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Kouki Kawakami

Section

Graduate School of Pharmaceutical Sciences

Job title

Associate Professor

Degree

Ph.D (Tohoku University)

researchmap

https://researchmap.jp/kkwkm

J-GLOBAL ID

202101001006737978

e-Rad No.

30908421

ORCID

https://orcid.org/0000-0002-2507-8619

Research History 8

2026/04 - Present

Tohoku University　Graduate School of Pharmaceutical Sciences
2026/04 - Present

The University of Tokyo　Research Center for Advanced Science and Technology
2024/02 - 2026/03

The University of Tokyo　Research Center for Advanced Science and Technology
2023/04 - 2026/03

Japan Society for the Promotion of Science
2023/04 - 2024/01

The University of Tokyo　Graduate School of Arts and Sciences
2022/04 - 2023/03

Tohoku University　Graduate School of Pharmaceutical Sciences　Assistant professor
2021/04 - 2022/03

Tohoku University　Graduate School of Pharmaceutical Sciences
2019/04 - 2021/03

Japan Society for the Promotion of Science

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Education 3

Tohoku University　Graduate School of Pharmaceutical Sciences　Division of Bio-Pharmaceutical Science

2018/04 - 2021/03
Tohoku University　Graduate School of Pharmaceutical Sciences　Division of Bio-Pharmaceutical Science

2016/04 - 2018/03
Tohoku University　Faculty of Pharmaceutical Sciences　Department of Pharmaceutical Sciences

2012/04 - 2016/03

Professional Memberships 2

日本薬学会
日本生化学会

Research Interests 1

G-protein-coupled receptor (GPCR)

Research Areas 3

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

Awards 6

井上研究奨励賞

2023/12　井上科学振興財団
日本生化学会東北支部優秀論文賞

2022/05　日本生化学会東北支部
若手優秀発表賞

2021/11　第94回日本生化学会大会
若手優秀発表賞

2019/09　第92回日本生化学会大会
松尾研究奨励賞

2018/07　International GPCR symposium
優秀発表賞

2017/09　第16回次世代を担う若手ファーマ・バイオフォーラム 2017

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Papers 43

Universal pipeline for high-resolution GPCR structure determination. International-journal Peer-reviewed

Asato Kojima, Kouki Kawakami, Naoya Kobayashi, Kazuhiro Kobayashi, Toshiki E Matsui, Kohei Uemoto, Yuzhong Gu, Tomohiro J Narita, Mai Kugawa, Masahiro Fukuda, Hideaki E Kato

Nature structural & molecular biology　2026/08/28

DOI： 10.1038/s41594-026-01869-6 　

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G protein-coupled receptors (GPCRs) regulate human physiology and are major drug targets. Although cryo-electron microscopy has accelerated GPCR structural biology, inactive-state structures remain difficult because current fusion-based strategies often require extensive experimental screening to identify rigid constructs suitable for high-resolution reconstruction. Here we introduce a universal pipeline that integrates an in silico fusion construct screening program, NOAH (nonexperimental, artificial-intelligence-assisted, high-throughput construct screening for structural analysis), with a de novo designed fusion protein, ARK1 (artificially designed fiducial marker). NOAH enabled structure determination of vasopressin V2 receptor bound to the antagonist tolvaptan or partial agonist OPC51803 and bradykinin B2 receptor bound to the antagonist icatibant, revealing receptor activation and inhibition mechanisms. Coupling NOAH to ARK1 improved the V2 receptor-tolvaptan map and enabled high-resolution structures of lysophosphatidic acid receptor 2 bound to Ki16425 and free fatty acid receptor 2 bound to GLPG0974. NOAH-ARK1 minimizes trial-and-error construct optimization and provides a broadly applicable route for GPCR structural analysis and drug discovery.
The dynamic basis of G-protein recognition and activation by a GPCR. International-journal Peer-reviewed

