Denawa, Isao Kii, Francois Marie Ngako Kadji, Takayuki Kishi, Dohyun Im, Tatsuro Shimamura, Hiroshi Onogi, Suguru Yoshida, So Iwata, Junken Aoki, Takamitsu Hosoya, Masatoshi Hagiwara

Cell chemical biology　28　(8)　1132-1144　2021/02/02

DOI： 10.1016/j.chembiol.2021.01.004 　

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Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G12-biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy.
Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359. International-journal

Cornelia Hermes, René Richarz, Daniel A Wirtz, Julian Patt, Wiebke Hanke, Stefan Kehraus, Jan Hendrik Voß, Jim Küppers, Tsubasa Ohbayashi, Vigneshwaran Namasivayam, Judith Alenfelder, Asuka Inoue, Peter Mergaert, Michael Gütschow, Christa E Müller, Evi Kostenis, Gabriele M König, Max Crüsemann

Nature communications　12　(1)　144-144　2021/01/08

DOI： 10.1038/s41467-020-20418-3 　

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The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.
An experimental strategy to probe Gq contribution to signal transduction in living cells. International-journal

Julian Patt, Judith Alenfelder, Eva Marie Pfeil, Jan Hendrik Voss, Nicole Merten, Funda Eryilmaz, Nina Heycke, Uli Rick, Asuka Inoue, Stefan Kehraus, Xavier Deupi, Christa E Müller, Gabriele M König, Max Crüsemann, Evi Kostenis

The Journal of biological chemistry　296　100472-100472　2021

DOI： 10.1016/j.jbc.2021.100472 　

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Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR-Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity.
A novel luminescence-based β-arrestin recruitment assay for unmodified receptors. International-journal

Maria Hauge Pedersen, Jennifer Pham, Helena Mancebo, Asuka Inoue, Wesley B Asher, Jonathan A Javitch

The Journal of biological chemistry　296　100503-100503　2021

DOI： 10.1016/j.jbc.2021.100503 　

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G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestin-signaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells.
Constitutive G protein coupling profiles of understudied orphan GPCRs. International-journal

Sumin Lu, Wonjo Jang, Asuka Inoue, Nevin A Lambert

PloS one　16　(4)　e0247743　2021

DOI： 10.1371/journal.pone.0247743 　

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A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered "orphan" receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and β-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively-active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.
Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors International-journal

Ben Jones, Emma Rose McGlone, Zijian Fang, Phil Pickford, Ivan R. Corrêa, Atsuro Oishi, Ralf Jockers, Asuka Inoue, Sunil Kumar, Frederik Görlitz, Chris Dunsby, Paul M.W. French, Guy A. Rutter, Tricia Tan, Alejandra Tomas, Stephen R. Bloom

Journal of Biological Chemistry　296　100133-100133　2021/01
Publisher: Elsevier BV
DOI： 10.1074/jbc.ra120.016334 　

ISSN： 0021-9258

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Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of β-arrestins. Indeed, recently described GLP-1R agonists with reduced β-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both β-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in β-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced β-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.
Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gβγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs International-journal

Eva Marie Pfeil, Julian Brands, Nicole Merten, Timo Vögtle, Maddalena Vescovo, Ulrike Rick, Ina Maria Albrecht, Nina Heycke, Kouki Kawakami, Yuki Ono, Francois Marie Ngako Kadji, Suzune Hiratsuka, Junken Aoki, Felix Häberlein, Michaela Matthey, Jaspal Garg, Stephanie Hennen, Marie Lise Jobin, Kerstin Seier, Davide Calebiro, Alexander Pfeifer, Akos Heinemann, Daniela Wenzel, Gabriele M. König, Bernhard Nieswandt, Bernd K. Fleischmann, Asuka Inoue, Katharina Simon, Evi Kostenis

Molecular Cell　80　(6)　940-954.e6　2020/12/17

DOI： 10.1016/j.molcel.2020.10.027 　

ISSN： 1097-2765

eISSN： 1097-4164
Publisher Correction: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3. International-journal

Signe Mathiasen, Tiago Palmisano, Nicole A Perry, Hannah M Stoveken, Alex Vizurraga, Dyke P McEwen, Najeah Okashah, Tobias Langenhan, Asuka Inoue, Nevin A Lambert, Gregory G Tall, Jonathan A Javitch

Nature chemical biology　16　(12)　1440-1440　2020/12

DOI： 10.1038/s41589-020-0649-z 　

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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone International-journal

Dohyun Im, Asuka Inoue, Takaaki Fujiwara, Takanori Nakane, Yasuaki Yamanaka, Tomoko Uemura, Chihiro Mori, Yuki Shiimura, Kanako Terakado Kimura, Hidetsugu Asada, Norimichi Nomura, Tomoyuki Tanaka, Ayumi Yamashita, Eriko Nango, Kensuke Tono, Francois Marie Ngako Kadji, Junken Aoki, So Iwata, Tatsuro Shimamura

Nature Communications　11　(1)　6442-6442　2020/12
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-020-20221-0 　

eISSN： 2041-1723

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<title>Abstract</title>In addition to the serotonin 5-HT_2A receptor (5-HT_2AR), the dopamine D₂ receptor (D₂R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D₂R have been described in complex with the inverse agonists risperidone (D₂R_ris) and haloperidol (D₂R_hal). Here we describe the structure of human D₂R in complex with spiperone (D₂R_spi). In D₂R_spi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D₂R_ris and D₂R_hal, demonstrating that ECL2 in D₂R is highly dynamic. Moreover, D₂R_spi exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D₂R and 5-HT_2AR. Together with D₂R_ris and D₂R_hal, the structural information of D₂R_spi should be of value for designing novel antipsychotics with improved safety and efficacy.
CXCR7: a β-arrestin-biased receptor that potentiates cell migration and recruits β-arrestin2 exclusively through Gβγ subunits and GRK2. International-journal

Huong Thi Nguyen, Arfaxad Reyes-Alcaraz, Hyo Jeong Yong, Lan Phuong Nguyen, Hee-Kyung Park, Asuka Inoue, Cheol Soon Lee, Jae Young Seong, Jong-Ik Hwang

Cell & bioscience　10　(1)　134-134　2020/11/23

DOI： 10.1186/s13578-020-00497-x 　

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BACKGROUND: Some chemokine receptors referred to as atypical chemokine receptors (ACKRs) are thought to non-signaling decoys because of their inability to activate typical G-protein signaling pathways. CXCR7, also known as ACKR3, binds to only two chemokines, SDF-1α and I-TAC, and recruits β-arrestins. SDF-1α also binds to its own conventional receptor, CXCR4, involving in homeostatic modulation such as development and immune surveillance as well as pathological conditions such as inflammation, ischemia, and cancers. Recently, CXCR7 is suggested as a key therapeutic target together with CXCR4 in such conditions. However, the molecular mechanisms underlying cellular responses and functional relation with CXCR7 and CXCR4 have not been elucidated, despite massive studies. Therefore, we aimed to reveal the molecular networks of CXCR7 and CXCR4 and compare their effects on cell migration. METHODS: Base on structural complementation assay using NanoBiT technology, we characterized the distinct mechanisms underlying β-arrestin2 recruitment by both CXCR4 and CXCR7. Crosslinking and immunoprecipitation were conducted to analyze complex formation of the receptors. Gene deletion using CRISPR and reconstitution of the receptors were applied to analysis of ligand-dependent ERK phosphorylation and cell migration. All experiments were performed in triplicate and repeated more than three times. Unpaired Student's t-tests or ANOVA using PRISM5 software were employed for statistical analyses. RESULTS: Ligand binding to CXCR7 does not result in activation of typical signaling pathways via Gα subunits but activation of GRK2 via βγ subunits and receptor phosphorylation with subsequent β-arrestin2 recruitment. In contrast, CXCR4 induced Gαi activation and recruited β-arrestin2 through C-terminal phosphorylation by both GRK2 and GRK5. SDF-1α-stimulated ERK phosphorylation was facilitated by CXCR4, but not CXCR7. Heterodimerization of CXCR4 and CXCR7 was not confirmed in this study, while homodimerization of them was verified by crosslinking experiment and NanoBiT assay. Regarding chemotaxis, SDF-1α-stimulated cell migration was mediated by both CXCR4 and CXCR7. CONCLUSION: This study demonstrates that SDF-1α-stimulated CXCR7 mediates β-arrestin2 recruitment via different molecular networking from that of CXCR4. CXCR7 may be neither a simple scavenger nor auxiliary receptor but plays an essential role in cell migration through cooperation with CXCR4.
Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists. International-journal

Ali Işbilir, Jan Möller, Marta Arimont, Vladimir Bobkov, Cristina Perpiñá-Viciano, Carsten Hoffmann, Asuka Inoue, Raimond Heukers, Chris de Graaf, Martine J Smit, Paolo Annibale, Martin J Lohse

Proceedings of the National Academy of Sciences of the United States of America　117　(46)　29144-29154　2020/11/17

DOI： 10.1073/pnas.2013319117 　

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Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.
Internalization-Dependent Free Fatty Acid Receptor 2 Signaling Is Essential for Propionate-Induced Anorectic Gut Hormone Release. International-journal

Natarin Caengprasath, Noemi Gonzalez-Abuin, Maria Shchepinova, Yue Ma, Asuka Inoue, Edward W Tate, Gary Frost, Aylin C Hanyaloglu

iScience　23　(9)　101449-101449　2020/09/25

DOI： 10.1016/j.isci.2020.101449 　

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The ability of propionate, a short-chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management, and glycemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2), a GPCR that activates Gαi and Gαq/11. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell whereby FFA2 is targeted to very early endosomes. Furthermore, propionate activates a Gαi/p38 signaling pathway, which requires receptor internalization and is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites engage membrane trafficking pathways and that receptor internalization could orchestrate complex GPCR pathways within the gut.
GPR101 drives growth hormone hypersecretion and gigantism in mice via constitutive activation of Gs and Gq/11. International-journal

Dayana Abboud, Adrian F Daly, Nadine Dupuis, Mohamed Ali Bahri, Asuka Inoue, Andy Chevigné, Fabien Ectors, Alain Plenevaux, Bernard Pirotte, Albert Beckers, Julien Hanson

Nature communications　11　(1)　4752-4752　2020/09/21

DOI： 10.1038/s41467-020-18500-x 　

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Growth hormone (GH) is a key modulator of growth and GH over-secretion can lead to gigantism. One form is X-linked acrogigantism (X-LAG), in which infants develop GH-secreting pituitary tumors over-expressing the orphan G-protein coupled receptor, GPR101. The role of GPR101 in GH secretion remains obscure. We studied GPR101 signaling pathways and their effects in HEK293 and rat pituitary GH3 cell lines, human tumors and in transgenic mice with elevated somatotrope Gpr101 expression driven by the rat Ghrhr promoter (GhrhrGpr101). Here, we report that Gpr101 causes elevated GH/prolactin secretion in transgenic GhrhrGpr101 mice but without hyperplasia/tumorigenesis. We show that GPR101 constitutively activates not only Gs, but also Gq/11 and G12/13, which leads to GH secretion but not proliferation. These signatures of GPR101 signaling, notably PKC activation, are also present in human pituitary tumors with high GPR101 expression. These results underline a role for GPR101 in the regulation of somatotrope axis function.
三量体G蛋白質-GEF-GAPシグナル:受容体シグナルの時空間的選択性と多様性の生化学 GPCRバイアスリガンドのシグナル機構の理解

川上耕季, 平塚寿々音, 井上飛鳥

日本生化学会大会プログラム・講演要旨集　93回　[2S03a-03]　2020/09
Publisher: (公社)日本生化学会
Agonist-induced formation of unproductive receptor-G<inf>12</inf> complexes International-journal

Najeah Okashah, Shane C. Wright, Kouki Kawakami, Signe Mathiasen, Joris Zhou, Sumin Lu, Jonathan A. Javitch, Asuka Inoue, Michel Bouvier, Nevin A. Lambert

Proceedings of the National Academy of Sciences of the United States of America　117　(35)　21723-21730　2020/09/01

DOI： 10.1073/pnas.2003787117 　

ISSN： 0027-8424

eISSN： 1091-6490
新たな酸化脂質研究の潮流酸化リン脂質とそのGPCR型受容体を介した痒みの知覚

岸貴之, 可野邦行, 大村谷昌樹, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　93回　[2S08m-05]　2020/09
Publisher: (公社)日本生化学会
G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3. International-journal Peer-reviewed

Signe Mathiasen, Tiago Palmisano, Nicole A Perry, Hannah M Stoveken, Alex Vizurraga, Dyke P McEwen, Najeah Okashah, Tobias Langenhan, Asuka Inoue, Nevin A Lambert, Gregory G Tall, Jonathan A Javitch

Nature chemical biology　16　(12)　1343-1350　2020/08/10

DOI： 10.1038/s41589-020-0617-7 　

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The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.
Delineation of molecular determinants for FR900359 inhibition of Gq/11 unlocks inhibition of Gαs. International-journal Peer-reviewed

Michael W Boesgaard, Kasper Harpsøe, Michelle Malmberg, Christina R Underwood, Asuka Inoue, Jesper M Mathiesen, Gabriele M König, Evi Kostenis, David E Gloriam, Hans Bräuner-Osborne

The Journal of biological chemistry　295　(40)　13850-13861　2020/08/04

DOI： 10.1074/jbc.RA120.013002 　

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Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11subfamily consists of Gq, G11, G14 and G16 proteins of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families; Gs, Gi/o and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359 binding site in Gq and Gs We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359 and a minimum of three mutations are necessary to introduce inhibition in Gs In all, this suggest that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved opening up for targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359 sensitive Gas mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.
Structural insights into differences in G protein activation by family A and family B GPCRs. International-journal Peer-reviewed

Daniel Hilger, Kaavya Krishna Kumar, Hongli Hu, Mie Fabricius Pedersen, Evan S O'Brien, Lise Giehm, Christine Jennings, Gözde Eskici, Asuka Inoue, Michael Lerch, Jesper Mosolff Mathiesen, Georgios Skiniotis, Brian K Kobilka

Science (New York, N.Y.)　369　(6503)　2020/07/31

DOI： 10.1126/science.aba3373 　

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Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β2 adrenergic receptor (β2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the β2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.
Non-naturally-occurring Regio Isomer of Lysophosphatidylserine Exhibits Potent Agonistic Activity Towards G Protein-coupled Receptors. International-journal Peer-reviewed

Sho Nakamura, Misa Sayama, Akiharu Uwamizu, Sejin Jung, Masaya Ikubo, Yuko Otani, Kuniyuki Kano, Jumpei Omi, Asuka Inoue, Junken Aoki, Tomohiko Ohwada

Journal of medicinal chemistry　63　(17)　9990-10029　2020/07/28

DOI： 10.1021/acs.jmedchem.0c01126 　

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Lysophosphatidylserine (LysoPS), an endogenous ligand of G protein-coupled receptors, consists of L-serine, glycerol and fatty acid moieties connected by phosphodiester and ester linkages, respectively. An ester linkage of phosphatidylserine (PS) can be hydrolyzed at the 1-position or at the 2-position, to give 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. 2-Acyl lysophospholipid is in non-enzymatic equilibrium with 1-acyl lysophospholipid in vivo. On the other hand, 3-acyl lysophospholipid is not found, at least in mammals, raising the question of whether the reason for this might be that the 3-acyl isomer lacks the biological activities of the other isomers. Here, to test this idea, we designed and synthesized a series of new 3-acyl lysophospholipids. Structure-activity relationship studies of more than 100 "glycol surrogates" derivatives led to the identification of potent and selective agonists for LysoPS receptors GPR34 and P2Y10. Thus, the non-natural 3-acyl compounds are indeed active, and appear to be biologically orthogonal with respect to the physiologically relevant 1- and 2-acyl lysophospholipids.
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation. International-journal Peer-reviewed

Mithu Baidya, Punita Kumari, Hemlata Dwivedi-Agnihotri, Shubhi Pandey, Madhu Chaturvedi, Tomasz Maciej Stepniewski, Kouki Kawakami, Yubo Cao, Stéphane A Laporte, Jana Selent, Asuka Inoue, Arun K Shukla

EMBO reports　21　(9)　e49886　2020/07/26

DOI： 10.15252/embr.201949886 　

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β-arrestins (βarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B2 R), analogous to that present in the vasopressin receptor (V2 R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys294 in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.
Structure and selectivity engineering of the M1 muscarinic receptor toxin complex. International-journal Peer-reviewed

Shoji Maeda, Jun Xu, Francois Marie N Kadji, Mary J Clark, Jiawei Zhao, Naotaka Tsutsumi, Junken Aoki, Roger K Sunahara, Asuka Inoue, K Christopher Garcia, Brian K Kobilka

Science (New York, N.Y.)　369　(6500)　161-167　2020/07/10

DOI： 10.1126/science.aax2517 　

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Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.
G protein-regulated endocytic trafficking of adenylyl cyclase type 9. International-journal Peer-reviewed

André M Lazar, Roshanak Irannejad, Tanya A Baldwin, Aparna B Sundaram, J Silvio Gutkind, Asuka Inoue, Carmen W Dessauer, Mark Von Zastrow

eLife　9　2020/06/09

DOI： 10.7554/eLife.58039 　

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GPCRs are increasingly recognized to initiate signaling via heterotrimeric G proteins as they move through the endocytic network, but little is known about how relevant G protein effectors are localized. Here we report selective trafficking of adenylyl cyclase type 9 (AC9) from the plasma membrane to endosomes while adenylyl cyclase type 1 (AC1) remains in the plasma membrane, and stimulation of AC9 trafficking by ligand-induced activation of Gs-coupled GPCRs. AC9 transits a similar, dynamin-dependent early endocytic pathway as ligand-activated GPCRs. However, unlike GPCR traffic control which requires β-arrestin but not Gs, AC9 traffic control requires Gs but not β-arrestin. We also show that AC9, but not AC1, mediates cAMP production stimulated by endogenous receptor activation in endosomes. These results reveal dynamic and isoform-specific trafficking of adenylyl cyclase in the endocytic network, and a discrete role of a heterotrimeric G protein in regulating the subcellular distribution of a relevant effector.
Signal profiling of the β1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β1AR and β2AR. International-journal Peer-reviewed

Viktoriya Lukasheva, Dominic Devost, Christian Le Gouill, Yoon Namkung, Ryan D Martin, Jean-Michel Longpré, Mohammad Amraei, Yuji Shinjo, Mireille Hogue, Monique Lagacé, Billy Breton, Junken Aoki, Jason C Tanny, Stéphane A Laporte, Graciela Pineyro, Asuka Inoue, Michel Bouvier, Terence E Hébert

Scientific reports　10　(1)　8779-8779　2020/05/29

DOI： 10.1038/s41598-020-65636-3 　

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A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β1-adrenergic receptor (β1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the β1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.
免疫応答におけるリゾリン脂質シグナルの機能解明

近江純平, 新上雄司, 可野邦行, 上水明治, 井上飛鳥, 青木淳賢

脂質生化学研究　62　192-193　2020/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone. International-journal Peer-reviewed

Hidetsugu Asada, Asuka Inoue, Francois Marie Ngako Kadji, Kunio Hirata, Yuki Shiimura, Dohyun Im, Tatsuro Shimamura, Norimichi Nomura, Hiroko Iwanari, Takao Hamakubo, Osamu Kusano-Arai, Hiromi Hisano, Tomoko Uemura, Chiyo Suno, Junken Aoki, So Iwata

Structure (London, England : 1993)　28　(4)　418-425　2020/04/07

DOI： 10.1016/j.str.2019.12.003 　

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Angiotensin II (AngII) is a peptide hormone that plays a key role in regulating blood pressure, and its interactions with the G protein-coupled receptors, AngII type-1 receptor (AT1R) and AngII type-2 receptor (AT2R), are central to its mechanism of action. We solved the crystal structure of human AT2R bound to AngII and its specific antibody at 3.2-Å resolution. AngII (full agonist) and [Sar1, Ile8]-AngII (partial agonist) interact with AT2R in a similar fashion, except at the bottom of the AT2R ligand-binding pocket. In particular, the residues including Met1283.36, which constitute the deep end of the cavity, play important roles in angiotensin receptor (ATR) activation upon AngII binding. These differences that occur upon endogenous ligand binding may contribute to a structural change in AT2R, leading to normalization of the non-canonical coordination of helix 8. Our results will inform the design of more effective ligands for ATRs.
Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors. International-journal Peer-reviewed

Markus Kuschak, Vigneshwaran Namasivayam, Muhammad Rafehi, Jan H Voss, Jaspal Garg, Jonathan G Schlegel, Aliaa Abdelrahman, Stefan Kehraus, Raphael Reher, Jim Küppers, Katharina Sylvester, Sonja Hinz, Michaela Matthey, Daniela Wenzel, Bernd K Fleischmann, Alexander Pfeifer, Asuka Inoue, Michael Gütschow, Gabriele M König, Christa E Müller

British journal of pharmacology　177　(8)　1898-1916　2020/04

DOI： 10.1111/bph.14960 　

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BACKGROUND AND PURPOSE: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. EXPERIMENTAL APPROACH: We have now developed Gq -specific, cell-permeable 3 H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. KEY RESULTS: FR and YM displayed low nanomolar affinity for Gαq , Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq -knockout cells, but not for Gα15 . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel Gq inhibitors, which inhibited Gq signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. CONCLUSIONS AND IMPLICATIONS: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.
Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR. International-journal Peer-reviewed

Alex D White, Frederic G Jean-Alphonse, Fei Fang, Karina A Peña, Shi Liu, Gabriele M König, Asuka Inoue, Despoina Aslanoglou, Samuel H Gellman, Evi Kostenis, Kunhong Xiao, Jean-Pierre Vilardaga

Proceedings of the National Academy of Sciences of the United States of America　117　(13)　7455-7460　2020/03/31

DOI： 10.1073/pnas.1918158117 　

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cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11 We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gβγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR-βarrestin-Gβγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.
Membrane Phospholipid Analogues as Molecular Rulers to Probe the Position of the Hydrophobic Contact Point of Lysophospholipid Ligands on the Surface of G-Protein-Coupled Receptor during Membrane Approach. International-journal Peer-reviewed

Misa Sayama, Akiharu Uwamizu, Yuko Otani, Asuka Inoue, Junken Aoki, Masakazu Sekijima, Tomohiko Ohwada

Biochemistry　59　(11)　1173-1201　2020/03/24

DOI： 10.1021/acs.biochem.0c00061 　

ISSN： 0006-2960

eISSN： 1520-4995
クライオ電子顕微鏡を用いたメラトニン受容体シグナル伝達複合体の立体構造解析

岡本紘幸, 宮内弘剛, 井上飛鳥, 志甫谷渉, 山下恵太郎, 草木迫司, 西澤知宏, 濡木理

日本薬学会年会要旨集　140年会　27H-pm10S　2020/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein. International-journal Peer-reviewed

Kazuhiro Kobayashi, Wataru Shihoya, Tomohiro Nishizawa, Francois Marie Ngako Kadji, Junken Aoki, Asuka Inoue, Osamu Nureki

Nature structural & molecular biology　27　(3)　274-280　2020/03

DOI： 10.1038/s41594-020-0386-8 　

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here, we present a cryo-EM structure of human PAC1R bound to PACAP and an engineered Gs heterotrimer. The structure revealed that transmembrane helix TM1 plays an essential role in PACAP recognition. The extracellular domain (ECD) of PAC1R tilts by ~40° compared with that of the glucagon-like peptide-1 receptor (GLP-1R) and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP-1R ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.
Structure of the neurotensin receptor 1 in complex with β-arrestin 1. International-journal Peer-reviewed

Weijiao Huang, Matthieu Masureel, Qianhui Qu, John Janetzko, Asuka Inoue, Hideaki E Kato, Michael J Robertson, Khanh C Nguyen, Jeffrey S Glenn, Georgios Skiniotis, Brian K Kobilka

Nature　579　(7798)　303-308　2020/03

DOI： 10.1038/s41586-020-1953-1 　

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Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with βarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.
Structural insight into small molecule action on Frizzleds. International-journal Peer-reviewed

Paweł Kozielewicz, Ainoleena Turku, Carl-Fredrik Bowin, Julian Petersen, Jana Valnohova, Maria Consuelo Alonso Cañizal, Yuki Ono, Asuka Inoue, Carsten Hoffmann, Gunnar Schulte

Nature communications　11　(1)　414-414　2020/01/21

DOI： 10.1038/s41467-019-14149-3 　

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WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.
Activation of the GLP-1 receptor by a non-peptidic agonist. International-journal Peer-reviewed

Peishen Zhao, Yi-Lynn Liang, Matthew J Belousoff, Giuseppe Deganutti, Madeleine M Fletcher, Francis S Willard, Michael G Bell, Michael E Christe, Kyle W Sloop, Asuka Inoue, Tin T Truong, Lachlan Clydesdale, Sebastian G B Furness, Arthur Christopoulos, Ming-Wei Wang, Laurence J Miller, Christopher A Reynolds, Radostin Danev, Patrick M Sexton, Denise Wootten

Nature　577　(7790)　432-436　2020/01

DOI： 10.1038/s41586-019-1902-z 　

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Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
Spatial regulation of GPR64/ADGRG2 signaling by β-arrestins and GPCR kinases. International-journal Peer-reviewed

Pedram Azimzadeh, Sarah C Talamantez-Lyburn, Katarina T Chang, Asuka Inoue, Nariman Balenga

Annals of the New York Academy of Sciences　1456　(1)　26-43　2019/11

DOI： 10.1111/nyas.14227 　

ISSN： 0077-8923

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Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 signaling and endocytosis, we generated mutants that lack either NTF alone (ΔNTF) or NTF and tethered agonist (P622). We discover that unlike full-length GPR64, ΔNTF and P622 mutants interact with β-arrestin1 and β-arrestins2 and are constitutively internalized in steady states. However, only ΔNTF shows exaggerated basal activation of the Gαs -cAMP-CRE signaling cascade. Neither ΔNTF nor P622 shows constitutive activation of the Gα13 -SRE pathway, but both mutants respond to exogenously added agonistic peptide via CRE and SRE. GPCR kinases and dynamin mediate the constitutive internalization of ΔNTF and P622 to early endosomes, where ΔNTF constantly induces CRE. These data suggest that NTF not only shields the tethered agonist to prevent G protein signaling but also confers a conformation that inhibits the interaction with β-arrestins and the consequent endocytosis and sustained signaling from endosomes.
High-endothelial cell-derived S1P regulates dendritic cell localization and vascular integrity in the lymph node. International-journal Peer-reviewed

Szandor Simmons, Naoko Sasaki, Eiji Umemoto, Yutaka Uchida, Shigetomo Fukuhara, Yusuke Kitazawa, Michiyo Okudaira, Asuka Inoue, Kazuo Tohya, Keita Aoi, Junken Aoki, Naoki Mochizuki, Kenjiro Matsuno, Kiyoshi Takeda, Masayuki Miyasaka, Masaru Ishii

eLife　8　2019/10/01

DOI： 10.7554/eLife.41239 　

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While the sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor-1 (S1PR1) axis is critically important for lymphocyte egress from lymphoid organs, S1PR1-activation also occurs in vascular endothelial cells (ECs), including those of the high-endothelial venules (HEVs) that mediate lymphocyte immigration into lymph nodes (LNs). To understand the functional significance of the S1P/S1PR1-Gi axis in HEVs, we generated Lyve1;Spns2Δ/Δ conditional knockout mice for the S1P-transporter Spinster-homologue-2 (SPNS2), as HEVs express LYVE1 during development. In these mice HEVs appeared apoptotic and were severely impaired in function, morphology and size; leading to markedly hypotrophic peripheral LNs. Dendritic cells (DCs) were unable to interact with HEVs, which was also observed in Cdh5CRE-ERT2;S1pr1Δ/Δ mice and wildtype mice treated with S1PR1-antagonists. Wildtype HEVs treated with S1PR1-antagonists in vitro and Lyve1-deficient HEVs show severely reduced release of the DC-chemoattractant CCL21 in vivo. Together, our results reveal that EC-derived S1P warrants HEV-integrity through autocrine control of S1PR1-Gi signaling, and facilitates concomitant HEV-DC interactions.
生理活性物質アンジオテンシンIIの結合によるアンジオテンシンII受容体活性化機構の構造基盤

浅田秀基, 井上飛鳥, Francois Kadji M.N., 平田邦生, 椎村祐樹, Dohyun Im, 青木淳賢, 岩田想

日本生化学会大会プログラム・講演要旨集　92回　[1T03m-04]　2019/09
Publisher: (公社)日本生化学会
新規酸化リン脂質受容体の呼吸制御機構の解析

岸貴之, 可野邦行, 井上飛鳥, 吉田美沙紀, 大村谷昌樹, 青木淳賢

日本生化学会大会プログラム・講演要旨集　92回　[2P-040]　2019/09
Publisher: (公社)日本生化学会
リゾホスファチジルセリンはオートクライン/パラクライン的に作用し活性化B細胞の凝集塊形成を抑制する

上水明治, 新上雄司, 井上飛鳥, 可野邦行, 青木淳賢

日本生化学会大会プログラム・講演要旨集　92回　[3T18m-369)]　2019/09
Publisher: (公社)日本生化学会
Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm. International-journal Peer-reviewed

Francesco Raimondi, Asuka Inoue, Francois M N Kadji, Ni Shuai, Juan-Carlos Gonzalez, Gurdeep Singh, Alicia Alonso de la Vega, Rocio Sotillo, Bernd Fischer, Junken Aoki, J Silvio Gutkind, Robert B Russell

Oncogene　38　(38)　6491-6506　2019/09

DOI： 10.1038/s41388-019-0895-2 　

ISSN： 0950-9232

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Oncodriver genes are usually identified when mutations recur in multiple tumours. Different drivers often converge in the activation or repression of key cancer-relevant pathways. However, as many pathways contain multiple members of the same gene family, individual mutations might be overlooked, as each family member would necessarily have a lower mutation frequency and thus not identified as significant in any one-gene-at-a-time analysis. Here, we looked for mutated, functional sequence positions in gene families that were mutually exclusive (in patients) with another gene in the same pathway, which identified both known and new candidate oncodrivers. For instance, many inactivating mutations in multiple G-protein (particularly Gi/o) coupled receptors, are mutually exclusive with Gαs oncogenic activating mutations, both of which ultimately enhance cAMP signalling. By integrating transcriptomics and interaction data, we show that the Gs pathway is upregulated in multiple cancer types, even those lacking known GNAS activating mutations. This suggests that cancer cells may develop alternative strategies to activate adenylate cyclase signalling in multiple cancer types. Our study provides a mechanistic interpretation for several rare somatic mutations in multi-gene oncodrivers, and offers possible explanations for known and potential off-label cancer treatments, suggesting new therapeutic opportunities.
WNT-3A-induced β-catenin signaling does not require signaling through heterotrimeric G proteins. International-journal Peer-reviewed

Carl-Fredrik Bowin, Asuka Inoue, Gunnar Schulte

The Journal of biological chemistry　294　(31)　11677-11684　2019/08/02

DOI： 10.1074/jbc.AC119.009412 　

ISSN： 0021-9258

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The network of Wingless/Int-1 (WNT)-induced signaling pathways includes β-catenin-dependent and -independent pathways. β-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, β-catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL receptor-related protein 5/6 (LRP5/6) receptor complex. FZDs are G protein-coupled receptors, but the question of whether heterotrimeric G proteins are involved in WNT/β-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/β-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of Gs/Golf/Gq/G11/G12/G13/Gz in HEK293 (ΔG7) cells, leaving the expression of pertussis toxin (PTX)-sensitive Gi/o proteins unchanged, to assess whether WNT-3A activates WNT/β-catenin signaling in WT and ΔG7 cells devoid of functional G protein signaling. We monitored WNT-3A-induced activation by detection of phosphorylation of LDL receptor-related protein 6 (LRP6), electrophoretic mobility shift of the phosphoprotein Dishevelled (DVL), β-catenin stabilization and dephosphorylation, and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/β-catenin signaling in ΔG7 cells in both the absence and presence of Gi/o-blocking PTX. Furthermore, cells completely devoid of G protein expression, so called Gα-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/β-catenin signaling. These findings corroborate the concept that heterotrimeric G proteins are not required for this FZD- and DVL-mediated signaling branch. Our observations agree with previous results arguing for FZD conformation-dependent functional selectivity between DVL and heterotrimeric G proteins. In conclusion, WNT/β-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins.
Conformational transitions of a neurotensin receptor 1-Gi1 complex. International-journal Peer-reviewed

Hideaki E Kato, Yan Zhang, Hongli Hu, Carl-Mikael Suomivuori, Francois Marie Ngako Kadji, Junken Aoki, Kaavya Krishna Kumar, Rasmus Fonseca, Daniel Hilger, Weijiao Huang, Naomi R Latorraca, Asuka Inoue, Ron O Dror, Brian K Kobilka, Georgios Skiniotis

Nature　572　(7767)　80-85　2019/08

DOI： 10.1038/s41586-019-1337-6 　

ISSN： 0028-0836

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Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells. International-journal Peer-reviewed

Teresa Buenaventura, Stavroula Bitsi, William E Laughlin, Thomas Burgoyne, Zekun Lyu, Affiong I Oqua, Hannah Norman, Emma R McGlone, Andrey S Klymchenko, Ivan R Corrêa Jr, Abigail Walker, Asuka Inoue, Aylin Hanyaloglu, Jak Grimes, Zsombor Koszegi, Davide Calebiro, Guy A Rutter, Stephen R Bloom, Ben Jones, Alejandra Tomas

PLoS biology　17　(8)　e3000097　2019/08

DOI： 10.1371/journal.pbio.3000097 　

ISSN： 1544-9173

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The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.
Illuminating the Onco-GPCRome: Novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy. International-journal Peer-reviewed

Victoria Wu, Huwate Yeerna, Nijiro Nohata, Joshua Chiou, Olivier Harismendy, Francesco Raimondi, Asuka Inoue, Robert B Russell, Pablo Tamayo, J Silvio Gutkind

The Journal of biological chemistry　294　(29)　11062-11086　2019/07/19

DOI： 10.1074/jbc.REV119.005601 　

ISSN： 0021-9258

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G protein-coupled receptors (GPCRs) are the largest gene family of cell membrane-associated molecules mediating signal transmission, and their involvement in key physiological functions is well-established. The ability of GPCRs to regulate a vast array of fundamental biological processes, such as cardiovascular functions, immune responses, hormone and enzyme release from endocrine and exocrine glands, neurotransmission, and sensory perception (e.g. vision, odor, and taste), is largely due to the diversity of these receptors and the layers of their downstream signaling circuits. Dysregulated expression and aberrant functions of GPCRs have been linked to some of the most prevalent human diseases, which renders GPCRs one of the top targets for pharmaceutical drug development. However, the study of the role of GPCRs in tumor biology has only just begun to make headway. Recent studies have shown that GPCRs can contribute to the many facets of tumorigenesis, including proliferation, survival, angiogenesis, invasion, metastasis, therapy resistance, and immune evasion. Indeed, GPCRs are widely dysregulated in cancer and yet are underexploited in oncology. We present here a comprehensive analysis of GPCR gene expression, copy number variation, and mutational signatures in 33 cancer types. We also highlight the emerging role of GPCRs as part of oncocrine networks promoting tumor growth, dissemination, and immune evasion, and we stress the potential benefits of targeting GPCRs and their signaling circuits in the new era of precision medicine and cancer immunotherapies.
PRECOG: PREdicting COupling probabilities of G-protein coupled receptors. International-journal Peer-reviewed

Gurdeep Singh, Asuka Inoue, J Silvio Gutkind, Robert B Russell, Francesco Raimondi

Nucleic acids research　47　(W1)　W395-W401-W401　2019/07/02

DOI： 10.1093/nar/gkz392 　

ISSN： 0305-1048

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G-protein coupled receptors (GPCRs) control multiple physiological states by transducing a multitude of extracellular stimuli into the cell via coupling to intra-cellular heterotrimeric G-proteins. Deciphering which G-proteins couple to each of the hundreds of GPCRs present in a typical eukaryotic organism is therefore critical to understand signalling. Here, we present PRECOG (precog.russelllab.org): a web-server for predicting GPCR coupling, which allows users to: (i) predict coupling probabilities for GPCRs to individual G-proteins instead of subfamilies; (ii) visually inspect the protein sequence and structural features that are responsible for a particular coupling; (iii) suggest mutations to rationally design artificial GPCRs with new coupling properties based on predetermined coupling features.
An accurate and versatile method for determining the acyl group-introducing position of lysophospholipid acyltransferases. International-journal Peer-reviewed

Hiroki Kawana, Kuniyuki Kano, Hideo Shindou, Asuka Inoue, Takao Shimizu, Junken Aoki

Biochimica et biophysica acta. Molecular and cell biology of lipids　1864　(7)　1053-1060　2019/07

DOI： 10.1016/j.bbalip.2019.02.008 　

ISSN： 1388-1981

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Lysophospholipid acyltransferases (LPLATs) incorporate a fatty acid into the hydroxyl group of lysophospholipids (LPLs) and are critical for determining the fatty acid composition of phospholipids. Previous studies have focused mainly on their molecular identification and their substrate specificity regarding the polar head groups and acyl-CoAs. However, little is known about the positional specificity of the hydroxyl group of the glycerol backbone (sn-2 or sn-1) at which LPLATs introduce a fatty acid. This is mainly due to the instability of LPLs used as an acceptor, especially for LPLs with a fatty acid at the sn-2 position of the glycerol backbone (sn-2-LPLs), which are essential for the enzymatic assay to determine the positional specificity. In this study, we established a method to determine the positional specificity of LPLAT by preparing stable sn-2-LPLs in combination with PLA2 digestion, and applied the method for determining the positional specificity of several LPLATs including LPCAT1, LYCAT and LPCAT3. We found that LPCAT1 introduced palmitic acid both at the sn-1 and sn-2 positions of palmitoyl-LPC, while LYCAT and LPCAT3 specifically introduced stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 position of LPC, respectively. The present method for evaluating the positional specificity could also be used for biochemical characterization of other LPLATs.
Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes. International-journal Peer-reviewed

Yu Hisano, Mari Kono, Andreane Cartier, Eric Engelbrecht, Kuniyuki Kano, Kouki Kawakami, Yanbao Xiong, Wenji Piao, Sylvain Galvani, Keisuke Yanagida, Andrew Kuo, Yuki Ono, Satoru Ishida, Junken Aoki, Richard L Proia, Jonathan S Bromberg, Asuka Inoue, Timothy Hla

The Journal of experimental medicine　216　(7)　1582-1598　2019/07/01

DOI： 10.1084/jem.20181895 　

ISSN： 0022-1007

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Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein-coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9-based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/β-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/β-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.
Variable G protein determinants of GPCR coupling selectivity. International-journal Peer-reviewed

Najeah Okashah, Qingwen Wan, Soumadwip Ghosh, Manbir Sandhu, Asuka Inoue, Nagarajan Vaidehi, Nevin A Lambert

Proceedings of the National Academy of Sciences of the United States of America　116　(24)　12054-12059　2019/06/11

DOI： 10.1073/pnas.1905993116 　

ISSN： 0027-8424

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G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the Gs- and Gq-coupled receptors we studied are relatively promiscuous and always couple to some extent to Gi1 heterotrimers. In contrast, Gi-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.
Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity. International-journal Peer-reviewed

Davide Malfacini, Julian Patt, Suvi Annala, Kasper Harpsøe, Funda Eryilmaz, Raphael Reher, Max Crüsemann, Wiebke Hanke, Hang Zhang, Daniel Tietze, David E Gloriam, Hans Bräuner-Osborne, Kristian Strømgaard, Gabriele M König, Asuka Inoue, Jesus Gomeza, Evi Kostenis

The Journal of biological chemistry　294　(15)　5747-5758　2019/04/12

DOI： 10.1074/jbc.RA118.007250 　

ISSN： 0021-9258

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Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.
Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation. International-journal Peer-reviewed

Amanda E Mackenzie, Tezz Quon, Li-Chiung Lin, Alexander S Hauser, Laura Jenkins, Asuka Inoue, Andrew B Tobin, David E Gloriam, Brian D Hudson, Graeme Milligan

FASEB journal : official publication of the Federation of American Societies for Experimental Biology　33　(4)　5005-5017　2019/04

DOI： 10.1096/fj.201801956R 　

ISSN： 0892-6638

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Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.
Direct targeting of Gαq and Gα11 oncoproteins in cancer cells. International-journal Peer-reviewed

Suvi Annala, Xiaodong Feng, Naveen Shridhar, Funda Eryilmaz, Julian Patt, JuHee Yang, Eva M Pfeil, Rodolfo Daniel Cervantes-Villagrana, Asuka Inoue, Felix Häberlein, Tanja Slodczyk, Raphael Reher, Stefan Kehraus, Stefania Monteleone, Ramona Schrage, Nina Heycke, Ulrike Rick, Sandra Engel, Alexander Pfeifer, Peter Kolb, Gabriele König, Moritz Bünemann, Thomas Tüting, José Vázquez-Prado, J Silvio Gutkind, Evelyn Gaffal, Evi Kostenis

Science signaling　12　(573)　2019/03/19

DOI： 10.1126/scisignal.aau5948 　

ISSN： 1945-0877

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Somatic gain-of-function mutations of GNAQ and GNA11, which encode α subunits of heterotrimeric Gαq/11 proteins, occur in about 85% of cases of uveal melanoma (UM), the most common cancer of the adult eye. Molecular therapies to directly target these oncoproteins are lacking, and current treatment options rely on radiation, surgery, or inhibition of effector molecules downstream of these G proteins. A hallmark feature of oncogenic Gαq/11 proteins is their reduced intrinsic rate of hydrolysis of guanosine triphosphate (GTP), which results in their accumulation in the GTP-bound, active state. Here, we report that the cyclic depsipeptide FR900359 (FR) directly interacted with GTPase-deficient Gαq/11 proteins and preferentially inhibited mitogenic ERK signaling rather than canonical phospholipase Cβ (PLCβ) signaling driven by these oncogenes. Thereby, FR suppressed the proliferation of melanoma cells in culture and inhibited the growth of Gαq-driven UM mouse xenografts in vivo. In contrast, FR did not affect tumor growth when xenografts carried mutated B-RafV600E as the oncogenic driver. Because FR enabled suppression of malignant traits in cancer cells that are driven by activating mutations at codon 209 in Gαq/11 proteins, we envision that similar approaches could be taken to blunt the signaling of non-Gαq/11 G proteins.
Non-Edg型LPA受容体LPA4-6及びGPR55の作動薬創製

金藤奨, 井上飛鳥, 可野邦行, 尾谷優子, 大和田智彦, 青木淳賢

日本薬学会年会要旨集　139年会　(3)　75-75　2019/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Gαiファミリー欠損細胞を用いたGPCR-Gαiサブタイプの共役の解析

小野雄基, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢

日本薬学会年会要旨集　139年会　(3)　64-64　2019/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Konjac ceramide (kCer) regulates keratinocyte migration by Sema3A-like repulsion mechanism. International-journal Peer-reviewed

Seigo Usuki, Noriko Tamura, Tomohiro Tamura, Shigeki Higashiyama, Kunikazu Tanji, Susumu Mitsutake, Asuka Inoue, Junken Aoki, Katsuyuki Mukai, Yasuyuki Igarashi

Biochemistry and biophysics reports　17　132-138　2019/03

DOI： 10.1016/j.bbrep.2018.11.004 　

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Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway.
Identification and biochemical characterization of a second zebrafish autotaxin gene. International-journal Peer-reviewed

Ryoji Kise, Ryohei Okasato, Kuniyuki Kano, Asuka Inoue, Atsuo Kawahara, Junken Aoki

Journal of biochemistry　165　(3)　269-275　2019/03/01

DOI： 10.1093/jb/mvy114 　

ISSN： 0021-924X

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Autotaxin (ATX) is a secreted enzyme that produces a bioactive lysophospholipid, lysophosphatidic acid (LPA). ATX plays a role in vascular and neural development in embryos but its mechanisms remain unclear. At the beginning of this study, only one zebrafish atx gene (atxa) was known and had been investigated. In this study, we generated ATX knockout (KO) fish by TALEN targeting atxa. Unexpectedly, atxa KO fish showed neither vascular defects nor reduction of ATX activity, implying the existence of one or more other ATXs in the genome. By a BLAST search using ATXa protein fragments as a query, we found a genomic sequence that closely resembled atxa exons 13, 14 and 15. Consequently, we cloned a cDNA encoding a second zebrafish autotaxin (ATXb), and found that it was transcribed in various tissues. The atxb gene encoded a protein of 832 amino acids (compared to 850 amino acids in ATXa) with 60% amino acid identity to ATXa and clustered with ATXs from other species. A recombinant ATXb protein showed lysophospholipase D (lysoPLD) activities with substrate specificities similar to those of ATXa and mammalian ATXs. These results indicate that ATXb is a second zebrafish ATX, which possibly shares redundant roles with ATXa in embryonic development.
Efficacy of iguratimod vs. salazosulfapyridine as the first-line csDMARD for rheumatoid arthritis. Peer-reviewed