Kazuhiro Kobayashi, Kouki Kawakami, Toshiki E Matsui, Shun Yokoi, Masahiro Fukuda, Tomohiro J Narita, Hiroki Arai, Mai Tambo, Takashi Sumikama, Manae Tatsumi, Keitaro Yamashita, Junki Koyanagi, Mai Kugawa, Hisako Ikeda, Ayumi Sumino, Ayori Mitsutake, Brian K Kobilka, Asuka Inoue, Hideaki E Kato

Nature　2026/03/11

DOI： 10.1038/s41586-026-10228-w 　

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G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes1. Although more than 200 GPCR-G protein complex structures have been determined2, these static snapshots provide limited insight into the dynamics of G-protein association and dissociation. Here we present cryo-electron microscopy structures of human neurotensin receptor type 1 (NTSR1) with minimally modified Go and Gq, showing how the receptor's intracellular surface dynamically rearranges to accommodate each G-protein subtype. Furthermore, time-resolved cryo-electron microscopy analyses of NTSR1-Gi visualized G-protein dissociation processes on GDP/GTP binding. Characterization of more than 20 intermediates, complemented by mutational and computational analyses, identifies four key mechanistic features. First, GDP/GTP induces Gi release from both canonical and non-canonical active conformations with distinct kinetics. Second, NTSR1 uses common intracellular rearrangements to recognize different G-protein subtypes and to promote activation of a single subtype. Third, separation from Gβγ involves stepwise remodelling of the Gα switches I-III. Finally, Gi dissociates from the receptor through a pathway that is distinct from that of Gs, and the canonical and non-canonical NTSR1-Gi complexes further diverge in their dissociation trajectories. These findings provide a comprehensive framework for understanding GPCR signalling dynamics and guiding signal-targeted therapeutic development.
Structural insights into lipid chain-length selectivity and allosteric regulation of FFA2. International-journal Peer-reviewed

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.
Structural basis for ligand recognition and signaling of hydroxy-carboxylic acid receptor 2. International-journal Peer-reviewed

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.
Class B1 GPCR activation by an intracellular agonist. International-journal Peer-reviewed

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.
Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying β-arrestin transducer bias International-journal Peer-reviewed

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

Nature Communications　13　(1)　487-487　2022/12

DOI： 10.1038/s41467-022-28056-7 　

eISSN： 2041-1723
A Rapid and Universal Pipeline for High-Resolution GPCR Structure Determination through In Silico Construct Optimization and de novo Protein Design

Asato Kojima, Kouki Kawakami, Naoya Kobayashi, Kazuhiro Kobayashi, Toshiki E. Matsui, Kohei Uemoto, Yuzhong Gu, Masahiro Fukuda, Hideaki E. Kato

2026/04/06
Publisher: openRxiv
DOI： 10.64898/2026.04.02.716066 　

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Abstract G protein-coupled receptors (GPCRs) are critical regulators of human physiology and major drug targets. Although structural studies have provided valuable insights, determining GPCR structures remains challenging, especially for inactive state receptors. Recent advances in cryo-electron microscopy (cryo-EM) have enabled structural determination of small GPCRs by using fusion partner proteins and binders to increase molecular weight. However, current methods require extensive experimental screening of fusion constructs. Widely adopted strategies, such as BRIL-Fab complexes, also face limitations due to inherent flexibility. Here, we introduce a streamlined and universal pipeline that integrates an in silico fusion construct screening program, NOAH (NOAH: NOn-experimental, AI-assisted High-throughput construct screening), with a de novo designed fusion protein called ARK1 (ARtificially-designed fiducial marKer). We validate the efficacy of NOAH by determining the structures of the vasopressin V2 receptor (V2R) bound to the clinical antagonist tolvaptan and the partial agonist OPC51803, as well as the bradykinin B2 receptor (B2R) bound to the clinical antagonist icatibant, thereby elucidating their activation and deactivation mechanisms. Furthermore, we demonstrate the capability of NOAH-ARK1 by solving the tolvaptan-bound V2R structure at higher resolution and showcase the method’s versatility by determining the structure of lysophosphatidic acid receptor 2 (LPA2) bound to the antagonist Ki16425. This approach eliminates the need for time-consuming and labor-intensive construct optimization, providing a rapid and widely applicable solution for high-resolution GPCR structure determination and drug discovery.
Membrane-domain compartmentalization of active GPCRs by β-arrestins through PtdIns(4,5)P2 binding. International-journal Peer-reviewed

Ritsuki Kuramoto, Tatsuya Ikuta, Carlo Marion C Carino, Kouki Kawakami, Miisha Kushiro, Chihiro Watanabe, Yasunori Uchida, Mitsuhiro Abe, Yasushi Sako, Tomohiko Taguchi, Masataka Yanagawa, Asuka Inoue

Nature chemical biology　2025/08/06

DOI： 10.1038/s41589-025-01967-4 　

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Upon ligand-induced activation, G-protein-coupled receptors (GPCRs) recruit β-arrestins (βarrs) to the plasma membrane, where phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) stabilizes the GPCR-βarr complex. Although PtdIns(4,5)P2 is reported to form nanoscale clusters, the spatiotemporal dynamics of how the GPCR-βarr-PtdIns(4,5)P2 complex assembles and organizes in living cells remain unexplored. Here we demonstrate that multiple PtdIns(4,5)P2-binding sites on βarrs cooperatively promote GPCR-βarr assembly in membrane domains. Using molecular dynamics simulations, we identify a noncanonical (NC) PtdIns(4,5)P2-binding site, distinct from the known canonical site. Biochemical assays confirm that both sites are essential for βarr binding to PtdIns(4,5)P2-containing liposomes, while NanoBiT assays reveal synergistic contributions of both sites for βarr recruitment in living cells. Notably, single-molecule imaging demonstrates that the NC site is required for the rapid accumulation of the GPCR-βarr-PtdIns(4,5)P2 complex into immobile membrane domains upon ligand stimulation. Collectively, our findings highlight how multivalent βarr-PtdIns(4,5)P2 interactions drive GPCR-βarr compartmentalization, adding complexity to GPCR signaling dynamics.
Insights into G-protein coupling preference from cryo-EM structures of Gq-bound PTH1R. International-journal Peer-reviewed

Fumiya K Sano, Kota Shimizume, Kazuhiro Kobayashi, Toshikuni Awazu, Kouki Kawakami, Hiroaki Akasaka, Takaaki A Kobayashi, Tatsuki Tanaka, Hiroyuki H Okamoto, Hisato Hirano, Tsukasa Kusakizako, Wataru Shihoya, Yoshiaki Kise, Yuzuru Itoh, Ryuichiro Ishitani, Yasushi Okada, Yasushi Sako, Masataka Yanagawa, Asuka Inoue, Osamu Nureki

Nature chemical biology　2025/06/26

DOI： 10.1038/s41589-025-01957-6 　

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The parathyroid hormone type 1 receptor (PTH1R) is a prototypical class B1 G-protein-coupled receptor that couples to both Gq and Gs, having a crucial role in calcium homeostasis and serving as a therapeutic target for osteoporosis. Therapies targeting PTH1R face challenges because of Gq-associated prolonged signaling, which leads to bone resorption. To address this, selective activation of Gs signaling is desirable. However, the structural basis of Gq-mediated signaling remains unclear, limiting the development of signal-selective drugs. Here, we present cryo-electron microscopy structures of the PTH1R-Gq complex in two distinct extracellular conformations, demonstrating the role of N-linked glycans at N1761.28 in stabilizing the ligand-tilted conformation. Comparison with a Gs-bound PTH1R structure highlights the role of key interactions involving both the C terminus of Gα and the receptor's intracellular loop 2 in Gq signaling. These structural insights provide a foundation for understanding the molecular mechanisms of PTH1R signaling.
A molecular mechanism to diversify Ca²⁺ signaling downstream of Gs protein-coupled receptors Peer-reviewed