Nozaki Y, Inoue A, Kinoshita K, Funauchi M, Matsumura I

Modern rheumatology　1-10　2019/03

DOI： 10.1080/14397595.2019.1572267 　

ISSN： 1439-7595
Identification of P2Y receptors involved in oleamide-suppressing inflammatory responses in murine microglia and human dendritic cells. International-journal Peer-reviewed

Masahiro Kita, Yasuhisa Ano, Asuka Inoue, Junken Aoki

Scientific reports　9　(1)　3135-3135　2019/02/28

DOI： 10.1038/s41598-019-40008-8 　

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Microglia, a type of immune cell in the central nervous system, are involved in inflammation leading to neurodegenerative diseases. We previously identified oleamide from fermented dairy products as a neuroprotective compound suppressing microglial inflammation. Oleamide is an endocannabinoid and displays anti-inflammatory activity via the cannabinoid-2 (CB2) receptor; however, the mechanism underlying this anti-inflammatory activity has not been fully elucidated. Here, we found that the suppressive effect of oleamide on microglial tumor necrosis factor-α (TNF-α) production was canceled by inhibitors of G-protein-coupled receptor (GPCR) downstream signaling but not by a CB2 antagonist, suggesting that GPCRs other than CB2 are involved in the anti-inflammatory effects of oleamide. An extensive screen for GPCRs using a transforming growth factor-α shedding assay system identified P2Y1, P2Y4, P2Y6, P2Y10, and P2Y11 as candidates for the oleamide target. P2Y1 and P2Y10 agonists suppressed microglial TNF-α production, while a pan P2 receptor antagonist canceled the suppressive effect. Furthermore, we observed a relationship between the P2Y1 agonistic activities and the suppressive activities of oleamide and its analogs. Taken together, our results suggest that, in addition to CB2, P2Y type receptors are the potential targets of oleamide, and P2Y1 plays a role in the suppression of microglial inflammatory responses by oleamide. (200/200 words).
Molecular mechanism of lysophosphatidic acid-induced hypertensive response. International-journal Peer-reviewed

Kuniyuki Kano, Hirotaka Matsumoto, Asuka Inoue, Hiroshi Yukiura, Motomu Kanai, Jerold Chun, Satoshi Ishii, Takao Shimizu, Junken Aoki

Scientific reports　9　(1)　2662-2662　2019/02/25

DOI： 10.1038/s41598-019-39041-4 　

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Lysophosphatidic acid (LPA) is a blood-derived bioactive lipid with numerous biological activities exerted mainly through six defined G protein-coupled receptors (LPA1-LPA6). LPA was first identified as a vasoactive compound because it induced transient hypertension when injected intravenously in rodents. Here, we examined the molecular mechanism underlying the LPA-induced hypertensive response. The LPA-induced hypertensive response was significantly attenuated by pretreatment with a Rho kinase inhibitor, which blocks Gα12/13 signaling. Consistent with this, the response was weakened in KO mice of LPA4, a Gα12/13-coupling LPA receptor. KO mice of another Gα12/13-coupling LPA receptor, LPA6, also showed an attenuated LPA-induced hypertensive response. However, LPA6 KO mice also displayed attenuated pressor responses to an adrenergic agent and abnormal blood vessel formation. Using several LPA analogs with varied affinity for each LPA receptor, we found a good correlation between the hypertensive and LPA4 agonistic activities. Incubated mouse plasma, which contained abundant LPA, also induced a hypertensive response. Interestingly the response was completely abolished when the plasma was incubated in the presence of an ATX inhibitor. Together, these results indicate that circulating LPA produced by ATX contributes to the elevation of blood pressure through multiple LPA receptors, mainly LPA4.
GPR31-dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial metabolites. International-journal Peer-reviewed

Naoki Morita, Eiji Umemoto, Setsuko Fujita, Akio Hayashi, Junichi Kikuta, Ikuo Kimura, Takeshi Haneda, Toshio Imai, Asuka Inoue, Hitomi Mimuro, Yuichi Maeda, Hisako Kayama, Ryu Okumura, Junken Aoki, Nobuhiko Okada, Toshiyuki Kida, Masaru Ishii, Ryusuke Nabeshima, Kiyoshi Takeda

Nature　566　(7742)　110-114　2019/02

DOI： 10.1038/s41586-019-0884-1 　

ISSN： 0028-0836

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Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.
Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine. International-journal Peer-reviewed

Kanako Terakado Kimura, Hidetsugu Asada, Asuka Inoue, Francois Marie Ngako Kadji, Dohyun Im, Chihiro Mori, Takatoshi Arakawa, Kunio Hirata, Yayoi Nomura, Norimichi Nomura, Junken Aoki, So Iwata, Tatsuro Shimamura

Nature structural & molecular biology　26　(2)　121-128　2019/02

DOI： 10.1038/s41594-018-0180-z 　

ISSN： 1545-9993

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Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs.
Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis. International-journal Peer-reviewed

Bradley L Hoare, Shoni Bruell, Ashish Sethi, Paul R Gooley, Michael J Lew, Mohammed A Hossain, Asuka Inoue, Daniel J Scott, Ross A D Bathgate

iScience　11　93-113　2019/01/25

DOI： 10.1016/j.isci.2018.12.004 　

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The peptide hormone H2 relaxin has demonstrated promise as a therapeutic, but mimetic development has been hindered by the poorly understood relaxin receptor RXFP1 activation mechanism. H2 relaxin is hypothesized to bind to two distinct ECD sites, which reorientates the N-terminal LDLa module to activate the transmembrane domain. Here we provide evidence for this model in live cells by measuring bioluminescence resonance energy transfer (BRET) between nanoluciferase-tagged RXFP1 constructs and fluorescently labeled H2 relaxin (NanoBRET). Additionally, we validate these results using the related RXFP2 receptor and chimeras with an inserted RXFP1-binding domain utilizing NanoBRET and nuclear magnetic resonance studies on recombinant proteins. We therefore provide evidence for the multi-component molecular mechanism of H2 relaxin binding to RXFP1 on the full-length receptor in cells. Also, we show the utility of NanoBRET real-time binding kinetics to reveal subtle binding complexities, which may be overlooked in traditional equilibrium binding assays.
Asymmetric Recruitment of β-Arrestin1/2 by the Angiotensin II Type I and Prostaglandin F2α Receptor Dimer. International-journal Peer-reviewed

Dany Fillion, Dominic Devost, Rory Sleno, Asuka Inoue, Terence E Hébert

Frontiers in endocrinology　10　162-162　2019

DOI： 10.3389/fendo.2019.00162 　

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Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells. Here, we hypothesize that both Ang II- and PGF2α-induced activation of the AT1R/FP dimer, or the parent receptors alone, differentially regulate signaling by distinct patterns of β-arrestin recruitment. Using BRET-based biosensors, we assessed the recruitment kinetics of β-arrestin1/2 to the AT1R/FP dimer, or the parent receptors alone, when stimulated by either Ang II or PGF2α. Using cell lines with CRISPR/Cas9-mediated gene deletion, we also examined the role of G proteins in such recruitment. We observed that Ang II induced a rapid, robust, and sustained recruitment of β-arrestin1/2 to AT1R and, to a lesser extent, the heterodimer, as expected, since AT1R is a strong recruiter of both β-arrestin subtypes. However, PGF2α did not induce such recruitment to FP alone, although it did when the AT1R is present as a heterodimer. β-arrestins were likely recruited to the AT1R partner of the dimer. Gαq, Gα11, Gα12, and Gα13 were all involved to some extent in PGF2α-induced β-arrestin1/2 recruitment to the dimer as their combined absence abrogated the response, and their separate re-expression was sufficient to partially restore it. Taken together, our data sheds light on a new mechanism whereby PGF2α specifically recruits and signals through β-arrestin but only in the context of the AT1R/FP dimer, suggesting that this may be a new allosteric signaling entity.
Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches. International-journal Peer-reviewed

Kyla Bourque, Dominic Devost, Asuka Inoue, Terence E Hébert

Methods in molecular biology (Clifton, N.J.)　1947　169-182　2019

DOI： 10.1007/978-1-4939-9121-1_9 　

ISSN： 1064-3745

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Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.
Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500. International-journal Peer-reviewed

Chisae Nagiri, Wataru Shihoya, Asuka Inoue, Francois Marie Ngako Kadji, Junken Aoki, Osamu Nureki

Communications biology　2　236-236　2019

DOI： 10.1038/s42003-019-0482-7 　

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Endothelin receptors (ETA and ETB) are G-protein-coupled receptors activated by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a potent ETB-selective antagonist, which has preventive effects against brain edema. Here, we report the crystal structure of the human ETB receptor in complex with IRL2500 at 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500 and endothelin-1, and provides structural insights into its ETB-selectivity. Notably, the biphenyl group of IRL2500 penetrates into the transmembrane core proximal to D2.50, thus stabilizing the inactive conformation. Using the newly-established constitutively active mutant, we clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor. The current findings will expand the chemical space of ETR antagonists and facilitate the design of inverse agonists for other class A GPCRs.
BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes. International-journal Peer-reviewed

Shannon L O'Brien, Elizabeth K M Johnstone, Dominic Devost, Jacinta Conroy, Melissa E Reichelt, Brooke W Purdue, Mohammed A Ayoub, Tatsuo Kawai, Asuka Inoue, Satoru Eguchi, Terence E Hébert, Kevin D G Pfleger, Walter G Thomas

Biochemical pharmacology　158　232-242　2018/12

DOI： 10.1016/j.bcp.2018.10.017 　

ISSN： 0006-2952

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The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of β-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.
Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation. International-journal Peer-reviewed

Wataru Shihoya, Tamaki Izume, Asuka Inoue, Keitaro Yamashita, Francois Marie Ngako Kadji, Kunio Hirata, Junken Aoki, Tomohiro Nishizawa, Osamu Nureki

Nature communications　9　(1)　4711-4711　2018/11/09

DOI： 10.1038/s41467-018-07094-0 　

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Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.
Toward Structural Understanding of Ligand Recognition by Lysophosphatidic Acid Receptor Lpa6 Peer-reviewed

Taniguchi Reiya, Inoue Asuka, Sayama Misa, Yamashita Keitaro, Hirata Kunio, Yoshida Masahito, Nakada-Nakura Yoshiko, Otani Yuko, Kato Hideaki, Nishizawa Tomohiro, Doi Takayuki, Ohwada Tomohiko, Ishitani Ryuichiro, Aoki Junken, Nureki Osamu

PROTEIN SCIENCE　27　235-236　2018/11

ISSN： 0961-8368
Neuromedin U directly induces degranulation of skin mast cells, presumably via MRGPRX2. International-journal Peer-reviewed

Yoshimi Matsuo, Yuhki Yanase, Reiko Irifuku, Shunsuke Takahagi, Shoji Mihara, Kaori Ishii, Tomoko Kawaguchi, Akio Tanaka, Kazumasa Iwamoto, Haruka Watanuki, Kazuyuki Furuta, Satoshi Tanaka, Asuka Inoue, Junken Aoki, Michihiro Hide

Allergy　73　(11)　2256-2260　2018/11

DOI： 10.1111/all.13555 　

ISSN： 0105-4538
The LPA2 receptor agonist Radioprotectin-1 spares Lgr5-positive intestinal stem cells from radiation injury in murine enteroids. International-journal Peer-reviewed

Bryan Kuo, Erzsébet Szabó, Sue Chin Lee, Andrea Balogh, Derek Norman, Asuka Inoue, Yuki Ono, Junken Aoki, Gábor Tigyi

Cellular signalling　51　23-33　2018/11

DOI： 10.1016/j.cellsig.2018.07.007 　

ISSN： 0898-6568
Real-time examination of cAMP activity at relaxin family peptide receptors using a BRET-based biosensor. International-journal Peer-reviewed

Adam L Valkovic, Miranda B Leckey, Alice R Whitehead, Mohammed A Hossain, Asuka Inoue, Martina Kocan, Ross A D Bathgate

Pharmacology research & perspectives　6　(5)　e00432　2018/10

DOI： 10.1002/prp2.432 　

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Relaxin family peptide (RXFPs) 1-4 receptors modulate the activity of cyclic adenosine monophosphate (cAMP) to produce a range of physiological functions. RXFP1 and RXFP2 increase cAMP via Gαs, whereas RXFP3 and RXFP4 inhibit cAMP via Gαi/o. RXFP1 also shows a delayed increase in cAMP downstream of Gαi3. In this study we have assessed whether the bioluminescence resonance energy transfer (BRET)-based biosensor CAMYEL (cAMP sensor using YFP-Epac-Rluc), which allows real-time measurement of cAMP activity in live cells, will aid in understanding ligand- and cell-specific RXFP signaling. CAMYEL detected concentration-dependent changes in cAMP activity at RXFP1-4 in recombinant cell lines, using a variety of ligands with potencies comparable to those seen in conventional cAMP assays. We used RXFP2 and RXFP3 antagonists to demonstrate that CAMYEL detects dynamic changes in cAMP by reversing cAMP activation or inhibition respectively, with real-time addition of antagonist after agonist stimulation. To demonstrate the utility of CAMYEL to detect cAMP activation in native cells expressing low levels of RXFP receptor, we cloned CAMYEL into a lentiviral vector and transduced THP-1 cells, which express low levels of RXFP1. THP-1 CAMYEL cells demonstrated robust cAMP activation in response to relaxin. However, the CAMYEL assay was unable to detect the Gαi3-mediated phase of RXFP1 cAMP activation in PTX-treated THP-1 cells or HEK293A cells with knockout of Gαs. Our data demonstrate that cytoplasmically-expressed CAMYEL efficiently detects real-time cAMP activation by Gαs or inhibition by Gαi/o but may not detect cAMP generated in specific intracellular compartments such as that generated by Gαi3 upon RXFP1 activation.
Stepwise phosphorylation of leukotriene B4 receptor 1 defines cellular responses to leukotriene B4. International-journal Peer-reviewed

Yoshimitsu Nakanishi, Modong Tan, Takako Ichiki, Asuka Inoue, Jun-Ichi Yoshihara, Naoto Maekawa, Itsuki Takenoshita, Keisuke Yanagida, Shinya Yamahira, Satoshi Yamaguchi, Junken Aoki, Teruyuki Nagamune, Takehiko Yokomizo, Takao Shimizu, Motonao Nakamura

Science signaling　11　(544)　2018/08/21

DOI： 10.1126/scisignal.aao5390 　

ISSN： 1945-0877
The 17,18-epoxyeicosatetraenoic acid-G protein-coupled receptor 40 axis ameliorates contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. International-journal Peer-reviewed

Takahiro Nagatake, Yumiko Shiogama, Asuka Inoue, Junichi Kikuta, Tetsuya Honda, Prabha Tiwari, Takayuki Kishi, Atsushi Yanagisawa, Yosuke Isobe, Naomi Matsumoto, Michiko Shimojou, Sakiko Morimoto, Hidehiko Suzuki, So-Ichiro Hirata, Pär Steneberg, Helena Edlund, Junken Aoki, Makoto Arita, Hiroshi Kiyono, Yasuhiro Yasutomi, Masaru Ishii, Kenji Kabashima, Jun Kunisawa

The Journal of allergy and clinical immunology　142　(2)　470-484　2018/08

DOI： 10.1016/j.jaci.2017.09.053 　

ISSN： 0091-6749
Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice Peer-reviewed

Hideyuki Yanai, Shiho Chiba, Sho Hangai, Kohei Kometani, Asuka Inoue, Yoshitaka Kimura, Takaya Abe, Hiroshi Kiyonari, Junko Nishio, Naoko Taguchi-Atarashi, Yu Mizushima, Hideo Negishi, Rudolf Grosschedl, Tadatsugu Taniguchi

Proceedings of the National Academy of Sciences of the United States of America　115　(20)　5253-5258　2018/05/15
Publisher: National Academy of Sciences
DOI： 10.1073/pnas.1803936115 　

ISSN： 1091-6490 0027-8424
Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells. International-journal Peer-reviewed

Qingwen Wan, Najeah Okashah, Asuka Inoue, Rony Nehmé, Byron Carpenter, Christopher G Tate, Nevin A Lambert

The Journal of biological chemistry　293　(19)　7466-7473　2018/05/11

DOI： 10.1074/jbc.RA118.001975 　

ISSN： 0021-9258
On the G protein-coupling selectivity of the native A2B adenosine receptor. International-journal Peer-reviewed

Zhan-Guo Gao, Asuka Inoue, Kenneth A Jacobson

Biochemical pharmacology　151　201-213　2018/05

DOI： 10.1016/j.bcp.2017.12.003 　

ISSN： 0006-2952
Genome Editing Provides New Insights into Receptor-Controlled Signalling Pathways. International-journal Peer-reviewed

Graeme Milligan, Asuka Inoue

Trends in pharmacological sciences　39　(5)　481-493　2018/05

DOI： 10.1016/j.tips.2018.02.005 　

ISSN： 0165-6147
Targeting GLP-1 receptor trafficking to improve agonist efficacy. International-journal Peer-reviewed

Ben Jones, Teresa Buenaventura, Nisha Kanda, Pauline Chabosseau, Bryn M Owen, Rebecca Scott, Robert Goldin, Napat Angkathunyakul, Ivan R Corrêa Jr, Domenico Bosco, Paul R Johnson, Lorenzo Piemonti, Piero Marchetti, A M James Shapiro, Blake J Cochran, Aylin C Hanyaloglu, Asuka Inoue, Tricia Tan, Guy A Rutter, Alejandra Tomas, Stephen R Bloom

Nature communications　9　(1)　1602-1602　2018/04/23

DOI： 10.1038/s41467-018-03941-2 　

ISSN： 2041-1723
Receptor- and cellular compartment-specific activation of the cAMP/PKA pathway by α1-adrenergic and ETA endothelin receptors. International-journal Peer-reviewed

Ryan D Martin, Yalin Sun, Kyla Bourque, Nicolas Audet, Asuka Inoue, Jason C Tanny, Terence E Hébert

Cellular signalling　44　43-50　2018/04

DOI： 10.1016/j.cellsig.2018.01.002 　

ISSN： 0898-6568
Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors. International-journal Peer-reviewed

Maria Buur Nordskov Gabe, Alexander Hovard Sparre-Ulrich, Mie Fabricius Pedersen, Lærke Smidt Gasbjerg, Asuka Inoue, Hans Bräuner-Osborne, Bolette Hartmann, Mette Marie Rosenkilde

Biochemical pharmacology　150　97-107　2018/04

DOI： 10.1016/j.bcp.2018.01.040 　

ISSN： 0006-2952
CRISPR/Cas9-mediated Knockout of G Proteins and beta-arrestins Determines Transducer Specific Contributions to Dopamine D1 Receptor Signaling Peer-reviewed

Jain Manish K, Nilson Ashley N, Felsing Daniel E, Inoue Asuka, Allen John A

FASEB JOURNAL　32　(1)　2018/04

ISSN： 0892-6638
The Role of Distal Helix 5 as a Determinant of GPCR-G protein Coupling Selectivity Peer-reviewed

Okashah Najeah, Wan Qingwen, Inoue Asuka, Lambert Nevin

FASEB JOURNAL　32　(1)　2018/04

ISSN： 0892-6638
G protein-dependent signaling triggers a β-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation. International-journal Peer-reviewed

Aurélie Tréfier, Astrid Musnier, Flavie Landomiel, Thomas Bourquard, Thomas Boulo, Mohammed Akli Ayoub, Kelly León, Gilles Bruneau, Manon Chevalier, Guillaume Durand, Marie-Claire Blache, Asuka Inoue, Joël Fontaine, Christophe Gauthier, Sophie Tesseraud, Eric Reiter, Anne Poupon, Pascale Crépieux

FASEB journal : official publication of the Federation of American Societies for Experimental Biology　32　(3)　1154-1169　2018/03

DOI： 10.1096/fj.201700763R 　

ISSN： 0892-6638
Lack of beta-arrestin signaling in the absence of active G proteins. International-journal Peer-reviewed

Manuel Grundmann, Nicole Merten, Davide Malfacini, Asuka Inoue, Philip Preis, Katharina Simon, Nelly Rüttiger, Nicole Ziegler, Tobias Benkel, Nina Katharina Schmitt, Satoru Ishida, Ines Müller, Raphael Reher, Kouki Kawakami, Ayumi Inoue, Ulrike Rick, Toni Kühl, Diana Imhof, Junken Aoki, Gabriele M König, Carsten Hoffmann, Jesus Gomeza, Jürgen Wess, Evi Kostenis

Nature communications　9　(1)　341-341　2018/01/23

DOI： 10.1038/s41467-017-02661-3 　

ISSN： 2041-1723
AP2σ Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling, and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity. International-journal Peer-reviewed

Caroline M Gorvin, Angela Rogers, Benoit Hastoy, Andrei I Tarasov, Morten Frost, Silvia Sposini, Asuka Inoue, Michael P Whyte, Patrik Rorsman, Aylin C Hanyaloglu, Gerda E Breitwieser, Rajesh V Thakker

Cell reports　22　(4)　1054-1066　2018/01/23

DOI： 10.1016/j.celrep.2017.12.089 　

ISSN： 2211-1247
Lysophosphatidylserine suppresses IL-2 production in CD4 T cells through LPS3/GPR174. International-journal Peer-reviewed

Yuji Shinjo, Kumiko Makide, Keita Satoh, Fumiya Fukami, Asuka Inoue, Kuniyuki Kano, Yuko Otani, Tomohiko Ohwada, Junken Aoki

Biochemical and biophysical research communications　494　(1-2)　332-338　2017/12/09

DOI： 10.1016/j.bbrc.2017.10.028 　

ISSN： 0006-291X

eISSN： 1090-2104
The Calcium-Sensing Receptor, a Class C GPCR, Spatially-Directs G-protein Selectivity via Endosomal Signaling. Peer-reviewed

Caroline Gorvin, Angela Rogers, Benoit Hastoy, Andrei Tarasov, Morten Frost, Asuka Inoue, Michael Whyte, Patrik Rorsman, Aylin Hanyaloglu, Gerda Breitwieser, Rajesh Thakker

JOURNAL OF BONE AND MINERAL RESEARCH　32　S8-S8　2017/12

ISSN： 0884-0431

eISSN： 1523-4681
A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias. International-journal Peer-reviewed

Eugenia Sergeev, Anders Højgaard Hansen, Daniele Bolognini, Kouki Kawakami, Takayuki Kishi, Junken Aoki, Trond Ulven, Asuka Inoue, Brian D Hudson, Graeme Milligan

Scientific reports　7　(1)　13741-13741　2017/10/23

DOI： 10.1038/s41598-017-14096-3 　

ISSN： 2045-2322
Genetic variants affecting equivalent protein family positions reflect human diversity. International-journal Peer-reviewed

Francesco Raimondi, Matthew J Betts, Qianhao Lu, Asuka Inoue, J Silvio Gutkind, Robert B Russell

Scientific reports　7　(1)　12771-12771　2017/10/06

DOI： 10.1038/s41598-017-12971-7 　

ISSN： 2045-2322
X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog. International-journal Peer-reviewed

Wataru Shihoya, Tomohiro Nishizawa, Keitaro Yamashita, Asuka Inoue, Kunio Hirata, Francois Marie Ngako Kadji, Akiko Okuta, Kazutoshi Tani, Junken Aoki, Yoshinori Fujiyoshi, Tomoko Doi, Osamu Nureki

Nature structural & molecular biology　24　(9)　758-764　2017/09

DOI： 10.1038/nsmb.3450 　

ISSN： 1545-9993

eISSN： 1545-9985
Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA6. International-journal Peer-reviewed

Reiya Taniguchi, Asuka Inoue, Misa Sayama, Akiharu Uwamizu, Keitaro Yamashita, Kunio Hirata, Masahito Yoshida, Yoshiki Tanaka, Hideaki E Kato, Yoshiko Nakada-Nakura, Yuko Otani, Tomohiro Nishizawa, Takayuki Doi, Tomohiko Ohwada, Ryuichiro Ishitani, Junken Aoki, Osamu Nureki

Nature　548　(7667)　356-360　2017/08/17

DOI： 10.1038/nature23448 　

ISSN： 0028-0836

eISSN： 1476-4687
Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors. International-journal Peer-reviewed

Susanne Prokop, Nicole A Perry, Sergey A Vishnivetskiy, Andras D Toth, Asuka Inoue, Graeme Milligan, Tina M Iverson, Laszlo Hunyady, Vsevolod V Gurevich

Cellular signalling　36　98-107　2017/08

DOI： 10.1016/j.cellsig.2017.04.021 　

ISSN： 0898-6568

eISSN： 1873-3913
C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways. International-journal Peer-reviewed

Jeffrey S Smith, Priya Alagesan, Nimit K Desai, Thomas F Pack, Jiao-Hui Wu, Asuka Inoue, Neil J Freedman, Sudarshan Rajagopal

Molecular pharmacology　92　(2)　136-150　2017/08

DOI： 10.1124/mol.117.108522 　

ISSN： 0026-895X

eISSN： 1521-0111
Probing the Hydrophobic Binding Pocket of G-Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS1 by Docking-Aided Structure-Activity Analysis. International-journal Peer-reviewed

Misa Sayama, Asuka Inoue, Sho Nakamura, Sejin Jung, Masaya Ikubo, Yuko Otani, Akiharu Uwamizu, Takayuki Kishi, Kumiko Makide, Junken Aoki, Takatsugu Hirokawa, Tomohiko Ohwada

Journal of medicinal chemistry　60　(14)　6384-6399　2017/07/27

DOI： 10.1021/acs.jmedchem.7b00693 　

ISSN： 0022-2623

eISSN： 1520-4804
Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors. International-journal Peer-reviewed

Rory Sleno, Dominic Devost, Darlaine Pétrin, Alice Zhang, Kyla Bourque, Yuji Shinjo, Junken Aoki, Asuka Inoue, Terence E Hébert

The Journal of biological chemistry　292　(29)　12139-12152　2017/07/21

DOI： 10.1074/jbc.M117.793877 　

ISSN： 0021-9258

eISSN： 1083-351X
Autotaxin-lysophosphatidic acid-LPA3 signaling at the embryo-epithelial boundary controls decidualization pathways. International-journal Peer-reviewed

Shizu Aikawa, Kuniyuki Kano, Asuka Inoue, Jiao Wang, Daisuke Saigusa, Takeshi Nagamatsu, Yasushi Hirota, Tomoyuki Fujii, Soken Tsuchiya, Yoshitaka Taketomi, Yukihiko Sugimoto, Makoto Murakami, Makoto Arita, Makoto Kurano, Hitoshi Ikeda, Yutaka Yatomi, Jerold Chun, Junken Aoki

The EMBO journal　36　(14)　2146-2160　2017/07/14

DOI： 10.15252/embj.201696290 　

ISSN： 0261-4189

eISSN： 1460-2075
Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK. International-journal Peer-reviewed

Morgan O'Hayre, Kelsie Eichel, Silvia Avino, Xuefeng Zhao, Dana J Steffen, Xiaodong Feng, Kouki Kawakami, Junken Aoki, Karen Messer, Roger Sunahara, Asuka Inoue, Mark von Zastrow, J Silvio Gutkind

Science signaling　10　(484)　2017/06/20

DOI： 10.1126/scisignal.aal3395 　

ISSN： 1945-0877

eISSN： 1937-9145
Identification and pharmacological characterization of succinate receptor agonists. International-journal Peer-reviewed

Pierre Geubelle, Julie Gilissen, Sébastien Dilly, Laurence Poma, Nadine Dupuis, Céline Laschet, Dayana Abboud, Asuka Inoue, François Jouret, Bernard Pirotte, Julien Hanson

British journal of pharmacology　174　(9)　796-808　2017/05

DOI： 10.1111/bph.13738 　

ISSN： 0007-1188

eISSN： 1476-5381
Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells. International-journal Peer-reviewed

Wayne Stallaert, Emma T van der Westhuizen, Anne-Marie Schönegge, Bianca Plouffe, Mireille Hogue, Viktoria Lukashova, Asuka Inoue, Satoru Ishida, Junken Aoki, Christian Le Gouill, Michel Bouvier

Molecular pharmacology　91　(5)　533-544　2017/05

DOI： 10.1124/mol.116.106419 　

ISSN： 0026-895X

eISSN： 1521-0111
Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context. International-journal Peer-reviewed

Dominic Devost, Rory Sleno, Darlaine Pétrin, Alice Zhang, Yuji Shinjo, Rakan Okde, Junken Aoki, Asuka Inoue, Terence E Hébert

The Journal of biological chemistry　292　(13)　5443-5456　2017/03/31

DOI： 10.1074/jbc.M116.763854 　

ISSN： 0021-9258

eISSN： 1083-351X
Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis. International-journal Peer-reviewed

Thomas J Cahill 3rd, Alex R B Thomsen, Jeffrey T Tarrasch, Bianca Plouffe, Anthony H Nguyen, Fan Yang, Li-Yin Huang, Alem W Kahsai, Daniel L Bassoni, Bryant J Gavino, Jane E Lamerdin, Sarah Triest, Arun K Shukla, Benjamin Berger, John Little 4th, Albert Antar, Adi Blanc, Chang-Xiu Qu, Xin Chen, Kouki Kawakami, Asuka Inoue, Junken Aoki, Jan Steyaert, Jin-Peng Sun, Michel Bouvier, Georgios Skiniotis, Robert J Lefkowitz

Proceedings of the National Academy of Sciences of the United States of America　114　(10)　2562-2567　2017/03/07

DOI： 10.1073/pnas.1701529114 　

ISSN： 0027-8424
Proliferation of mouse endometrial stromal cells in culture is highly sensitive to lysophosphatidic acid signaling. International-journal Peer-reviewed

Shizu Aikawa, Kuniyuki Kano, Asuka Inoue, Junken Aoki

Biochemical and biophysical research communications　484　(1)　202-208　2017/02/26

DOI： 10.1016/j.bbrc.2016.12.154 　

ISSN： 0006-291X

eISSN： 1090-2104
Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors. International-journal Peer-reviewed

Kelly L Roszko, Ruiye Bi, Caroline M Gorvin, Hans Bräuner-Osborne, Xiao-Feng Xiong, Asuka Inoue, Rajesh V Thakker, Kristian Strømgaard, Thomas Gardella, Michael Mannstadt

JCI insight　2　(3)　e91079　2017/02/09

DOI： 10.1172/jci.insight.91079 　

ISSN： 2379-3708
Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function. International-journal Peer-reviewed

Stine Jørgensen, Christian Theil Have, Christina Rye Underwood, Lars Dan Johansen, Petrine Wellendorph, Anette Prior Gjesing, Christinna V Jørgensen, Shi Quan, Gao Rui, Asuka Inoue, Allan Linneberg, Niels Grarup, Wang Jun, Oluf Pedersen, Torben Hansen, Hans Bräuner-Osborne

The Journal of biological chemistry　292　(4)　1524-1534　2017/01/27

DOI： 10.1074/jbc.M116.756577 　

ISSN： 0021-9258

eISSN： 1083-351X
1-Fluoro-2,4-dinitrobenzene and its derivatives act as secretagogues on rodent mast cells. International-journal Peer-reviewed

Yohei Manabe, Marie Yoshimura, Kazuma Sakamaki, Asuka Inoue, Aya Kakinoki, Satoshi Hokari, Mariko Sakanaka, Junken Aoki, Hiroyuki Miyachi, Kazuyuki Furuta, Satoshi Tanaka

European journal of immunology　47　(1)　60-67　2017/01

DOI： 10.1002/eji.201646536 　

ISSN： 0014-2980

eISSN： 1521-4141
Epidermal loss of Gαq confers a migratory and differentiation defect in keratinocytes. International-journal Peer-reviewed

Colleen L Doçi, Constantinos M Mikelis, Juan Luis Callejas-Valera, Karina K Hansen, Alfredo A Molinolo, Asuka Inoue, Stefan Offermanns, J Silvio Gutkind

PloS one　12　(3)　e0173692　2017

DOI： 10.1371/journal.pone.0173692 　

ISSN： 1932-6203
Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling. International-journal Peer-reviewed

Elisa Alvarez-Curto, Asuka Inoue, Laura Jenkins, Sheikh Zahir Raihan, Rudi Prihandoko, Andrew B Tobin, Graeme Milligan

The Journal of biological chemistry　291　(53)　27147-27159　2016/12/30

DOI： 10.1074/jbc.M116.754887 　

ISSN： 0021-9258

eISSN： 1083-351X
Identification of lysophosphatidylthreonine with an aromatic fatty acid surrogate as a potent inducer of mast cell degranulation. International-journal Peer-reviewed

Takayuki Kishi, Hiroki Kawana, Misa Sayama, Kumiko Makide, Asuka Inoue, Yuko Otani, Tomohiko Ohwada, Junken Aoki

Biochemistry and biophysics reports　8　346-351　2016/12

DOI： 10.1016/j.bbrep.2016.09.013 　

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Upon various stimulations, mast cells (MCs) release a wide variety of chemical mediators stored in their cytoplasmic granules, which then initiates subsequent allergic reactions. Lysophosphatidylserine (LysoPS), a kind of lysophospholipid, potentiates the histamine release from MCs triggered by antigen stimulation. We previously showed through structure-activity studies of LysoPS analogs that LysoPS with a methyl group at the carbon of the serine residue, i.e., lysophosphatidylthreonine (LysoPT), is extremely potent in stimulating the MC degranulation. In this study, as our continuing study to identify more potent LysoPS analogs, we developed LysoPS analogs with fatty acid surrogates. We found that the substitution of oleic acid to an aromatic fatty acid surrogate (C3-pH-p-O-C11) in 2-deoxy-1-LysoPS resulted in significant increase in the ability to induce MCs degranulation compared with 2-deoxy-1-LysoPS with oleic acid. Conversion of the serine residue into the threonine residue further increased the activity of MC degranulation both in vitro and in vivo. The resulting super agonist, 2-deoxy-LysoPT with C3-pH-p-O-C11, will be a useful tool to elucidate the mechanisms of stimulatory effect of LysoPS on MC degranulation.
Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy. International-journal Peer-reviewed

Sebastian George Barton Furness, Yi-Lynn Liang, Cameron James Nowell, Michelle Louise Halls, Peter John Wookey, Emma Dal Maso, Asuka Inoue, Arthur Christopoulos, Denise Wootten, Patrick Michael Sexton

Cell　167　(3)　739-749　2016/10/20

DOI： 10.1016/j.cell.2016.09.021 　

ISSN： 0092-8674

eISSN： 1097-4172
Regulation of the lysophosphatidylserine and sphingosine 1-phosphate levels in autologous whole blood by the pre-storage leukocyte reduction Peer-reviewed

Y. Nagura, N. H. Tsuno, K. Kano, A. Inoue, J. Aoki, Y. Hirowatari, M. Kaneko, M. Kurano, M. Matsuhashi, R. Ohkawa, M. Tozuka, Y. Yatomi, H. Okazaki

Transfusion Medicine　26　(5)　365-372　2016/10/01
Publisher: Blackwell Publishing Ltd
DOI： 10.1111/tme.12326 　

ISSN： 1365-3148 0958-7578
Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: A tumor suppressor function for the Gα13/RhoA axis in B cells Peer-reviewed

M. O'Hayre, A. Inoue, I. Kufareva, Z. Wang, C. M. Mikelis, R. A. Drummond, S. Avino, K. Finkel, K. W. Kalim, G. Dipasquale, F. Guo, J. Aoki, Y. Zheng, M. S. Lionakis, A. A. Molinolo, J. S. Gutkind

Oncogene　35　(29)　3771-3780　2016/07/21
Publisher: Nature Publishing Group
DOI： 10.1038/onc.2015.442 　

ISSN： 1476-5594 0950-9232
Conformational Constraint of the Glycerol Moiety of Lysophosphatidylserine Affords Compounds with Receptor Subtype Selectivity. International-journal Peer-reviewed

Sejin Jung, Asuka Inoue, Sho Nakamura, Takayuki Kishi, Akiharu Uwamizu, Misa Sayama, Masaya Ikubo, Yuko Otani, Kuniyuki Kano, Kumiko Makide, Junken Aoki, Tomohiko Ohwada

Journal of medicinal chemistry　59　(8)　3750-76　2016/04/28

DOI： 10.1021/acs.jmedchem.5b01925 　

ISSN： 0022-2623

eISSN： 1520-4804
A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways. International-journal Peer-reviewed

Jianxin Hu, Matthew Stern, Luis E Gimenez, Lizzy Wanka, Lu Zhu, Mario Rossi, Jaroslawna Meister, Asuka Inoue, Annette G Beck-Sickinger, Vsevolod V Gurevich, Jürgen Wess

The Journal of biological chemistry　291　(15)　7809-20　2016/04/08

DOI： 10.1074/jbc.M115.702282 　

ISSN： 0021-9258

eISSN： 1083-351X
ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation. International-journal Peer-reviewed

Tatsuji Nishioka, Naoaki Arima, Kuniyuki Kano, Kotaro Hama, Eriko Itai, Hiroshi Yukiura, Ryoji Kise, Asuka Inoue, Seok-Hyung Kim, Lilianna Solnica-Krezel, Wouter H Moolenaar, Jerold Chun, Junken Aoki

Scientific reports　6　23433-23433　2016/03/23

DOI： 10.1038/srep23433 　

ISSN： 2045-2322
Fibroblastic reticular cell-derived lysophosphatidic acid regulates confined intranodal T-cell motility. International-journal Peer-reviewed

Akira Takeda, Daichi Kobayashi, Keita Aoi, Naoko Sasaki, Yuki Sugiura, Hidemitsu Igarashi, Kazuo Tohya, Asuka Inoue, Erina Hata, Noriyuki Akahoshi, Haruko Hayasaka, Junichi Kikuta, Elke Scandella, Burkhard Ludewig, Satoshi Ishii, Junken Aoki, Makoto Suematsu, Masaru Ishii, Kiyoshi Takeda, Sirpa Jalkanen, Masayuki Miyasaka, Eiji Umemoto

eLife　5　e10561　2016/02/02

DOI： 10.7554/eLife.10561 　

ISSN： 2050-084X
The experimental power of FR900359 to study Gq-regulated biological processes. International-journal Peer-reviewed

Ramona Schrage, Anna-Lena Schmitz, Evelyn Gaffal, Suvi Annala, Stefan Kehraus, Daniela Wenzel, Katrin M Büllesbach, Tobias Bald, Asuka Inoue, Yuji Shinjo, Ségolène Galandrin, Naveen Shridhar, Michael Hesse, Manuel Grundmann, Nicole Merten, Thomas H Charpentier, Matthew Martz, Adrian J Butcher, Tanja Slodczyk, Sylvain Armando, Maike Effern, Yoon Namkung, Laura Jenkins, Velten Horn, Anne Stößel, Harald Dargatz, Daniel Tietze, Diana Imhof, Céline Galés, Christel Drewke, Christa E Müller, Michael Hölzel, Graeme Milligan, Andrew B Tobin, Jesús Gomeza, Henrik G Dohlman, John Sondek, T Kendall Harden, Michel Bouvier, Stéphane A Laporte, Junken Aoki, Bernd K Fleischmann, Klaus Mohr, Gabriele M König, Thomas Tüting, Evi Kostenis

Nature communications　6　10156-10156　2015/12/14

DOI： 10.1038/ncomms10156 　

ISSN： 2041-1723
Blood levels of serotonin are specifically correlated with plasma lysophosphatidylserine among the glycero-lysophospholipids. International-journal Peer-reviewed

Makoto Kurano, Tomotaka Dohi, Takahiro Nojiri, Tamaki Kobayashi, Yuji Hirowatari, Asuka Inoue, Kuniyuki Kano, Hirotaka Matsumoto, Koji Igarashi, Masako Nishikawa, Katsumi Miyauchi, Hiroyuki Daida, Hitoshi Ikeda, Junken Aoki, Yutaka Yatomi

BBA clinical　4　92-8　2015/12

DOI： 10.1016/j.bbacli.2015.08.003 　

ISSN： 2214-6474
LPP3 localizes LPA6 signalling to non-contact sites in endothelial cells. International-journal Peer-reviewed

Hiroshi Yukiura, Kuniyuki Kano, Ryoji Kise, Asuka Inoue, Junken Aoki

Journal of cell science　128　(21)　3871-7　2015/11/01

DOI： 10.1242/jcs.172098 　

ISSN： 0021-9533

eISSN： 1477-9137
NEURONAL DEVELOPMENT. Glycerophospholipid regulation of modality-specific sensory axon guidance in the spinal cord. International-journal Peer-reviewed

Adam T Guy, Yasuko Nagatsuka, Noriko Ooashi, Mariko Inoue, Asuka Nakata, Peter Greimel, Asuka Inoue, Takuji Nabetani, Akiho Murayama, Kunihiro Ohta, Yukishige Ito, Junken Aoki, Yoshio Hirabayashi, Hiroyuki Kamiguchi

Science (New York, N.Y.)　349　(6251)　974-7　2015/08/28

DOI： 10.1126/science.aab3516 　

ISSN： 0036-8075

eISSN： 1095-9203
Comprehensive analysis of sphingosine-1-phosphate receptor mutants during zebrafish embryogenesis. International-journal Peer-reviewed

Yu Hisano, Asuka Inoue, Kiyohito Taimatsu, Satoshi Ota, Rie Ohga, Hirohito Kotani, Michiko Muraki, Junken Aoki, Atsuo Kawahara

Genes to cells : devoted to molecular & cellular mechanisms　20　(8)　647-58　2015/08

DOI： 10.1111/gtc.12259 　

ISSN： 1356-9597

eISSN： 1365-2443
Novel roles for GNA13 and RHOA as tumor suppressor genes Peer-reviewed

Morgan O'hayre, Asuka Inoue, Katiuchia Sales, Irina Kufareva, Juan Luis Callejas Valera, Fukun Guo, Constantinos Mikelis, Giovanni DiPasquale, Kira Finkel, Junken Aoki, Yi Zheng, Thomas H. Bugge, J. Silvio Gutkind

CANCER RESEARCH　75　2015/08

DOI： 10.1158/1538-7445.AM2015-2059 　

ISSN： 0008-5472

eISSN： 1538-7445
Quantitative evaluation of G protein coupling with GPCRs Peer-reviewed

Inoue Asuka, Aoki Junken

JOURNAL OF PHARMACOLOGICAL SCIENCES　128　(3)　S28　2015/07

ISSN： 1347-8613
Maternal and Zygotic Sphingosine Kinase 2 Are Indispensable for Cardiac Development in Zebrafish. International-journal Peer-reviewed

Yu Hisano, Asuka Inoue, Michiyo Okudaira, Kiyohito Taimatsu, Hirotaka Matsumoto, Hirohito Kotani, Rie Ohga, Junken Aoki, Atsuo Kawahara

The Journal of biological chemistry　290　(24)　14841-51　2015/06/12

DOI： 10.1074/jbc.M114.634717 　

ISSN： 0021-9258

eISSN： 1083-351X
Analysis of unique mutations in the LPAR6 gene identified in a Japanese family with autosomal recessive woolly hair/hypotrichosis: Establishment of a useful assay system for LPA6. International-journal Peer-reviewed

Ryota Hayashi, Asuka Inoue, Yasushi Suga, Junken Aoki, Yutaka Shimomura

Journal of dermatological science　78　(3)　197-205　2015/06

DOI： 10.1016/j.jdermsci.2015.03.006 　

ISSN： 0923-1811

eISSN： 1873-569X
Structure-activity relationships of lysophosphatidylserine analogs as agonists of G-protein-coupled receptors GPR34, P2Y10, and GPR174. International-journal Peer-reviewed

Masaya Ikubo, Asuka Inoue, Sho Nakamura, Sejin Jung, Misa Sayama, Yuko Otani, Akiharu Uwamizu, Keisuke Suzuki, Takayuki Kishi, Akira Shuto, Jun Ishiguro, Michiyo Okudaira, Kuniyuki Kano, Kumiko Makide, Junken Aoki, Tomohiko Ohwada

Journal of medicinal chemistry　58　(10)　4204-19　2015/05/28

DOI： 10.1021/jm5020082 　

ISSN： 0022-2623

eISSN： 1520-4804
Lysophosphatidylserine analogues differentially activate three LysoPS receptors. International-journal Peer-reviewed

Akiharu Uwamizu, Asuka Inoue, Kensuke Suzuki, Michiyo Okudaira, Akira Shuto, Yuji Shinjo, Jun Ishiguro, Kumiko Makide, Masaya Ikubo, Sho Nakamura, Sejin Jung, Misa Sayama, Yuko Otani, Tomohiko Ohwada, Junken Aoki