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)　2024/12

DOI： 10.1038/s41467-024-51991-6 　

eISSN： 2041-1723
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　177151-177151　2024/11/21

DOI： 10.1016/j.ejphar.2024.177151 　

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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.
A cAMP-biosensor-based assay for measuring plasma arginine-vasopressin levels. International-journal Peer-reviewed

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.
Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor. International-journal Peer-reviewed

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.
Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants. International-journal Peer-reviewed

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.
Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system. International-journal Peer-reviewed

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.
Structural basis of CXC chemokine receptor 1 ligand binding and activation. International-journal Peer-reviewed

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.
Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor. International-journal Peer-reviewed

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.
Phosphorylation barcodes direct biased chemokine signaling at CXCR3. International-journal Peer-reviewed

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.
Structural basis of lysophosphatidylserine receptor GPR174 ligand recognition and activation. International-journal Peer-reviewed

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.
Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs. International-journal Peer-reviewed

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.
Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels. International-journal Peer-reviewed

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.
Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody International-journal Peer-reviewed

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
Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics International-journal Peer-reviewed

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
Endogenous ligand recognition and structural transition of a human PTH receptor. International-journal Peer-reviewed

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.e5　2022/08/04

DOI： 10.1016/j.molcel.2022.07.003 　

ISSN： 1097-2765

eISSN： 1097-4164
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 Peer-reviewed

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 　

ISSN： 1544-9173

eISSN： 1545-7885
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 Peer-reviewed

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
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 Peer-reviewed

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
An intrabody sensor to monitor conformational activation of β-arrestins International-journal Peer-reviewed

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
Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism International-journal Peer-reviewed

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
Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors International-journal Peer-reviewed

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, Jagannath Maharana, Ashutosh Ranjan, Madhu Chaturvedi, Gagan Deep Jhingan, Stéphane A. Laporte, Trent M. Woodruff, Asuka Inoue, Arun K. Shukla

Molecular Cell　81　(22)　4605-4621.e11　2021/11
Publisher: Elsevier BV
DOI： 10.1016/j.molcel.2021.09.007 　

ISSN： 1097-2765

eISSN： 1097-4164
Smoothened transduces hedgehog signals via activity-dependent sequestration of PKA catalytic subunits International-journal Peer-reviewed

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
N⁶-methyladenosine (m⁶A) is an endogenous A3 adenosine receptor ligand Peer-reviewed

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.e7　2021/02/18

DOI： 10.1016/j.molcel.2020.12.038 　

ISSN： 1097-2765

eISSN： 1097-4164
Toward understanding the role of G-protein signaling Peer-reviewed

Ryoji Kise, Yuki Ono, Kouki Kawakami, Asuka Inoue

Current Opinion in Endocrine and Metabolic Research　16　51-55　2021/02

DOI： 10.1016/j.coemr.2020.08.006 　

eISSN： 2451-9650
Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gβγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs Peer-reviewed

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
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation 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)　2020/09/03

DOI： 10.15252/embr.201949886 　

ISSN： 1469-221X

eISSN： 1469-3178
Agonist-induced formation of unproductive receptor-G<inf>12</inf> complexes Peer-reviewed

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
Illuminating G-Protein-Coupling Selectivity of GPCRs 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.e25　2019/06/13

DOI： 10.1016/j.cell.2019.04.044 　

ISSN： 0092-8674

eISSN： 1097-4172
Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes 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

Journal of Experimental Medicine　216　(7)　1582-1598　2019

DOI： 10.1084/jem.20181895 　

ISSN： 0022-1007

eISSN： 1540-9538
Lack of beta-arrestin signaling in the absence of active G proteins 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)　2018/12/01

DOI： 10.1038/s41467-017-02661-3 　

eISSN： 2041-1723
A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias 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)　2017/12/01