Journal of biochemistry　157　(3)　151-60　2015/03

DOI： 10.1093/jb/mvu060 　

ISSN： 0021-924X

eISSN： 1756-2651
Phosphatidylserine-specific phospholipase A1 (PS-PLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis. International-journal Peer-reviewed

Yuuki Iida, Eiji Sunami, Hiroharu Yamashita, Masaya Hiyoshi, Soichiro Ishihara, Hironori Yamaguchi, Asuka Inoue, Kumiko Makide, Nelson H Tsuno, Junken Aoki, Joji Kitayama, Toshiaki Watanabe

Anticancer research　35　(3)　1459-64　2015/03

ISSN： 0250-7005

eISSN： 1791-7530
Possible involvement of minor lysophospholipids in the increase in plasma lysophosphatidic Acid in acute coronary syndrome. Peer-reviewed

Makoto Kurano, Akiko Suzuki, Asuka Inoue, Yasunori Tokuhara, Kuniyuki Kano, Hirotaka Matsumoto, Koji Igarashi, Ryunosuke Ohkawa, Kazuhiro Nakamura, Tomotaka Dohi, Katsumi Miyauchi, Hiroyuki Daida, Kazuhisa Tsukamoto, Hitoshi Ikeda, Junken Aoki, Yutaka Yatomi

Arterioscler. Thromb. Vasc. Biol.　35　(2)　463-470　2015/02

DOI： 10.1161/ATVBAHA.114.304748 　

ISSN： 1524-4636

More details Close

Lysophosphatidic acids (LPA) have important roles in the field of vascular biology and are derived mainly from lysophosphatidylcholine via autotaxin. However, in our previous study, only the plasma LPA levels, and not the serum autotaxin levels, increased in patients with acute coronary syndrome (ACS). The aim of this study was to elucidate the pathway by which LPA is increased in patients with ACS.<br /> We measured the plasma lysophospholipids species in 141 consecutive patients undergoing coronary angiography (ACS, n=38; stable angina pectoris, n=71; angiographically normal coronary arteries, n=32) using a liquid chromatography-tandem mass spectrometry analysis. Among the ACS subjects, notable increases in the 22:6 LPA, 18:2 LPA, and 20:4 LPA levels were observed. The in vitro experiments revealed that serum incubation mainly increased the 18:2 LPA level, whereas platelet activation increased the 20:4 LPA level. Minor lysophospholipids other than LPA were also elevated in ACS subjects and were well correlated with the corresponding LPA species, including 22:6 LPA. A multiple regression analysis also revealed that lysophosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethan
Possible involvement of minor lysophospholipids in the increase in plasma lysophosphatidic acid in acute coronary syndrome. International-journal Peer-reviewed

Makoto Kurano, Akiko Suzuki, Asuka Inoue, Yasunori Tokuhara, Kuniyuki Kano, Hirotaka Matsumoto, Koji Igarashi, Ryunosuke Ohkawa, Kazuhiro Nakamura, Tomotaka Dohi, Katsumi Miyauchi, Hiroyuki Daida, Kazuhisa Tsukamoto, Hitoshi Ikeda, Junken Aoki, Yutaka Yatomi

Arteriosclerosis, thrombosis, and vascular biology　35　(2)　463-70　2015/02

DOI： 10.1161/ATVBAHA.114.304748 　

ISSN： 1079-5642

eISSN： 1524-4636
Detection of Physiological Activities of G Protein-Coupled Receptor-Acting Pharmaceuticals in Wastewater Peer-reviewed

Masaru Ihara, Asuka Inoue, Seiya Hanamoto, Han Zhang, Junken Aoki, Hiroaki Tanaka

ENVIRONMENTAL SCIENCE & TECHNOLOGY　49　(3)　1903-1911　2015/02

DOI： 10.1021/es505349s 　

ISSN： 0013-936X

eISSN： 1520-5851
Measuring Activation of Lipid G Protein- Coupled Receptors Using the TGF-á Shedding Assay Peer-reviewed

Asuka Inoue, Junken Aoki

Bioactive Lipid Mediators: Current Reviews and Protocols　393-408　2015/01/01
Publisher: Springer Japan
DOI： 10.1007/978-4-431-55669-5_28 　
Autotaxin overexpression causes embryonic lethality and vascular defects. International-journal Peer-reviewed

Hiroshi Yukiura, Kuniyuki Kano, Ryoji Kise, Asuka Inoue, Junken Aoki

PloS one　10　(5)　e0126734　2015

DOI： 10.1371/journal.pone.0126734 　

ISSN： 1932-6203
Novel lysophosphoplipid receptors: their structure and function. International-journal Peer-reviewed

Kumiko Makide, Akiharu Uwamizu, Yuji Shinjo, Jun Ishiguro, Michiyo Okutani, Asuka Inoue, Junken Aoki

Journal of lipid research　55　(10)　1986-95　2014/10

DOI： 10.1194/jlr.R046920 　

ISSN： 0022-2275

eISSN： 1539-7262
Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS. International-journal Peer-reviewed

Michiyo Okudaira, Asuka Inoue, Akira Shuto, Keita Nakanaga, Kuniyuki Kano, Kumiko Makide, Daisuke Saigusa, Yoshihisa Tomioka, Junken Aoki

Journal of lipid research　55　(10)　2178-92　2014/10

DOI： 10.1194/jlr.D048439 　

ISSN： 0022-2275

eISSN： 1539-7262
Regulation of intestinal IgA responses by dietary palmitic acid and its metabolism. International-journal Peer-reviewed

Jun Kunisawa, Eri Hashimoto, Asuka Inoue, Risa Nagasawa, Yuji Suzuki, Izumi Ishikawa, Shiori Shikata, Makoto Arita, Junken Aoki, Hiroshi Kiyono

Journal of immunology (Baltimore, Md. : 1950)　193　(4)　1666-71　2014/08/15

DOI： 10.4049/jimmunol.1302944 　

ISSN： 0022-1767

eISSN： 1550-6606
Overexpression of autotaxin, a lysophosphatidic acid-producing enzyme, enhances cardia bifida induced by hypo-sphingosine-1-phosphate signaling in zebrafish embryo. International-journal Peer-reviewed

Keita Nakanaga, Kotaro Hama, Kuniyuki Kano, Takanao Sato, Hiroshi Yukiura, Asuka Inoue, Daisuke Saigusa, Hidetoshi Tokuyama, Yoshihisa Tomioka, Hiroshi Nishina, Atsuo Kawahara, Junken Aoki

Journal of biochemistry　155　(4)　235-41　2014/04

DOI： 10.1093/jb/mvt114 　

ISSN： 0021-924X

eISSN： 1756-2651
[TGFalpha shedding assay is useful for identifying ligands for orphan GPCRs]. Peer-reviewed

Asuka Inoue, Junken Aoki

Seikagaku. The Journal of Japanese Biochemical Society　85　(11)　1029-33　2013/11

ISSN： 0037-1017
A novel mutation, c.699C > G (p.C233W), in the LIPH gene leads to a loss of the hydrolytic activity and the LPA(6) activation ability of PA-PLA(1)alpha in autosomal recessive wooly hair/hypotrichosis Peer-reviewed

Mayuko Yoshizawa, Motonobu Nakamura, Muhammad Farooq, Asuka Inoue, Junken Aoki, Yutaka Shimomura

JOURNAL OF DERMATOLOGICAL SCIENCE　72　(1)　61-64　2013/10

DOI： 10.1016/j.jdermsci.2013.05.001 　

ISSN： 0923-1811
A novel heterozygous deletion-insertion mutation in the mouse Krt71 gene underlies wavy coat phenotype Peer-reviewed

A. Fujimoto, A. Inoue, M. Farooq, H. Fujikawa, J. Aoki, M. Ito, Y. Shimomura

JOURNAL OF INVESTIGATIVE DERMATOLOGY　133　(1)　S80-S80　2013/05

ISSN： 0022-202X
Mitochondria-type GPAT is required for mitochondrial fusion Peer-reviewed

Yohsuke Ohba, Takeshi Sakuragi, Eriko Kage-Nakadai, Naoko H. Tomioka, Nozomu Kono, Rieko Imae, Asuka Inoue, Junken Aoki, Naotada Ishihara, Takao Inoue, Shohei Mitani, Hiroyuki Arai

EMBO JOURNAL　32　(9)　1265-1279　2013/05

DOI： 10.1038/emboj.2013.77 　

ISSN： 0261-4189

eISSN： 1460-2075
Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): Metabolically stabilized LPA receptor agonists Peer-reviewed

Guowei Jiang, Asuka Inoue, Junken Aoki, Glenn D. Prestwich

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS　23　(6)　1865-1869　2013/03

DOI： 10.1016/j.bmcl.2013.01.002 　

ISSN： 0960-894X
Pharmacological characterization of zebrafish Ca2+-mobilizing prostanoid receptors Peer-reviewed

Ryo Iwasaki, Atsuo Kawahara, Asuka Inoue, Junken Aoki, Soken Tsuchiya, Yukihiko Sugimoto

JOURNAL OF PHARMACOLOGICAL SCIENCES　121　(1)　72P-72P　2013

ISSN： 1347-8613
A missense mutation within the helix initiation motif of the keratin K71 gene underlies autosomal dominant woolly hair/hypotrichosis. International-journal Peer-reviewed

Atsushi Fujimoto, Muhammad Farooq, Hiroki Fujikawa, Asuka Inoue, Manabu Ohyama, Ritsuko Ehama, Jotaro Nakanishi, Motofumi Hagihara, Tokuro Iwabuchi, Junken Aoki, Masaaki Ito, Yutaka Shimomura

The Journal of investigative dermatology　132　(10)　2342-2349　2012/10

DOI： 10.1038/jid.2012.154 　

ISSN： 0022-202X
The beta 9 Loop Domain of PA-PLA(1)alpha Has a Crucial Role in Autosomal Recessive Woolly Hair/Hypotrichosis Peer-reviewed

Satoru Shinkuma, Asuka Inoue, Junken Aoki, Wataru Nishie, Ken Natsuga, Hideyuki Ujiie, Toshifumi Nomura, Riichiro Abe, Masashi Akiyama, Hiroshi Shimizu

JOURNAL OF INVESTIGATIVE DERMATOLOGY　132　(8)　2093-2095　2012/08

DOI： 10.1038/jid.2012.96 　

ISSN： 0022-202X
Synthesis and biological evaluation of optically active Ki16425. International-journal Peer-reviewed

Takanao Sato, Kenji Sugimoto, Asuka Inoue, Shinichi Okudaira, Junken Aoki, Hidetoshi Tokuyama

Bioorganic & medicinal chemistry letters　22　(13)　4323-6　2012/07/01

DOI： 10.1016/j.bmcl.2012.05.012 　

ISSN： 0960-894X
Simultaneous quantitation of sphingoid bases and their phosphates in biological samples by liquid chromatography/electrospray ionization tandem mass spectrometry. International-journal Peer-reviewed

Daisuke Saigusa, Kanako Shiba, Asuka Inoue, Kotaro Hama, Michiyo Okutani, Nagisa Iida, Masayoshi Saito, Kaori Suzuki, Tohru Kaneko, Naoto Suzuki, Hiroaki Yamaguchi, Nariyasu Mano, Junichi Goto, Takanori Hishinuma, Junken Aoki, Yoshihisa Tomioka

Analytical and bioanalytical chemistry　403　(7)　1897-905　2012/06

DOI： 10.1007/s00216-012-6004-9 　

ISSN： 1618-2642
GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the sn-2 position. International-journal Peer-reviewed

Hajime Kitamura, Kumiko Makide, Akira Shuto, Masaya Ikubo, Asuka Inoue, Kensuke Suzuki, Yusuke Sato, Sho Nakamura, Yuko Otani, Tomohiko Ohwada, Junken Aoki

Journal of biochemistry　151　(5)　511-8　2012/05

DOI： 10.1093/jb/mvs011 　

ISSN： 0021-924X
A missense mutation within the helix initiation motif of the keratin 71 (KRT71) gene underlies autosomal dominant woolly hair/hypotrichosis Peer-reviewed

A. Fujimoto, A. Inoue, M. Ohyama, H. Fujikawa, M. Farooq, R. Ehama, J. Nakanishi, M. Hagihara, T. Iwabuchi, J. Aoki, M. Ito, Y. Shimomura

JOURNAL OF INVESTIGATIVE DERMATOLOGY　132　(1)　S67-S67　2012/05

DOI： 10.1038/jid.2012.154 　

ISSN： 0022-202X
The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice Peer-reviewed

Shigetomo Fukuhara, Szandor Simmons, Shunsuke Kawamura, Asuka Inoue, Yasuko Orba, Takeshi Tokudome, Yuji Sunden, Yuji Arai, Kazumasa Moriwaki, Junji Ishida, Akiyoshi Uemura, Hiroshi Kiyonari, Takaya Abe, Akiyoshi Fukamizu, Masanori Hirashima, Hirofumi Sawa, Junken Aoki, Masaru Ishii, Naoki Mochizuki

JOURNAL OF CLINICAL INVESTIGATION　122　(4)　1416-1426　2012/04

DOI： 10.1172/JCI60746 　

ISSN： 0021-9738
Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids. International-journal Peer-reviewed

Naoaki Arima, Asuka Inoue, Kumiko Makide, Takamasa Nonaka, Junken Aoki

Journal of lipid research　53　(3)　513-21　2012/03

DOI： 10.1194/jlr.M022400 　

ISSN： 0022-2275
Regulation of Peroxisomal Lipid Metabolism by Catalytic Activity of Tumor Suppressor H-rev107 Peer-reviewed

Toru Uyama, Ikuyo Ichi, Nozomu Kono, Asuka Inoue, Kazuhito Tsuboi, Xing-Hua Jin, Nobukazu Araki, Junken Aoki, Hiroyuki Arai, Natsuo Ueda

JOURNAL OF BIOLOGICAL CHEMISTRY　287　(4)　2706-2718　2012/01

DOI： 10.1074/jbc.M111.267575 　

ISSN： 0021-9258
Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish. International-journal Peer-reviewed

Hiroshi Yukiura, Kotaro Hama, Keita Nakanaga, Masayuki Tanaka, Yoichi Asaoka, Shinichi Okudaira, Naoaki Arima, Asuka Inoue, Takafumi Hashimoto, Hiroyuki Arai, Atsuo Kawahara, Hiroshi Nishina, Junken Aoki

The Journal of biological chemistry　286　(51)　43972-83　2011/12/23

DOI： 10.1074/jbc.M111.301093 　

ISSN： 0021-9258
[Physiological functions of lysophosphatidylserine]. Peer-reviewed

Asuka Inoue, Michiyo Okutani, Junken Aoki

Seikagaku. The Journal of Japanese Biochemical Society　83　(6)　518-24　2011/06

ISSN： 0037-1017
Crystal structure of autotaxin and insight into GPCR activation by lipid mediators. International-journal Peer-reviewed

Hiroshi Nishimasu, Shinichi Okudaira, Kotaro Hama, Emiko Mihara, Naoshi Dohmae, Asuka Inoue, Ryuichiro Ishitani, Junichi Takagi, Junken Aoki, Osamu Nureki

Nature structural & molecular biology　18　(2)　205-12　2011/02
Publisher: 2
DOI： 10.1038/nsmb.1998 　

ISSN： 1545-9993

More details Close

Autotaxin (ATX, also known as Enpp2) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine to generate lysophosphatidic acid (LPA), a lipid mediator that activates G protein-coupled receptors to evoke various cellular responses. Here, we report the crystal structures of mouse ATX alone and in complex with LPAs with different acyl-chain lengths and saturations. These structures reveal that the multidomain architecture helps to maintain the structural rigidity of the lipid-binding pocket, which accommodates the respective LPA molecules in distinct conformations. They indicate that a loop region in the catalytic domain is a major determinant for the substrate specificity of the Enpp family enzymes. Furthermore, along with biochemical and biological data, these structures suggest that the produced LPAs are delivered from the active site to cognate G protein-coupled receptors through a hydrophobic channel.
Crystal structure of autotaxin and insight into GPCR activation by lipid mediators Peer-reviewed

Hiroshi Nishimasu, Shinichi Okudaira, Kotaro Hama, Emiko Mihara, Naoshi Dohmae, Asuka Inoue, Ryuichiro Ishitani, Junichi Takagi, Junken Aoki, Osamu Nureki

NATURE STRUCTURAL & MOLECULAR BIOLOGY　18　(2)　205-U271　2011/02

DOI： 10.1038/nsmb.1998 　

ISSN： 1545-9985
Prevalent LIPH Founder Mutations Lead to Loss of P2Y5 Activation Ability of PA-PLA(1)alpha in Autosomal Recessive Hypotrichosis Peer-reviewed

Satoru Shinkuma, Masashi Akiyama, Asuka Inoue, Junken Aoki, Ken Natsuga, Toshifumi Nomura, Ken Arita, Riichiro Abe, Kei Ito, Hideki Nakamura, Hideyuki Ujiie, Akihiko Shibaki, Hiraku Suga, Yuichiro Tsunemi, Wataru Nishie, Hiroshi Shimizu

HUMAN MUTATION　31　(5)　602-610　2010/05

DOI： 10.1002/humu.21235 　

ISSN： 1059-7794
Two pathways for lysophosphatidic acid production. International-journal Peer-reviewed

Junken Aoki, Asuka Inoue, Shinichi Okudaira

Biochimica et biophysica acta　1781　(9)　513-8　2008/09

DOI： 10.1016/j.bbalip.2008.06.005 　

ISSN： 1388-1981
Embryo spacing and implantation timing are differentially regulated by LPA3-mediated lysophosphatidic acid signaling in mice Peer-reviewed

Kotaro Hama, Junken Aoki, Asuka Inoue, Tomoko Endo, Tomokazu Amano, Rie Motoki, Motomu Kanai, Xiaoqin Ye, Jerold Chun, Norio Matsuki, Hiroshi Suzuki, Masakatsu Shibasaki, Hiroyuki Arai

BIOLOGY OF REPRODUCTION　77　(6)　954-959　2007/12

DOI： 10.1095/biolreprod.107.060293 　

ISSN： 0006-3363
Structure and function of extracellular phospholipase A(1) belonging to the pancreatic lipase gene family Peer-reviewed

Junken Aoki, Asuka Inoue, Kumiko Makide, Naoya Saiki, Hiroyuki Arai

BIOCHIMIE　89　(2)　197-204　2007/02

DOI： 10.1016/j.biochi.2006.09.021 　

ISSN： 0300-9084
Phospholipase A(1): structure, distribution and function Peer-reviewed

Asuka Inoue, Junken Aoki

FUTURE LIPIDOLOGY　1　(6)　687-700　2006/12

DOI： 10.2217/17460875.1.6.687 　

ISSN： 1746-0875
Lysophosphatidic receptor, LPA(3), is positively and negatively regulated by progesterone and estrogen in the mouse uterus Peer-reviewed

Kotaro Hama, Junken Aoki, Koji Bandoh, Asuka Inoue, Tomoko Endo, Tomokazu Amano, Hiroshi Suzuki, Hiroyuki Arai

LIFE SCIENCES　79　(18)　1736-1740　2006/09

DOI： 10.1016/j.lfs.2006.06.002 　

ISSN： 0024-3205
LPA(3)-mediated lysophosphatidic acid signalling in embryo implantation and spacing Peer-reviewed

XQ Ye, K Hama, JJA Contos, B Anliker, A Inoue, MK Skinner, H Suzuki, T Amano, G Kennedy, H Arai, J Aoki, J Chun

NATURE　435　(7038)　104-108　2005/05

DOI： 10.1038/nature03505 　

ISSN： 0028-0836

Show all ︎Show first 5

Misc. 235

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CARINO Carlo Marion Codog, 桑原莉来, 川上耕季, 柳川正隆, 井上飛鳥, 井上飛鳥

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Analysis of the Molecular Basis of Adverse Effects of Mu-opioid Receptor Agonists

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日本薬学会年会要旨集(Web)　144th　2024

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Ligand recognition and activation mechanisms of Free Fatty Acid Receptor 2

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日本薬学会年会要旨集(Web)　144th　2024

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構造生物学から見たバソプレシン・オキシトシンの分子理解

福永彩美, 川上耕季, 小柳淳暉, 福田昌弘, 小林和弘, 松井俊貴, SCOTT Daniel J., 井上飛鳥, 加藤英明, 加藤英明

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新井大輝, 辰己茉菜絵, 生田達也, 川上耕季, 井上飛鳥

日本薬学会年会要旨集(Web)　143rd　2023

ISSN： 0918-9823
Analysis of biased-signal creation substrates and mechanisms in GPCRs

中村楽, 川上耕季, 生田達也, 井上飛鳥

日本薬学会年会要旨集(Web)　143rd　2023

ISSN： 0918-9823
Novel PIP₂ binding site of β arrestin and its function

CARINO Carlo Marion Codog, 生田達也, 倉本律輝, 川上耕季, 内田安則, 田口友彦, 井上飛鳥

日本薬学会年会要旨集(Web)　143rd　2023

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Clinical Neuroscience　40　(4)　2022

ISSN： 0289-0585
分子から迫る神経薬理学 GPCRシグナル検出系の開発

川上耕季, 井上飛鳥

Clinical Neuroscience　40　(5)　2022

ISSN： 0289-0585
Toward understanding the role of G-protein signaling

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Current Opinion in Endocrine and Metabolic Research　16　51-55　2021/02

DOI： 10.1016/j.coemr.2020.08.006 　

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ISSN： 0918-9823
NanoBiT-BRET法によるGPCRシグナル複合体解析

川上耕季, 平塚寿々音, 桑原莉来, 吉田美沙紀, 井上飛鳥

日本生化学会大会(Web)　94th　2021
GRK3選択的活性化機構の生化学的解析

カリニョカーロマリオンコドッグ, 生田達也, 志甫谷渉, 川上耕季, 平塚寿々音, 濡木理, 井上飛鳥

日本生化学会大会(Web)　94th　2021
ミューオピオイド受容体作動薬の副作用発現を担うシグナル制御因子

平塚寿々音, 川上耕季, 井上飛鳥

日本生化学会大会(Web)　94th　2021
バソプレシンV2受容体に対するβアレスチンの結合様式と構造変化

倉本律輝, 生田達也, 川上耕季, 井上飛鳥

日本生化学会大会(Web)　94th　2021
βアレスチンの機能多様性の分子機構の解析

川上耕季, 井上飛鳥

GPCR研究会プログラム・抄録集　16th (Web)　2021
RNA修飾代謝物のN6-methyladenosine(m6A)は受容体応答を引き起こす新規液性因子である

小川亜希子, 小川亜希子, 名切千彩恵, 志甫谷渉, 井上飛鳥, 川上耕季, 平塚寿々音, 青木淳賢, 青木淳賢, 伊藤康裕, 鈴木健夫, 鈴木勉, 井上俊洋, 濡木理, 富澤一仁, 魏范研, 魏范研

日本生化学会大会(Web)　94th　2021
βアレスチンのGPCRへの結合様式の違いによる機能の差異の解明

桑原莉来, 川上耕季, 吉田美沙紀, 平塚寿々音, 辰己茉菜絵, 井上飛鳥

日本生化学会大会(Web)　94th　2021
GPCRバイアスリガンドのシグナル機構の理解

川上耕季, 平塚寿々音, 井上飛鳥, 井上飛鳥

日本生化学会大会(Web)　93rd　2020
Gタンパク質依存的なβアレスチン制御メカニズムの発見と解析

川上耕季, 柳川正隆, 平塚寿々音, SHUKLA Arun K., 佐甲靖志, 青木淳賢, 青木淳賢, 青木淳賢, 井上飛鳥, 井上飛鳥, 井上飛鳥

日本生化学会大会(Web)　93rd　2020
μオピオイド受容体におけるGPCRキナーゼの選択的活性化機構の解明

平塚寿々音, 川上耕季, 吉田美沙紀, 青木淳賢, 青木淳賢, 井上飛鳥, 井上飛鳥

日本生化学会大会(Web)　93rd　2020
NanoBiTを用いたGαq-PLCβ相互作用解析手法の開発とGq共役型受容体の機能解析

西郷雄貴, 川上耕季, 平塚寿々音, 青木淳賢, 青木淳賢, 井上飛鳥, 井上飛鳥, 井上飛鳥

日本生化学会大会(Web)　93rd　2020
βアレスチン構造センサーを用いたバイアスリガンドとシグナル伝達の解明

吉田美沙紀, 川上耕季, 平塚寿々音, SHUKLA Arun K., 青木淳賢, 青木淳賢, 井上飛鳥, 井上飛鳥

日本生化学会大会(Web)　93rd　2020
リゾリン脂質の疎水性部は膜受容体結合時にどこに位置するか

佐山美紗, 上水明治, 井上飛鳥, 青木淳賢, 関嶋政和, 尾谷優子, 大和田智彦

日本薬学会年会要旨集　139年会　(2)　98-98　2019/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Gαiファミリー欠損細胞を用いたGPCR-Gαiサブタイプの共役の解析

小野雄基, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢

日本薬学会年会要旨集　139年会　(3)　64-64　2019/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Non-Edg型LPA受容体LPA4-6及びGPR55の作動薬創製

金藤奨, 井上飛鳥, 可野邦行, 尾谷優子, 大和田智彦, 青木淳賢

日本薬学会年会要旨集　139年会　(3)　75-75　2019/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
かゆみや呼吸を制御する酸化リン脂質とその受容体の同定

吉田美沙紀, 岸貴之, 川名裕己, 川名裕己, 可野邦行, 可野邦行, 仲川清隆, 井上飛鳥, 井上飛鳥, 井上飛鳥, 青木淳賢, 青木淳賢

次世代を担う若手ファーマ・バイオフォーラム講演要旨集　18th　2019
GRK欠損HEK293細胞の作製と各種リガンドのGRKサプタイプ選択性評価

平塚寿々音, 川上耕季, 井上飛鳥, 井上飛鳥, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　92nd　2019
GPCRキナーゼ解析ツールの開発とバイアス型リガンドの解析

川上耕季, 川上耕季, 井上飛鳥, 井上飛鳥, 井上飛鳥, 平塚寿々音, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　92nd　2019
GRK解析ツールの開発とバイアス型リガンドの理解

川上耕季, 川上耕季, 井上飛鳥, 井上飛鳥, 平塚寿々音, 青木淳賢, 青木淳賢

GPCR研究会プログラム・抄録集　15th　2019
Gタンパク質共役選択性の理解

井上飛鳥, 井上飛鳥, 井上飛鳥, RAIMONDI Francesco, KADJI Francois Marie Ngako, SINGH Gurdeep, 岸貴之, 上水明治, 小野雄基, 新上雄司, 石田覚, ARANG Nadia, 川上耕季, GUTKIND J.Silvio, 青木淳賢, 青木淳賢, RUSSELL Robert B.

GPCR研究会プログラム・抄録集　15th　2019
GPCR-Gタンパク質共役データベースを利用したG12選択的DREADDの作製

小野雄基, 井上飛鳥, 井上飛鳥, 井上飛鳥, RAIMONDI Francesco, KADJI Francois Marie Ngako, SINGH Gurdeep, 岸孝之, 上水明治, 新上雄司, 石田覚, ARANG Nadia, 川上耕季, GUTKIND J Silvio, 青木淳賢, RUSSELL Roberet B.

GPCR研究会プログラム・抄録集　15th　2019
【リゾリン脂質メディエーター研究の最前線】生理活性脂質LysoPSの受容体と代謝酵素

井上飛鳥, 上水明治, 青木淳賢

生化学　90　(5)　614-619　2018/10
Publisher: (公社)日本生化学会
ISSN： 0037-1017
スプリットルシフェラーゼを用いた三量体Gタンパク質活性化の検出法の確立とGPCR研究への応用

井上飛鳥, Kadji Francois, Marie Ngako, 青木淳賢

日本生化学会大会プログラム・講演要旨集　91回　[1T12m-02(1P　2018/09
Publisher: (公社)日本生化学会
脂質酸化を起点とするバイオロジーの新展開酸化リン脂質の新規薬理作用とその受容体

岸貴之, 井上飛鳥, 石黒純, 大村谷昌樹, 青木淳賢

日本生化学会大会プログラム・講演要旨集　91回　[1S07e-04]　2018/09
Publisher: (公社)日本生化学会
ロイコトリエンB4受容体のリン酸化修飾に関する解析昨年からの続報

竹之下逸樹, 相原咲希, 井上飛鳥, 青木淳賢, 中村元直

日本生化学会大会プログラム・講演要旨集　91回　[1P-208]　2018/09
Publisher: (公社)日本生化学会
オーファンGPCRに対するGαタンパク質共役解析

川上耕季, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　91回　[1P-216]　2018/09
Publisher: (公社)日本生化学会
Gαi欠損HEK293細胞の作製と評価

小野雄基, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢

日本生化学会大会プログラム・講演要旨集　91回　[1P-219]　2018/09
Publisher: (公社)日本生化学会
ATX-LPAシグナルによる内分細胞間接着制御機構の解析

木瀬亮次, 可野邦行, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　91回　[2T11a-01(2P　2018/09
Publisher: (公社)日本生化学会
LPAによる昇圧作用メカニズムの解析

可野邦行, 松本宏隆, 井上飛鳥, 雪浦弘志, Chun Jerold, 石井聡, 清水孝雄, 青木淳賢

脂質生化学研究　60　209-210　2018/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
LPA6およびPA‐PLA1aは毛包の特定の層に発現し正常な体毛形成に寄与する

片寄愛望, 可野邦行, 可野邦行, 井上飛鳥, 井上飛鳥, 青木淳賢, 青木淳賢, 青木淳賢

日本薬学会年会要旨集(CD-ROM)　138th　(3)　ROMBUNNO.28PA‐am053-211　2018/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
脂質リガンドのGPCRへの結合機構の解明のための誘導体合成と計算科学の融合研究

佐山美紗, 井上飛鳥, 青木淳賢, 広川貴次, 関嶋政和, 尾谷優子, 大和田智彦

日本薬学会年会要旨集　138年会　(2)　110-110　2018/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
アミノ酸点変異導入による新規GPCR活性化手法の開発

柴田剛明, 井上飛鳥, 川上耕季, 木瀬亮次, 青木淳賢

日本薬学会年会要旨集　138年会　(3)　100-100　2018/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
初代培養リンパ球を用いたリゾホスファチジルセリン産生機構の解析

佐藤慧太, 新上雄司, 尾谷優子, 大和田智彦, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　138年会　(3)　211-211　2018/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
LPA6およびPA-PLA1aは毛包の特定の層に発現し正常な体毛形成に寄与する

片寄愛望, 可野邦行, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　138年会　(3)　211-211　2018/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
全Giファミリー分子欠損細胞の作製とGi共役型受容体シグナルの解析

小野雄基, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢

次世代を担う若手ファーマ・バイオフォーラム講演要旨集　17th　2018
TGFα切断アッセイ系を活用したロイコトリエンB4受容体のシグナル伝達解析について

瀬川美奈, 井上飛鳥, 青木淳賢, 中村元直

生命科学系学会合同年次大会　2017年度　[1P-0475]　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
Gαi欠損HEK293細胞の作成

小野雄基, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢

生命科学系学会合同年次大会　2017年度　[1P-0516]　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
Non-Edg型LPA受容体LPA4-6の作動薬創製

金藤奨, 井上飛鳥, 尾谷優子, 大和田智彦, 青木淳賢

生命科学系学会合同年次大会　2017年度　[1LBA-017]　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
ゼブラフィッシュにおけるGPCR活性化検出系の確立

柴田剛明, 井上飛鳥, 木瀬亮次, 川上耕季, 青木淳賢

生命科学系学会合同年次大会　2017年度　[2P-0455]　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
ATX-LPAシグナルは細胞間接着の形成に重要である

木瀬亮次, 新上雄司, 可野邦行, 井上飛鳥, 青木淳賢

生命科学系学会合同年次大会　2017年度　[3PT12-13(3P　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
がん細胞の代謝がもたらす組織微小環境を標的にしたがん治療

豊本雅靖, 井上飛鳥, 飯田慶, 出縄政嗣, 喜井勲, 林佳子, 岸貴之, 小野木博, 吉田優, 細谷孝充, 青木淳賢, 萩原正敏

生命科学系学会合同年次大会　2017年度　[3AT25-1419)]　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
三量体Gタンパク質の活性化センサーの開発

井上飛鳥, Francois Marie Kadji, 川上耕季, 青木淳賢

生命科学系学会合同年次大会　2017年度　[3AT25-11(3P　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
酸化脂質に応答する霊長類特異的受容体、MRGX4のマウス機能的ホモログの同定

岸貴之, 井上飛鳥, 青木淳賢

生命科学系学会合同年次大会　2017年度　[3P-0056]　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
3.43Q変異に起因する恒常活性化GPCRの性状解析

川上耕季, 井上飛鳥, 柴田剛明, 青木淳賢

生命科学系学会合同年次大会　2017年度　[4LT10-02(3P　2017/12
Publisher: 生命科学系学会合同年次大会運営事務局
LPAシグナルは細胞形態の維持に重要である

木瀬亮次, 川上耕季, 可野邦行, 井上飛鳥, 青木淳賢

日本細胞生物学会大会講演要旨集　69回　55-55　2017/05
Publisher: (一社)日本細胞生物学会
常染色体劣性縮毛症/乏毛症(ARWH/H)の遺伝子解析診断に有用なアッセイ系の確立

林良太, 阿部理一郎, 下村裕, 井上飛鳥, 青木淳賢, 須賀康, 赤坂俊英

日本皮膚科学会雑誌　127　(4)　635-635　2017/04
Publisher: (公社)日本皮膚科学会
ISSN： 0021-499X
酸化脂質に応答するオーファンGPCR、MRGX4のマウスにおける機能的ホモログの同定

岸貴之, 井上飛鳥, 石黒純, 青木淳賢

日本薬学会年会要旨集　137年会　(3)　55-55　2017/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
ATX-LPAシグナルはVE-cadherin機能調節を介して血管新生を制御する

木瀬亮次, 可野邦行, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　137年会　(3)　56-56　2017/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
PA-PLA1aおよびLPA6は毛包の特定の層に発現し正常な体毛形成に寄与する

片寄愛望, 可野邦行, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　137年会　(3)　56-56　2017/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
恒常活性がみられるGPCRの発現解析

柴田剛明, 井上飛鳥, 川上耕季, 青木淳賢

日本薬学会年会要旨集　137年会　(3)　90-90　2017/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
多数のGPCRを共通に活性化させる変異の解析

川上耕季, 井上飛鳥, 柴田剛明, 金藤奨, 青木淳賢

日本薬学会年会要旨集　137年会　(3)　94-94　2017/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
ポストGPCRシグナル創薬全盛期世代の研究戦略 GPCRシグナルを基軸とした創薬基礎研究

井上飛鳥, 青木淳賢

日本薬学会年会要旨集　137年会　(1)　116-116　2017/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
3.43Q変異に起因する恒常活性化GPCRの解析

川上耕季, 井上飛鳥, 井上飛鳥, 井上飛鳥, 柴田剛明, 青木淳賢, 青木淳賢

GPCR研究会プログラム・抄録集　14th　2017
恒常活性化変異GPCRの性状解析

川上耕季, 井上飛鳥, 井上飛鳥, 井上飛鳥, 柴田剛明, 青木淳賢, 青木淳賢

次世代を担う若手ファーマ・バイオフォーラム講演要旨集　16th　2017
高輝度スプリットルシフェラーゼを用いたGタンパク質センサー

井上飛鳥, 井上飛鳥, 井上飛鳥, KADJI Francois Marie, 川上耕季, 青木淳賢, 青木淳賢

GPCR研究会プログラム・抄録集　14th　2017
恒常活性がみられる点変異GPCRの発現解析

柴田剛明, 井上飛鳥, 井上飛鳥, 井上飛鳥, 川上耕季, 青木淳賢, 青木淳賢

GPCR研究会プログラム・抄録集　14th　2017
酸化リン脂質により活性化する3種のオーファンGPCRの同定

岸貴之, 井上飛鳥, 石黒純, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[1T10-02(1P　2016/09
Publisher: (公社)日本生化学会
アカデミア発創薬探索研究 GPCR解析ツールの開発とGPCRを標的とするアカデミア創薬

井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[1S04-1]　2016/09
Publisher: (公社)日本生化学会
全LPA受容体欠損細胞の作製とLPA研究への応用

川上耕季, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[2T13-01(2P　2016/09
Publisher: (公社)日本生化学会
PA-PLA1αおよびLPA6は毛包の特定の層に発現し正常な体毛形成に寄与する

片寄愛望, 可野邦行, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[2T09-05(2P　2016/09
Publisher: (公社)日本生化学会
LPA6の欠損は精原細胞の減少を引き起こす

岸田真輝, 可野邦行, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[2T09-06(2P　2016/09
Publisher: (公社)日本生化学会
新規LysoPS受容体の抗体産生における役割

新上雄司, 巻出久美子, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[2T16-06(1P　2016/09
Publisher: (公社)日本生化学会
腫瘍免疫におけるリゾホスファチジルセリンの役割

巻出久美子, 新上雄司, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[2T09-07(2P　2016/09
Publisher: (公社)日本生化学会
腸内細菌の長鎖不飽和脂肪酸代謝物によるGPR120活性化の検討

本郷翔子, 森本育美, 山上小百合, 古田美咲, 滝澤祥恵, 井上飛鳥, 青木淳賢, 東山繁樹, 吉田守克, 宮里幹也, 岸野重信, 小川順, 中田理恵子, 井上裕康

日本生化学会大会プログラム・講演要旨集　89回　[2P-106]　2016/09
Publisher: (公社)日本生化学会
生命科学研究の必要条件としての構造生物学オートタキシン-ENPP2シグナリングの構造と機能、そして構造に基づいた創薬

濡木理, 谷口怜哉, 加藤一希, 西増弘志, 井上飛鳥, 青木淳賢, 石谷隆一郎

日本生化学会大会プログラム・講演要旨集　89回　[3S06-1]　2016/09
Publisher: (公社)日本生化学会
GPR35を介したマスト細胞の脱顆粒応答抑制機構の解明

水粉翔也, 古田和幸, 井上飛鳥, 青木淳賢, 田中智之

日本生化学会大会プログラム・講演要旨集　89回　[3T16-04(2P　2016/09
Publisher: (公社)日本生化学会
リゾホスファチジルセリン受容体LPS1/GPR34に対する強力且つ選択的アゴニストの開発とその応用

上水明治, 巻出久美子, 中村翔, 井上飛鳥, 尾谷優子, 大和田智彦, 青木淳賢

日本生化学会大会プログラム・講演要旨集　89回　[3P-088]　2016/09
Publisher: (公社)日本生化学会
Implications of Spns2-deficiency on S1P-driven lymphocytes egress, HEV-integrity and immunity

S. Simmons, N. Sasaki, E. Umemoto, N. Yoshizumi, S. Fukuhara, Y. Kitazawa, M. Okudaira, A. Inoue, D. Motooka, S. Nakamura, T. Iida, J. Aoki, N. Mochizuki, K. Matsuno, K. Takeda, M. Miyasaka, M. Ishii

EUROPEAN JOURNAL OF IMMUNOLOGY　46　267-267　2016/08

ISSN： 0014-2980

eISSN： 1521-4141
長鎖不飽和脂肪酸とその腸内細菌代謝物によるGPR120活性化の検討

本郷翔子, 森本育美, 山上小百合, 古田美咲, 滝澤祥恵, 井上飛鳥, 青木淳賢, 東山繁樹, 吉田守克, 宮里幹也, 岸野重信, 小川順, 中田理恵子, 井上裕康

脂質生化学研究　58　53-54　2016/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
内皮細胞におけるATX-LPAシグナルの解析

木瀬亮次, 可野邦行, 井上飛鳥, 青木淳賢

脂質生化学研究　58　159-160　2016/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
ヒト先天性乏毛症患者由来の変異LPA6受容体の機能解析

上水明治, 井上飛鳥, 下村裕, 青木淳賢

脂質生化学研究　58　161-164　2016/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
マスト細胞上のLysoPS受容体探索ツールの開発

岸貴之, 佐山美沙, 巻出久美子, 川名裕己, 井上飛鳥, 尾谷優子, 大和田知彦, 青木淳賢

脂質生化学研究　58　165-167　2016/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
「疾患代謝」から解明される生命現象と創薬研究への応用代謝物が作用するGPCRの同定と機能解析

井上飛鳥, 青木淳賢

日本薬学会年会要旨集　136年会　(1)　258-258　2016/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
リゾリン脂質のGPCRへの結合様式解明のための誘導体合成

佐山美紗, ジョン・セジン, 中村翔, 井久保仁也, 尾谷優子, 上水明治, 岸貴之, 井上飛鳥, 巻出久美子, 青木淳賢, 広川貴次, 大和田智彦

日本薬学会年会要旨集　136年会　(2)　126-126　2016/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
β-arrestin欠損細胞の作製とGPCR研究への応用

川上耕季, 井上飛鳥, 井上飛鳥, 石田覚, 青木淳賢

GPCR研究会プログラム・抄録集　13th　2016
【GPCR研究の最前線2016】GPCR研究のあらたな基盤と視点構造・機能解析の進歩オーファンGPCRのリガンド同定と新規GPCR活性化検出法

井上飛鳥, 青木淳賢

医学のあゆみ　256　(5)　373-378　2016/01
Publisher: 医歯薬出版(株)
ISSN： 0039-2359
CRISPR/Cas9システムを用いたLysoPS受容体TKOマウスの作製

新上雄司, 井上飛鳥, 巻出久美子, 中島修, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[1P0181]-[1P0181]　2015/12
Publisher: (公社)日本生化学会
TGFα切断アッセイを用いたGPCRリガンドスクリーニング

岸貴之, 井上飛鳥, 石黒純, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[2T21-01(2P0178)]　2015/12
Publisher: (公社)日本生化学会
LysoPS受容体のサブタイプ選択的アゴニストの開発

上水明治, 井上飛鳥, 井久保仁也, 中村翔, ジョン・セジン, 佐山美紗, 岸貴之, 巻出久美子, 尾谷優子, 大和田智彦, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[2P0147]-[2P0147]　2015/12
Publisher: (公社)日本生化学会
ATX-LPAシグナルによる血管形成制御機構の解析

木瀬亮次, 可野邦行, 井上飛鳥, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[2P0347]-[2P0347]　2015/12
Publisher: (公社)日本生化学会
GPR120活性化を指標とする新規食品機能評価系の検討

森本育美, 滝澤祥恵, 本郷翔子, 井上飛鳥, 青木淳賢, 東山繁樹, 吉田守克, 宮里幹也, 中田理恵子, 井上裕康

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[2P1232]-[2P1232]　2015/12
Publisher: (公社)日本生化学会
GPCRの共役するGタンパク質の網羅的解析とその応用

井上飛鳥, 新上雄司, 岸貴之, 石田覚, 上水明治, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[3P0844]-[3P0844]　2015/12
Publisher: (公社)日本生化学会
リゾホスファチジン酸の昇圧作用メカニズムの解析

可野邦行, 井上飛鳥, 松本宏隆, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[4T4L-10(3P0308)]　2015/12
Publisher: (公社)日本生化学会
GPCRシグナル解析ツールの開発

石田覚, 井上飛鳥, 新上雄司, 川上耕季, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[4T12L-06(3P0830)]　2015/12
Publisher: (公社)日本生化学会
第二世代の脂質メディエーター・リゾリン脂質の生理・病態機能

青木淳賢, 青木淳賢, 可野邦行, 井上飛鳥, 井上飛鳥

生体膜と薬物の相互作用シンポジウム講演要旨集　37th　35-36　2015/11/19

ISSN： 0919-2131
【代謝疾患とメタボローム】代謝物が作用するGPCRをどのように同定するか?