DOI： 10.1038/s41598-017-14096-3 　

eISSN： 2045-2322
Genetic evidence that b-arrestins are dispensable for the initiation of b<inf>2</inf>-adrenergic receptor signaling to ERK 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
Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis Peer-reviewed

Thomas J. Cahill, 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, 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

eISSN： 1091-6490

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Misc. 78

Development of a Comprehensive GPCR Screening System for Ligand Deorphanization

松下葵, 川上耕季, 福永彩美, 加藤英明, 加藤英明

日本薬学会年会要旨集(Web)　146th　2026

ISSN： 0918-9823
Comprehensive analysis toward understanding the molecular basis of GPCR internalization pathways

成田智裕, 成田智裕, 川上耕季, 福永彩美, 加藤英明, 加藤英明

日本薬学会年会要旨集(Web)　146th　2026

ISSN： 0918-9823
Innovations in GPCR structural pharmacology: Accelerating structure determination and unraveling signaling dynamics

川上耕季, 加藤英明

日本薬学会年会要旨集(Web)　146th　2026

ISSN： 0918-9823
Development of novel small-molecule bradykinin 2 receptor agonists via structure-based antagonist-to-agonist switching

池田恵美, 池田恵美, 新開有紗, 新開有紗, GU Yuzhong, 小林和弘, 小林和弘, 川上耕季, 川上耕季, 加藤英明, 加藤英明, 斉藤毅, 斉藤毅

日本薬学会年会要旨集(Web)　146th　2026

ISSN： 0918-9823
Structural insights into allosteric activation and inactivation mechanisms of FFA2

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

PROTEIN SCIENCE　34　2025/11/27

ISSN： 0961-8368

eISSN： 1469-896X
GPCR内在化経路の再評価とその薬理学的意義

成田智裕, 成田智裕, 川上耕季, 加藤英明, 加藤英明

日本生化学会大会(Web)　98th　2025
社会性行動を制御するバソプレシン・オキシトシン交差認識の理解

福永彩美, 福永彩美, 川上耕季, 度会晃行, 小柳淳暉, 福田昌弘, 小林和弘, 松井俊貴, GENG Lang, SCOTT Daniel, LI Yulong, 奧山輝大, 奧山輝大, 奧山輝大, 加藤英明, 加藤英明

日本生化学会大会(Web)　98th　2025
GPCR内在化経路の再評価とその薬理学的意義

成田智裕, 成田智裕, 川上耕季, 加藤英明, 加藤英明

日本生化学会大会(Web)　98th　2025
Analysis of GPCR internalization pathways

成田智裕, 川上耕季, 加藤英明, 加藤英明

日本薬学会年会要旨集(Web)　145th　2025

ISSN： 0918-9823
14-3-3-GPCR間相互作用誘導機構およびその機能解析

カリニョカーロマリオンコドッグ, 桑原莉来, 川上耕季, 柳川正隆, 井上飛鳥, 井上飛鳥

日本生化学会大会(Web)　97th　2024
14-3-3とGPCRの相互作用の制御機構とその機能解析

CARINO Carlo Marion Codog, 桑原莉来, 川上耕季, 柳川正隆, 井上飛鳥, 井上飛鳥

日本分子生物学会年会プログラム・要旨集(Web)　47th　2024
Molecular mechanism of neuropeptide cross-recognition by the vasopressin receptor

福永彩美, 川上耕季, 小柳淳暉, 福田昌弘, 辻村真樹, 小林和弘, 松井俊貴, SCOTT Daniel J., 加藤英明, 加藤英明

日本薬学会年会要旨集(Web)　144th　2024

ISSN： 0918-9823
Analysis of the Molecular Basis of Adverse Effects of Mu-opioid Receptor Agonists

CARINO Carlo Marion Codog, 平塚寿々音, 中村楽, 川上耕季, 柳川正隆, 井上飛鳥

日本薬学会年会要旨集(Web)　144th　2024

ISSN： 0918-9823
Ligand recognition and activation mechanisms of Free Fatty Acid Receptor 2