岸貴之, 井上飛鳥, 青木淳賢

内分泌・糖尿病・代謝内科　41　(5)　347-353　2015/11
Publisher: (有)科学評論社
ISSN： 1884-2917
【脂質疾患学なぜ"あぶら"の異常が病気を引き起こすのか?その質的量的変化と肥満、がん、不妊症、免疫・皮膚・神経疾患】(第2章)多彩な疾患における脂質の量的・質的変化皮膚疾患リゾホスファチジン酸と先天性乏毛症

下村裕, 井上飛鳥, 青木淳賢

実験医学　33　(15)　2451-2455　2015/09
Publisher: (株)羊土社
ISSN： 0288-5514
酸化リン脂質に応答するGPCRの同定および機能解析

青木淳賢, 石黒純, 井上飛鳥

ビタミン　89　(7)　359-360　2015/07
Publisher: (公社)日本ビタミン学会
DOI： 10.20632/vso.89.7_359 　

ISSN： 0006-386X
ATX-LPAシグナルによる血管形成制御機構の解析

木瀬亮次, 雪浦弘志, 可野邦行, 井上飛鳥, 青木淳賢

脂質生化学研究　57　97-99　2015/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
4価型人工ペプチドを用いたホスファチジン酸結合プローブの開発

小川莉奈, 長尾耕治郎, 原雄二, 田村朋則, 浜地格, 井上飛鳥, 青木淳賢, 高橋美帆, 西川喜代孝, 梅田真郷

脂質生化学研究　57　170-173　2015/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
TGFα切断アッセイを用いたω-3系脂肪酸によるGPR120活性化

森本育美, 滝澤祥恵, 本郷翔子, 井上飛鳥, 青木淳賢, 東山繁樹, 吉田守克, 宮里幹也, 中田理恵子, 井上裕康

ビタミン　89　(4)　223-223　2015/04
Publisher: (公社)日本ビタミン学会
ISSN： 0006-386X
リゾホスファチジルセリン分解系の解析

川名裕己, 奥平倫世, 井上飛鳥, 巻出久美子, 三枝大輔, 青木淳賢

日本薬学会年会要旨集　135年会　(3)　53-53　2015/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
Gタンパク質欠損細胞を用いたGPCR-Gαサブユニットの共役の解析

岸貴之, 井上飛鳥, 新上雄司, 上水明治, 石田覚, 青木淳賢

日本薬学会年会要旨集　135年会　(3)　67-67　2015/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
リゾホスファチジルセリン特異的GPCR-リガンド結合モデルの構築と合成リガンドによる検証

佐山美紗, ジョンセジン, 中村翔, 井久保仁也, 尾谷優子, 上水明治, 岸貴之, 井上飛鳥, 巻出久美子, 青木淳賢, 広川貴次, 大和田智彦

日本薬学会年会要旨集(CD-ROM)　135th　2015
β-arrestin欠損細胞の作製

川上耕季, 井上飛鳥, 井上飛鳥, 石田覚, 青木淳賢, 青木淳賢

日本薬学会東北支部大会講演要旨集　54th　2015
Lysophospholipid mediators: Their receptors and synthetic pathways

Kuniyuki Kano, Kumiko Makide, Jun Ishiguro, Hiroshi Yukiura, Shizu Aikawa, Akiharu Uwamizu, Yuji Shinjo, Kahori Namiki, Hiroki Kawana, Saki Nemoto, Hirotaka Matsumoto, Ryoji Kise, Asuka Inoue, Junken Aoki

Bioactive Lipid Mediators: Current Reviews and Protocols　109-126　2015/01/01

DOI： 10.1007/978-4-431-55669-5_8 　
ヒト先天性脱毛症モデルマウスLPA6KOマウスにおける毛包の解析

片寄愛望, 可野邦行, 可野邦行, 井上飛鳥, 青木淳賢, 青木淳賢

日本薬学会東北支部大会講演要旨集　54th　56　2015
LPA₆欠損マウスにおける精巣の形成不全の解析

岸田真輝, 可野邦行, 可野邦行, 井上飛鳥, 青木淳賢, 青木淳賢

日本薬学会東北支部大会講演要旨集　54th　55　2015
下水からの医薬品生理活性の検出

井原賢, 井上飛鳥, 花本征也, Zhang Han, 青木淳賢, 田中宏明

環境ホルモン学会研究発表会要旨集　17回　44-44　2014/12
Publisher: 環境ホルモン学会(日本内分泌撹乱化学物質学会)
LysoPS受容体アゴニストの探索 LysoPS類似体を用いたアプローチ

上水明治, 井上飛鳥, 井久保仁也, 中村翔, セジン・ジョン, 佐山美紗, 岸貴之, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[2T09p-04]　2014/10
Publisher: (公社)日本生化学会
CRISPR-Cas9システムを用いたLysoPS受容体変異Jurkat T cellの作製

新上雄司, 井上飛鳥, 石黒純, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[2T09p-05]　2014/10
Publisher: (公社)日本生化学会
TALENによるLPA4欠損ゼブラフィッシュの作製

木瀬亮次, 雪浦弘志, 可野邦行, 井上飛鳥, 久野悠, 川原敦雄, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[3T09a-09]　2014/10
Publisher: (公社)日本生化学会
リゾホスファチジルセリンはTリンパ球の活性化を抑制する

石黒純, 新上雄司, 北村一, 井上飛鳥, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[3T09a-12]　2014/10
Publisher: (公社)日本生化学会
自己免疫性リンパ球増殖症におけるP2Y10の機能解析

奥平倫世, 根本祥李, 井上飛鳥, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[3T10a-09]　2014/10
Publisher: (公社)日本生化学会
三量体Gタンパク質欠損細胞を用いたGPCRのシグナル解析手法の確立

井上飛鳥, 新上雄司, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[4T13a-08]　2014/10
Publisher: (公社)日本生化学会
コンカナバリンA誘発性肝炎におけるリゾホスファチジルセリン産生機構の解析

丹羽祐介, 鈴木健介, 奥平倫世, 井上飛鳥, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　87回　[2P-083]　2014/10
Publisher: (公社)日本生化学会
【GPCR研究の熱い潮流】新しいGPCR活性化測定法"TGFα切断アッセイ" その発見の経緯と薬学研究における応用

井上飛鳥, 青木淳賢

ファルマシア　50　(9)　862-866　2014/09
Publisher: (公社)日本薬学会
ISSN： 0014-8601
Lipase H(LIPH)の新しい遺伝子変異c.699C>G(p.C233W)による先天性縮毛症・乏毛症の1例

吉岡学, 吉澤真裕子, 日野亮介, 小林美和, 中村元信, Muhammmmad Farooq, 下村裕, 井上飛鳥, 青木淳賢

西日本皮膚科　76　(3)　294-295　2014/06
Publisher: 日本皮膚科学会-西部支部
ISSN： 0386-9784
コンカナバリンA誘発性肝炎におけるリゾホスファチジルセリンの役割

巻出久美子, 鈴木健介, 奥平倫世, 井上飛鳥, 青木淳賢

脂質生化学研究　56　143-145　2014/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
血小板活性化の血漿リゾフォスファチジルセリン上昇への関与

蔵野信, 廣渡祐史, 徳原康哲, 土肥智貴, 宮内克己, 代田浩之, 五十嵐浩二, 井上飛鳥, 青木淳賢, 矢冨裕

日本血栓止血学会誌　25　(2)　252-252　2014/04
Publisher: (一社)日本血栓止血学会
ISSN： 0915-7441
Physiological roles of lysophosphatidic acid (LPA)

248　(13)　963-969　2014/03/29
Publisher: 医歯薬出版
ISSN： 0039-2359
創薬標的分子の同定を目指す新しい脂質マシナリー研究免疫系を制御する新たな脂質メディエーターリゾホスファチジルセリン

青木淳賢, 巻出久美子, 井上飛鳥

日本薬学会年会要旨集　134年会　(1)　234-234　2014/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
TGFα切断アッセイのハイスループットスクリーニングへの応用

上水明治, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　134年会　(3)　63-63　2014/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
血中におけるリゾホスファチジルセリンの安定性の解析

奥平倫世, 飯田奈紀沙, 井上飛鳥, 巻出久美子, 中村翔, 尾谷優子, 大和田智彦, 青木淳賢

日本薬学会年会要旨集　134年会　(3)　72-72　2014/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
リゾリン脂質受容体における疎水性ポケット構造の推定

佐山美紗, セジンジョン, 中村翔, 井久保仁也, 尾谷優子, 上水明治, 井上飛鳥, 巻出久美子, 青木淳賢, 広川貴次, 大和田智彦

日本薬学会関東支部大会講演要旨集　58th　2014
新規GPCR活性化検出法を利用したオーファン受容体のリガンドの同定

井上飛鳥, 青木淳賢

生化学　85　(11)　1029-1033　2013/11
Publisher: (公社)日本生化学会
ISSN： 0037-1017
【ホスホリパーゼの機能・応用】ホスホリパーゼA1の構造と機能

中永景太, 井上飛鳥, 青木淳賢

オレオサイエンス　13　(10)　493-500　2013/10
Publisher: (公社)日本油化学会
DOI： 10.5650/oleoscience.13.493 　

ISSN： 1345-8949
細胞内型ホスホリパーゼA1の機能解析

中永景太, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　1LBA-015　2013/09
Publisher: (公社)日本生化学会
軟骨形成におけるATX-LPA-LPA1 axisの役割

西岡達二, 板井恵理子, 有馬直明, 井上飛鳥, Chun Jerold, Moolenaar Wouter, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　1LBA-016　2013/09
Publisher: (公社)日本生化学会
コンカナバリンA誘導肝炎におけるリゾホスファチジルセリンの機能解析

鈴木健介, 奥谷倫世, 井上飛鳥, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　1LBA-017　2013/09
Publisher: (公社)日本生化学会
LysoPSはGPR174を介してIL-2産生を抑制する

新上雄司, 北村一, 石黒純, 奥谷倫世, 井上飛鳥, 巻出久美子, 井久保仁也, 大和田智彦, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　1T10a-08　2013/09
Publisher: (公社)日本生化学会
リゾホスファチジルセリンは活性化リンパ球の増殖を抑制する

石黒純, 北村一, 首藤啓明, 新上雄司, 井上飛鳥, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　1T10a-09　2013/09
Publisher: (公社)日本生化学会
LPAはLPA6を介してVE-cadherinによる血管内皮細胞間接着を抑制する

雪浦弘志, 中永景太, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　1T10a-13　2013/09
Publisher: (公社)日本生化学会
生体膜におけるリン脂質分子種の非対称分布

石川真実, 加藤詩子, 芦本徹, 井上飛鳥, 青木淳賢, 梅田真郷

日本生化学会大会プログラム・講演要旨集　86回　2T10a-03　2013/09
Publisher: (公社)日本生化学会
オートファゴソーム形成とグリセロリン脂質生合成系の密接な関係

西村多喜, 田村律人, 中永景太, 井上飛鳥, 青木淳賢, 水島昇

日本生化学会大会プログラム・講演要旨集　86回　2T11a-01　2013/09
Publisher: (公社)日本生化学会
TGFα切断アッセイのハイスループットスクリーニングへの応用

上水明治, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　2T14p-09　2013/09
Publisher: (公社)日本生化学会
血中におけるリゾホスファチジルセリンの安定性の解析

奥平倫世, 飯田奈紀沙, 井上飛鳥, 巻出久美子, 中村翔, 尾谷優子, 大和田智彦, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　2P-057　2013/09
Publisher: (公社)日本生化学会
機能性酸化脂質研究の新展開酸化リン脂質によるGタンパク質共役型受容体の活性化とその意義

井上飛鳥, 石黒純, 青木淳賢

日本生化学会大会プログラム・講演要旨集　86回　3S02p-5　2013/09
Publisher: (公社)日本生化学会
細胞内型ホスホリパーゼA1の機能解析

中永景太, 井上飛鳥, 青木淳賢

生化学　85　(8)　714-714　2013/08
Publisher: (公社)日本生化学会
ISSN： 0037-1017
LysoPSはGPR174を介してT細胞のIL-2産生を抑制する

新上雄司, 奥谷倫世, 石黒純, 北村一, 井上飛鳥, 巻出久美子, 青木淳賢

生化学　85　(8)　719-719　2013/08
Publisher: (公社)日本生化学会
ISSN： 0037-1017
LRATにホモロジーを示す新規リン脂質代謝酵素による細胞内ペルオキシソーム含量の調節

宇山徹, 坪井一人, 金星華, 上田夏生, 市育代, 河野望, 新井洋由, 井上飛鳥, 青木淳賢, 荒木伸一

ビタミン　87　(5-6)　305-305　2013/06
Publisher: (公社)日本ビタミン学会
DOI： 10.20632/vso.87.5-6_305_1 　

ISSN： 0006-386X
Close Up実験法(Series 238) TGFα切断を用いたGPCRの高感度・高精度な活性化検出法

井上飛鳥, 青木淳賢

実験医学　31　(8)　1305-1312　2013/05
Publisher: (株)羊土社
ISSN： 0288-5514
【最新生理活性脂質研究-実験手法、基礎的知識とその応用-】(第1章)技術編リゾリン脂質の質量分析解析

奥平倫世, 井上飛鳥, 青木淳賢

遺伝子医学MOOK　(24)　44-48　2013/05
Publisher: (株)メディカルドゥ
ISSN： 1349-2527
【最新生理活性脂質研究-実験手法、基礎的知識とその応用-】(第3章)基礎編リゾリン脂質に対する受容体

可野邦行, 井上飛鳥, 石黒純, 青木淳賢

遺伝子医学MOOK　(24)　162-168　2013/05
Publisher: (株)メディカルドゥ
ISSN： 1349-2527
酸化脂質研究の最前線疾患シグナルとしての酸化リン脂質と新たな解析法酸化リン脂質を認識するGタンパク質共役型受容体

井上飛鳥, 石黒純, 青木淳賢

脂質生化学研究　55　11-11　2013/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
細胞内型ホスホリパーゼA1の機能解析

中永景太, 井上飛鳥, 青木淳賢

脂質生化学研究　55　74-76　2013/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
LysoPSの検出系の確立と生体内におけるLysoPSの検出

奥平倫世, 井上飛鳥, 三枝大輔, 富岡佳久, 巻出久美子, 青木淳賢

脂質生化学研究　55　128-130　2013/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
LRATに相同性を示す新規リン脂質代謝酵素によるペルオキシソームの機能調節

宇山徹, 市育代, 河野望, 井上飛鳥, 坪井一人, 荒木伸一, 青木淳賢, 新井洋由, 上田夏生

ビタミン　87　(4)　253-253　2013/04
Publisher: (公社)日本ビタミン学会
ISSN： 0006-386X
keratin71遺伝子に病的変異を同定し得た常染色体優性縮毛症/乏毛症(autosomal dominant woolly hair/hypotrichosis)の1家系

藤本篤, 藤川大基, 伊藤雅章, ファルーク・モハメド, 下村裕, 井上飛鳥, 青木淳賢, 大山学, 江浜律子, 仲西城太郎, 萩原基文, 岩渕徳郎

日本皮膚科学会雑誌　123　(4)　461-461　2013/04
Publisher: (公社)日本皮膚科学会
ISSN： 0021-499X

eISSN： 1346-8146
脂質バイオロジーの最前線 Lipid phophate phosphatase 3による血管形成制御

雪浦弘志, 濱弘太郎, 中永景太, 井上飛鳥, 奥平真一, 青木淳賢

日本薬学会年会要旨集　133年会　(1)　188-188　2013/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
リゾホスファチジルセリン受容体アゴニストの配座に関する研究

中村翔, 鄭世珍, 井久保仁也, 尾谷優子, 上水明治, 井上飛鳥, 巻出久美子, 青木淳賢, 大和田智彦

日本薬学会年会要旨集　133年会　(2)　88-88　2013/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
自己免疫性肝炎モデルにおけるリゾホスファチジルセリンの薬理作用

巻出久美子, 鈴木健介, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　133年会　(3)　70-70　2013/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
TGFα切断を用いたGPCRと三量体Gタンパク質の共役活性の評価系の確立

井上飛鳥, 青木淳賢

日本薬学会年会要旨集　133年会　(3)　161-161　2013/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
リゾホスファチジン酸LPAの昇圧作用メカニズムの解析

松本宏隆, 可野邦行, 井上飛鳥, 青木淳賢

日本薬学会東北支部大会講演要旨集　52nd　59　2013
生体膜リン脂質の新たな個性に迫るセリンリゾリン脂質を認識する新規GPCRとその機能

青木淳賢, 巻出久美子, 井上飛鳥

日本生化学会大会プログラム・講演要旨集　85回　1S15-6　2012/12
Publisher: (公社)日本生化学会
新規脂質メディエーターが制御する多彩な機能毛髪形成メカニズムから派生したGタンパク質共役型受容体(GPCR)の新規検出系の開発とその応用

井上飛鳥, 石黒純, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　85回　2S03-6　2012/12
Publisher: (公社)日本生化学会
細胞内型ホスホリパーゼA1の機能解析

中永景太, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　85回　2T03-09　2012/12
Publisher: (公社)日本生化学会
Lipid phosphate phosphatase 3(LPP3)による血管形成制御機構の解析

雪浦弘志, 濱弘太郎, 奥平真一, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　85回　2T04-10　2012/12
Publisher: (公社)日本生化学会
PS特異的ホスホリパーゼA1(PS-PLA1)を用いた新規細胞表面ホスファチジルセリン検出系の確立

橋本崇史, 井上飛鳥, 奥谷倫世, 有馬直明, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　85回　2P-120　2012/12
Publisher: (公社)日本生化学会
Phospholipase A/Acyltransferaseファミリーによる細胞内ペルオキシソーム含量の制御

宇山徹, 市育代, 河野望, 井上飛鳥, 井上愛美, 坪井一人, 金星華, 荒木伸一, 青木淳賢, 徳村彰, 新井洋由, 上田夏生

日本生化学会大会プログラム・講演要旨集　85回　2P-129　2012/12
Publisher: (公社)日本生化学会
新規リゾホスファチジルセリン受容体の発現解析

石黒純, 新上雄司, 北村一, 井上飛鳥, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　85回　3T06-01　2012/12
Publisher: (公社)日本生化学会
ゼブラフィッシュカルシウム動員系プロスタノイド受容体の性状解析

岩崎亮, 川原敦雄, 井上飛鳥, 青木淳賢, 土屋創建, 杉本幸彦

日本生化学会大会プログラム・講演要旨集　85回　3P-098　2012/12
Publisher: (公社)日本生化学会
軟骨形成におけるLPA signalの機能解析

板井恵理子, 有馬直明, 濱弘太郎, 井上飛鳥, 奥平真一, 青木淳賢

日本生化学会大会プログラム・講演要旨集　85回　3P-111　2012/12
Publisher: (公社)日本生化学会
急性冠症候群における血漿リゾホスファチジン酸増加機序の検討 LC/MSによるリゾリン脂質分子種解析

蔵野信, 鈴木明子, 大川龍之介, 中村和宏, 土肥智貴, 宮内克己, 代田浩之, 井上飛鳥, 青木淳賢, 池田均, 矢冨裕

臨床病理　60　(補冊)　194-194　2012/10
Publisher: 日本臨床検査医学会
ISSN： 0047-1860
LC/MS/MSを用いたスフィンゴシン-1-リン酸の超高感度分析法について

高橋友大, 三枝大輔, 陣野大輔, 井上飛鳥, 奥谷倫代, 青木淳賢, 鈴木直人, 富岡佳久

JSBMS Letters　37　(Suppl.)　65-65　2012/09
Publisher: (一社)日本医用マススペクトル学会
ISSN： 1881-5464
LC/MS/MSを用いたマウス脳組織中スフィンゴシン一リン酸の超高感度定量分析について

三枝大輔, 高橋友大, 大宅史織, 井上飛鳥, 奥谷倫代, 鈴木直人, 眞野成康, 青木淳賢, 富岡佳久

JSBMS Letters　37　(Suppl.)　66-66　2012/09
Publisher: (一社)日本医用マススペクトル学会
ISSN： 1881-5464
体毛形成に関与する生理活性脂質リゾホスファチジン酸の機能解明

井上飛鳥, 有馬直明, 石黒純, Prestwich GD, 新井洋由, 青木淳賢

生化学　84　(6)　501-501　2012/06
Publisher: (公社)日本生化学会
ISSN： 0037-1017
生理活性脂質リゾホスファチジルセリンによる免疫抑制作用

巻出久美子, 北村一, 井上飛鳥, 青木淳賢

生化学　84　(6)　502-502　2012/06
Publisher: (公社)日本生化学会
ISSN： 0037-1017
新規脂質代謝酵素H-rev107の発現はペルオキシソームの機能異常を引き起こす

宇山徹, 市育代, 河野望, 井上飛鳥, 坪井一人, 金星華, 荒木伸一, 青木淳賢, 新井洋由, 上田夏生

脂質生化学研究　54　104-107　2012/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
細胞内リゾホスファチジン酸によるミトコンドリアの形態制御

大場陽介, 井上貴雄, 櫻木健司, 原直子, 中臺枝里子, 井上飛鳥, 石原直忠, 青木淳賢, 三谷昌平, 新井洋由

脂質生化学研究　54　224-226　2012/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
TGFα切断を用いた脂質メディエーター受容体の検出法の開発

井上飛鳥, 石黒純, 巻出久美子, 青木淳賢

脂質生化学研究　54　272-275　2012/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
酸化リン脂質に応答する新規GPCRの同定

石黒純, 井上飛鳥, 青木淳賢

脂質生化学研究　54　276-279　2012/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
リゾホスファチジルセリンは自己免疫性肝炎を緩和させる

鈴木健介, 北村一, 奥谷倫世, 首藤啓明, 巻出久美子, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　132年会　(3)　102-102　2012/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
酸化リン脂質に応答する新規GPCRの同定

石黒純, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　132年会　(3)　105-105　2012/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
生体内におけるリゾホスファチジルセリンの検出

奥谷倫世, 井上飛鳥, 巻出久美子, 三枝大輔, 富岡佳久, 青木淳賢

日本薬学会年会要旨集　132年会　(3)　121-121　2012/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
LC/MS/MSを用いた生体試料中スフィンゴ脂質同時定量法の構築

芝可奈子, 三枝大輔, 井上飛鳥, 濱弘太郎, 奥谷倫世, 鈴木千登世, 佐藤恵美子, 眞野成康, 菱沼隆則, 鈴木直人, 佐藤博, 阿部高明, 青木淳賢, 富岡佳久

日本薬学会年会要旨集　132年会　(4)　138-138　2012/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
常染色体劣性乏毛症患者に高頻度に認められたLIPH founder変異とその機能解析

新熊悟, 秋山真志, 夏賀健, 乃村俊史, 有田賢, 西江渉, 清水宏, 井上飛鳥, 青木淳賢

日本皮膚科学会雑誌　122　(2)　379-379　2012/02
Publisher: (公社)日本皮膚科学会
ISSN： 0021-499X
ゼブラフィッシュプロスタノイド受容体の性状解析

告恭史郎, 岩崎亮, 川原敦雄, 井上飛鳥, 青木淳賢, 杉本幸彦

日本生化学会大会プログラム・講演要旨集　84回　2P-0111　2011/09
Publisher: (公社)日本生化学会
ホスホリパーゼA1(PLA1)によって産生される2-アシル型リゾリン脂質の性状解析

首藤啓明, 井上飛鳥, 奥谷倫世, 北村一, 有馬直明, 巻出久美子, 青木淳賢

日本生化学会大会プログラム・講演要旨集　84回　2P-0122　2011/09
Publisher: (公社)日本生化学会
グリセロ脂質de novo合成系で産生されるLPAはミトコンドリアの形態を制御する

大場陽介, 井上貴雄, 櫻木健司, 原直子, 井上飛鳥, 岡敏彦, 石原直忠, 中臺枝里子, 青木淳賢, 三谷昌平, 新井洋由

日本生化学会大会プログラム・講演要旨集　84回　2P-0350　2011/09
Publisher: (公社)日本生化学会
生理活性リゾリン脂質の定量系の開発と産生機構の解析

井上飛鳥, 奥谷倫世, 三枝大輔, 奥平真一, 鈴木直人, 富岡佳久, 青木淳賢

日本生化学会大会プログラム・講演要旨集　84回　3P-0068　2011/09
Publisher: (公社)日本生化学会
ISSN： 0037-1017
新規GPCRシグナル検出法-TGFα切断アッセイ-の開発

石黒純, 井上飛鳥, 有馬直明, 首藤啓明, 青木淳賢

日本生化学会大会プログラム・講演要旨集　84回　2T16a-6　2011/09
Publisher: (公社)日本生化学会
骨形成におけるLPAシグナリングの役割の解明

板井恵理子, 有馬直明, 濱弘太郎, 井上飛鳥, 奥平真一, Chun Jerold, Moolenaar Wouter, 青木淳賢

日本生化学会大会プログラム・講演要旨集　84回　3T8p-16　2011/09
Publisher: (公社)日本生化学会
リゾホスファチジルセリンの細胞接着抑制作用

北村一, 巻出久美子, 井上飛鳥, 青木淳賢

日本生化学会大会プログラム・講演要旨集　84回　3T8p-17　2011/09
Publisher: (公社)日本生化学会
リゾホスファチジルセリンの免疫抑制作用

奥谷倫世, 巻出久美子, 井上飛鳥, 中嶋千紗, 椛島健治, 青木淳賢

日本生化学会大会プログラム・講演要旨集　84回　3T8p-18　2011/09
Publisher: (公社)日本生化学会
LC/MS/MSを用いた脂質関連物質分離分析系の構築

三枝大輔, 芝可奈子, 井上飛鳥, 濱弘太郎, 青木淳賢, 鈴木直人, 富岡佳久

JSBMS Letters　36　(Suppl.)　71-71　2011/08
Publisher: (一社)日本医用マススペクトル学会
ISSN： 1881-5464
【リン脂質代謝と脂質メディエーター研究の最新の成果】 (第2部)リゾリン脂質を中心とした脂質メディエーター新しいリゾリン脂質メディエーターリゾホスファチジルセリン

井上飛鳥, 奥谷, 倫世, 青木淳賢

生化学　83　(6)　518-524　2011/06

ISSN： 0037-1017
リゾホスファチジン酸産生酵素オートタキシンの結晶構造解析

奥平真一, 西増弘志, 濱弘太郎, 三原恵美子, 堂前直, 井上飛鳥, 石谷隆一郎, 高木淳一, 濡木理, 青木淳賢

脂質生化学研究　53　97-100　2011/04
Publisher: 日本脂質生化学会
ISSN： 0285-1520
骨形成におけるLPAシグナリングの役割の解明

板井恵理子, 有馬直明, 濱弘太郎, 井上飛鳥, 奥平真一, Chun Jerold, Moolenaar Wouter, 青木淳賢

脂質生化学研究　53　105-107　2011/04
Publisher: 日本脂質生化学会
ISSN： 0285-1520
ホスホリパーゼA1(PLA1)により産生される2-アシル型リゾリン脂質の分離とその性状解析

首藤啓明, 井上飛鳥, 奥谷倫世, 北村一, 有馬直明, 巻出久美子, 青木淳賢

脂質生化学研究　53　259-263　2011/04
Publisher: 日本脂質生化学会
ISSN： 0285-1520
新規GPCRシグナル検出法-TGFα切断アッセイ-の開発

石黒純, 井上飛鳥, 有馬直明, 首藤啓明, 青木淳賢

日本薬学会年会要旨集　131年会　(3)　82-82　2011/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
生体内におけるリゾホスファチジルセリンの検出

奥谷倫世, 井上飛鳥, 巻出久美子, 青木淳賢

日本薬学会年会要旨集　131年会　(3)　84-84　2011/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
モデル生物を用いたLPAシグナルの作用機序の解析

濱弘太郎, 中永景太, 雪浦弘志, 有馬直明, 奥平真一, 井上飛鳥, 青木淳賢

日本薬学会年会要旨集　131年会　(3)　112-112　2011/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
LC-ESI-MS/MSを用いた生理活性リゾリン脂質の定量系の開発

井上飛鳥, 奥谷倫世, 三枝大輔, 奥平真一, 鈴木直人, 富岡佳久, 青木淳賢

日本薬学会年会要旨集　131年会　(3)　114-114　2011/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
生命応答を制御する脂質マシナリーゼブラフィッシュにおけるリゾホスファチジン酸シグナル(Lipid machineries that regulate biological responses Lysophosphatidic acid signaling in zebra fish)

濱弘太郎, 中永景太, 井上飛鳥, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　4W14-5　2010/12
Publisher: (公社)日本生化学会
LC-ESI-MS/MSをリゾリン脂質定量系の開発

井上飛鳥, 三枝大輔, 奥谷倫世, 鈴木直人, 富岡佳久, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　4T15-1　2010/12
Publisher: (公社)日本生化学会
ISSN： 0037-1017
リゾホスファチジルセリンの活性化リンパ球に対する効果

北村一, 巻出久美子, 井上飛鳥, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　4T15-4　2010/12
Publisher: (公社)日本生化学会
LC-ESI-MS/MSを用いたリゾホスファチジルセリンおよびリゾホスファチジルスレオニンの検出

奥谷倫世, 井上飛鳥, 巻出久美子, 三枝大輔, 富岡佳久, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　3P-0130　2010/12
Publisher: (公社)日本生化学会
ISSN： 0037-1017
炎症反応のマスト細胞活性化におけるリゾホスファチジルセリンの役割

巻出久美子, 奥谷倫世, 井上飛鳥, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　3P-0131　2010/12
Publisher: (公社)日本生化学会
PLA1型リゾリン脂質産生酵素の受容体活性化機構の解析

首藤啓明, 井上飛鳥, 北村一, 石黒純, 有馬直明, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　3P-0133　2010/12
Publisher: (公社)日本生化学会
広範なGPCRシグナルを検出する新規測定法-TGFα切断アッセイ-の開発

石黒純, 井上飛鳥, 有馬直明, 首藤啓明, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　3P-0134　2010/12
Publisher: (公社)日本生化学会
胎生期骨形成におけるLPAシグナリングの役割の解明

板井恵理子, 有馬直明, 濱弘太郎, 井上飛鳥, 奥平真一, Chun Jerold, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　83回・33回　3P-0146　2010/12
Publisher: (公社)日本生化学会
リゾ脂質受容体研究の新展開リゾホスファチジルセリンの産生酵素と受容体

青木淳賢, 北村一, 奥谷倫世, 佐藤侑介, 巻出久美子, 井上飛鳥

脂質生化学研究　52　12-12　2010/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
LC-ESI-MS/MSを用いたリゾリン脂質定量系の開発

井上飛鳥, 小野めぐみ, 三枝大輔, 奥谷倫世, 奥平真一, 鈴木直人, 富岡佳久, 青木淳賢

脂質生化学研究　52　187-190　2010/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
TGFα切断アッセイによるGPCRシグナルの検出

石黒純, 井上飛鳥, 有馬直明, 東山繁樹, 青木淳賢

脂質生化学研究　52　221-224　2010/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
オートタキシントランスジェニックマウスの解析

雪浦弘志, 奥平真一, 大上満, 小野めぐみ, 井上飛鳥, 濱弘太郎, 青木淳賢

脂質生化学研究　52　229-231　2010/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
LC-ESI-MS-MSを用いたリゾホスファチジルセリンの検出

奥谷倫世, 井上飛鳥, 巻出久美子, 三枝大輔, 富岡佳久, 青木淳賢, 青木淳賢

日本薬学会東北支部大会講演要旨集　49th　2010
コンディショナルノックアウトマウスを用いたオートタキシンの機能解析

有馬直明, 大上満, 奥平真一, 井上飛鳥, Moolenaar Wouter, 青木淳賢

日本生化学会大会プログラム・講演要旨集　82回　2T20a-14　2009/09
Publisher: (公社)日本生化学会
リゾホスファチジルセリン受容体P2Y10のリガンド特異性の解析

北村一, 巻出久美子, 奥谷倫世, 井上飛鳥, 有馬直明, 尾谷優子, 大和田智彦, 青木淳賢

日本生化学会大会プログラム・講演要旨集　82回　2T20p-4　2009/09
Publisher: (公社)日本生化学会
膜結合型TGFα切り出しを利用した新規GPCR活性化評価法の開発

井上飛鳥, 石黒純, 有馬直明, 東山繁樹, 青木淳賢

日本生化学会大会プログラム・講演要旨集　82回　2T20p-6　2009/09
Publisher: (公社)日本生化学会
TGFα切断アッセイによるGPCRシグナルの検出

石黒純, 井上飛鳥, 有馬直明, 東山繁樹, 青木淳賢

日本生化学会大会プログラム・講演要旨集　82回　2P-115　2009/09
Publisher: (公社)日本生化学会
新規リゾホスファチジルセリン受容体の同定

奥谷倫世, 北村一, 井上飛鳥, 有馬直明, 尾谷優子, 巻出久美子, 大和田智彦, 青木淳賢

日本生化学会大会プログラム・講演要旨集　82回　2P-117　2009/09
Publisher: (公社)日本生化学会
【情報伝達をつかさどる生理活性物質脂質メディエーターの機能】第二世代の生理活性脂質としてのリゾリン脂質

井上飛鳥, 青木, 淳賢

実験医学　27　(13)　2059-2067　2009/08
模型プロテアーゼ活性化を指標にした新規リゾホスファチジルセリン受容体の同定

奥谷倫世, 北村一, 有馬直明, 井上飛鳥, 巻出久美子, 青木淳賢

生化学　81　(6)　541-541　2009/06
Publisher: (公社)日本生化学会
ISSN： 0037-1017
体毛形成におけるホスファチジン酸特異的ホスホリパーゼA1αの機能解析

有馬直明, 井上飛鳥, 新井洋由, 青木淳賢

日本薬学会年会要旨集　129年会　(3)　74-74　2009/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
リゾリン脂質の新しい機能体毛形成におけるリゾホスファチジン酸の機能解明

井上飛鳥, 有馬直明, 新井洋由, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　81回・31回　1S22-3　2008/11
Publisher: (公社)日本生化学会
ホスファチジン酸特異的ホスホリパーゼA1の体毛形成への関与

井上飛鳥, 有馬直明, 新井洋由, 青木淳賢

脂質生化学研究　50　239-242　2008/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
PS-PLA1とmPA-PLA1αの基質特異性におけるループ構造の関与

有馬直明, 井上飛鳥, 巻出久美子, 青木淳賢

脂質生化学研究　50　243-246　2008/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520
リピッドメディエーターの最前線育毛におけるリゾホスファチジン酸の新規機能

青木淳賢, 井上飛鳥, 新井洋由

日本薬学会年会要旨集　128年会　(1)　110-110　2008/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
ホスファチジン酸特異的ホスホリパーゼA1の体毛形成への関与

井上飛鳥, 有馬直明, 新井洋由, 青木淳賢

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　80回・30回　4T2-3　2007/11
Publisher: (公社)日本生化学会
【脂質の新たな機能を追う】リゾリン脂質メディエーターの作用と生理的役割

巻出久美子, 奥平, 真一, 井上, 飛鳥, 遠藤, 智子, 田中, 将之, 新井, 洋由, 青木淳賢

細胞工学　26　(11)　1251-1259　2007/10
新規ホスファチジン酸特異的ホスホリパーゼA1の着床促進効果

井上飛鳥, 濱弘太郎, 青木淳賢, 新井洋由

脂質生化学研究　48　95-97　2006/05
Publisher: 日本脂質生化学会
ISSN： 0285-1520

Show all ︎Show first 5

Research Projects 15

GPCRシグナルの自在な切り分けから目指す安全性の高い創薬

井上飛鳥

Offer Organization: 科学技術振興機構

System: 戦略的な研究開発の推進創発的研究支援事業

Institution: 東北大学

2022 - 2028

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Gタンパク質共役型受容体(GPCR)は創薬開発の最も重要な作用標的として知られています。GPCRは多様な細胞内情報(シグナル)伝達を引き起こしますが、薬効を担う経路は一部であり他の経路は副作用につながります。本研究では、薬効と副作用のシグナル伝達を分子レベルで理解することを目指します。この研究成果は、薬効を維持しつつ副作用のシグナル伝達を低減した薬、すなわち安全性の高い薬の開発に貢献します。
Structural dynamics of GPCR in cells

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (S)

Institution: The University of Tokyo

2021/07/05 - 2026/03/31
Manipulation of G12/13 signaling and and drug development strategy

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (A)

Institution: Tohoku University

2021/04/05 - 2025/03/31
ミトラガイナアルカロイドを基盤とする副作用を示さないオピオイド作動性鎮痛薬の創製

石川勇人, 井上飛鳥

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 挑戦的研究(萌芽)

Institution: 千葉大学

2023/06 - 2025/03
Multi-scale platform for untangling physiological complexity

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Transformative Research Areas (B)

Institution: University of Tsukuba

2021/08/23 - 2024/03/31
オピオイド受容体の網羅的シグナル解析による薬理経路の同定

井上飛鳥, 生田達也

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 学術変革領域研究(B)

Institution: 東北大学

2021/08/23 - 2024/03/31

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本研究課題の対象の１つであるカッパーオピオイド受容体（KOR）に関しては、基準リガンドであるNalfurafineと比較して、YNT-1612はGタンパク質サブタイプ間でのシグナルバイアスは見られず、アレスチンリクルート活性が低下していた。アレスチンの構造変化やシグナル因子との結合の測定を試みたが、KORの基準リガンド自体がこれらの応答が微弱であり、YNT-1612のバイアス活性を評価することができなかった。また、今後のモデル動物でのYNT-1612の薬効評価を見据えて、マウスとラットのKORに対する活性を評価したところ、ヒトを含めたいずれの動物種のKORに対しても、アレスチン活性が弱く、Gタンパク質バイアス型アゴニストであることがわかった。他方の対象であるデルタオピオイド受容体（DOR）に関しては、基準リガンドであるメチオニンエンケファリンやSNC-80と比べて、KNT-127はGi活性はほぼ同等である一方、他のGタンパク質、特にGqとG12の活性が強いことがわかった。また、アレスチン経路については、KNT-127はアレスチンリクルート活性が弱く、IB30センサーで評価した場合のアレスチンの活性型構造変化が小さいことがわかった。一方、KNT-127はいずれのGRKサブタイプを介してアレスチンをリクルートする活性を示すことがわかった。また、X線結晶構造の安定型改変体として報告のあるDORの九重変異体に関して、KNT-127のGi活性に対する個々の変異の効果を調べたところ、2種類は野生型DORと同等の応答を示し、2種類が活性が低下し、残りの5種類が大きく活性が減弱することがわかった。
A study of novel lysophospholipids and exploration of their targets

Hasegawa Junya

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Tokyo Medical and Dental University

2020/04/01 - 2023/03/31

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For three years, we have been analyzing the physiological and pathophysiological functions of the novel phospholipids "LysoPIPs" which were discovered by the applicants in this research project. We have found several phospholipases A that produce LysoPIPs. We have also devised a method for the preparation of LysoPIPs that is not commercially available, and have now succeeded in purifying LysoPIPs with high purity. Using the purified LysoPIPs, my collaborators screened about 250 G protein-coupled receptors (GPCRs) whose ligands are LysoPIP3, one of the LysoPIPs. As a result, we found that two receptors showed high reactivity.
Study of drug discovery and biomarker for depression based on glial antidepressant receptor (LPA1)

Takebayashi Minoru

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

2018/04/01 - 2023/03/31

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In patients with depression,docosahexaenoic acid-lysophosphatidic acid (DHA-LPA), a lysophospholipid, and autotaxin, an LPA synthase, were decreased and correlated with depressive symptoms. On the other hand, preclinical studies revealed that LPA receptor 1 (LPA1) agonists have antidepressant effects, and a library of existing therapeutic agents was used for primary screening with the LPA1 assay. In summary, we discovered the possibility of abnormal lysophospholipid metabolism in the pathogenesis of depression, and furthermore, identified LPA1 agonists as new drug target molecules, creating the basis for drug repositioning.
Signaling basis of GPCR-acting drugs and their therapeutic mechanisms

Inoue Asuka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tohoku University

2017/04/01 - 2020/03/31

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G-protein-coupled receptors (GPCRs) represent the largest class of human membrane proteins. They are also regarded as important targets for drug development. GPCRs mediate signal information from external stimuli such as hormones and drugs to intracellular responses. Characterizing signal profiling of GPCRs is critical for understanding not only GPCR biology, but also GPCR-targeting drugs. In this project, we developed various methods to assess G-protein-coupling profiles of GPCRs and characterized more than 100 GPCR signaling. Through bioinformatics analysis, this experimental database enabled us to develop an algorithm to score signaling profile based on a given GPCR sequence. These achievement will contribute to development of GPCR-targeting drugs including a signal-biased ligand, which is expected to serve as a safe drug with a reduced side effect.
Understanding lipid-regulated TRP channels and their roles in thermal biology

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Institution: Tohoku University

2016/04/01 - 2018/03/31
A role of lysophosphatidic acid signaling in male organ

Asuka Inoue, KANO Kuniyuki

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tohoku University

2014/04/01 - 2017/03/31

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Lysophoshatidic acid (LPA) is a lipid mediator and regulates various cellular responses via acting on LPA-specific G protein-coupled receptors (LPA1-6). We have previously found that mice deficient for PA-PLA1β, an LPA-producing enzyme, show abnormal structures in male organs including testes and vesicular glands. In this study, we aimed at identifying a LPA receptor that is involved in the PA-PLA1β-LPA axis in the male organogenesis. We also examined defects at a cellular level in the LPA-regulated male organs. We identified approximately a half of LPA6-deficient mice has smaller testes. We also found that mRNA for LPA6 is localized to the spermatogonium, Sertoli cells and Leydig cells in juvenile mice and that the expression decreases as mice become aged. Since the phenotype of PA-PLA1β-deficient mice and LPA6-deficient mice is different from a reported triple LPA1-3-deficient mice, LPA signal has at least two distinct roles in development of male organs.
Gタンパク質共役型受容体の活性化に影響を及ぼす代謝物の同定

井上飛鳥

Offer Organization: 科学技術振興機構

System: 戦略的な研究開発の推進戦略的創造研究推進事業さきがけ

Institution: 東北大学

2013 - 2016

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Gタンパク質共役型受容体(GPCR)は、ヒトにおいて約900種類も存在し、各々が異なる代謝物を認識して様々な生命現象や疾患に関わります。本研究では、GPCRの結合分子を網羅的に探索し、疾患関連分子の作用標的を明らかにします。得られた知見は、疾患のメカニズムを解明するとともに、創薬に大きく貢献することが期待されます。
Screening for ligands of G protein-coupled receptors using the newly developed TGFalpha shedding assay

INOUE Asuka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Young Scientists (B)

Institution: Tohoku University

2012/04/01 - 2014/03/31

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In this KAKENHI project, by employing the newly developed transforming growth factor-alpha (TGFalpha) shedding assay, I screened for G protein-coupled receptors (GPCRs) that respond to bioactive molecules and developed an assay system that can quantify coupling efficiency of heterotrimeric G proteins. I identified three orphan GPCRs (P2Y10, GPR174, A630033H20) as lysophoshadylserine-specific receptors, one GPCR as a lysophoshadylglucoside receptor and two GPCRs as oxidized phospholipid receptors. I generated G protein-deficient HEK293 cells using the CRISPR-Cas9 system and successfully applied them to analyze G protein coupling of GPCRs.
Lysophosphatidic acid regulates hair follicle development

INOUE Asuka

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Young Scientists (B)

Institution: Tohoku University

2009 - 2010

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Lysophosphatidic acid has been established as an important mediator that is involved in various physiological events. Our work is to examine novel roles of lysophosphatidic acid by utilizing genetically engineered mice. Here we found that mice lacking production of lysophophatidic acid exhibited hair defects. We further identified mechanisms underlying the lysophophatidic acid-induced hair formation. Our work will be beneficial to development of medicine for hair disorders.
新規リン脂質分解酵素ホスホリパーゼA1のノックアウトマウスを用いた生理機能の解明

井上飛鳥

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 特別研究員奨励費

2006 - 2008

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リゾホスファチジン酸(LPA)はリゾリン脂質骨格を有する生理活性脂質であり、Gタンパク質共役型受容体を介して様々な細胞応答を引き起こす。これまでに7種類のLPA特異的受容体が報告されており、これらの遺伝子欠損(KO)マウスの解析から、LPAが個体レベルで脳神経系の発達や受精卵の着床過程を制御することが明らかになってきている。最近、LPA受容体のP2Y5およびLPA産生酵素のPA-PLA_1α/LIPHの遺伝子欠損患者が報告され、ともに先天性の貧毛症となることが明らかになった。しかし、どのようなメカニズムで貧毛になるかはまったくわかってない。そこで私は、PA-PLA_1αのKOマウスを用いて、体毛形成におけるLPAの機能の解明を目指して研究を行っている。採用初年度は、KOマウスを作製し全身の体毛が縮れるという形態異常を発見した。2年度は、毛包内のPA-PLA_1αの機能を詳細に検討し毛包組織の内根鞘と呼ばれる層の形成に関与することを見出した。当該年度の3年度は、LPAの下流でEGF受容体シグナルが活性化されることに着目して研究をおこなった。毛包角化細胞の初代培養系を用いてLPAがEGF受容体の活性化を引き起こすこと、P2Y5がEGFリガンドの切断に関与すること、さらにKOマウスの皮膚ではEGF受容体のリン酸化レベルが減少していることを見出した。二の研究を通じて、毛包内の内根鞘でPA-PLA_1αが産生したLPAがP2Y5を介して、EGF受容体シグナルを活性化して体毛の形成に関与するという、新規のメカニズムを明らかにした。

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