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

日本薬学会年会要旨集(Web)　144th　2024

ISSN： 0918-9823
新規水溶性ブラジキニン2受容体作動薬の創製

新開有紗, 新開有紗, GU Yuzhong, 川上耕季, 加藤英明, 斉藤毅, 斉藤毅

GPCR研究会プログラム・抄録集　18th　2024
β-arrestinのPIP₂新規結合部位の機能解析

倉本律輝, 生田達也, コドックカリニョ・カーロ・マリオン, 川上耕季, 内田安則, 田口友彦, 柳川正隆, 井上飛鳥

GPCR研究会プログラム・抄録集　18th　2024
ニューロテンシン受容体によるGタンパク質活性化過程の可視化

川上耕季, 小林和弘, 松井俊貴, 福田昌弘, 新井大輝, 辰己茉菜絵, 小柳淳暉, 草木迫司, JANETZKO John, 山下恵太郎, 濡木理, KOBILKA Brian, 井上飛鳥, 加藤英明

GPCR研究会プログラム・抄録集　18th　2024
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
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(Web)　96th　[1T07a-346)]　2023
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, 加藤英明, 加藤英明

日本生化学会大会(Web)　96th　[2P-080]　2023
Publisher: (公社)日本生化学会
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

バソプレシン・オキシトシン研究会プログラム・抄録集　33rd　2023
Development of a rapid and universal method to determine GPCR structures

Asato Kojima, Matsui Toshiki, Kobayashi Naoya, Fukuda Masahiro, Seiwa Nakamura, Kawakami Kouki, Kobayashi Kazuhiro, Kato Hideaki

Proceedings for Annual Meeting of The Japanese Pharmacological Society　97　1-B-SS04-5　2023
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.97.0_1-b-ss04-5 　

eISSN： 2435-4953

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More than 30% of drugs exert their effects by modulating the activity of G protein-coupled receptors (GPCRs) as agonists or antagonists. To understand the working mechanism of drugs and design new ones, the 3D structural information on therapeutic target proteins is crucial. However, every previous approach to determine the structures of GPCRs requires time-consuming experimental screenings of the expression construct for each target. This process significantly hinders high-throughput structural analysis of GPCRs. Moreover, there is no universal strategy for cryo-EM analysis of GPCRs in both agonist- and antagonist-bound forms. Here, I present a new method for rapid cryo-EM structure determination of GPCRs, called NOAH (NOvel AI-assisted High-throughput construct screening for structural analysis). NOAH is a program that automatically generates the expression constructs of soluble protein-fused GPCRs suitable for cryo-EM analysis. By employing the NOAH pipeline, we can skip the process of experimental screening, saving a significant amount of time and resources on the project. As a proof-of-concept experiment, I applied this method to three GPCRs and determined not only the antagonist-bound structures but also an agonist-bound structure, demonstrating NOAH’s potential to facilitate GPCR structural biology and drug discovery.
Analysis of αN helix function in Gα protein activation

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

ISSN： 0918-9823
短鎖脂肪酸受容体FFAR2のリガンド認識機構と活性化機構の解明

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, 加藤英明

日本蛋白質科学会年会プログラム・要旨集　23rd (CD-ROM)　2023
バソプレシン受容体1aによる神経ペプチドホルモン認識の構造基盤

福永彩美, 小柳淳暉, 福田昌弘, 川上耕季, 松井俊貴, 小林和弘, SCOTT Daniel J., 井上飛鳥, 加藤英明, 加藤英明

日本蛋白質科学会年会プログラム・要旨集　23rd (CD-ROM)　2023
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
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
Structural and functional basis of β-arrestin engagement with GPCRs

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(Web)　142年会　26N-am07S　2022/03
Publisher: (公社)日本薬学会
ISSN： 0918-9823
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　95th　2T12a-07　2022
Publisher: (公社)日本生化学会
Gαタンパク質を起点したGRK-βアレスチン機能制御機構

川上耕季, 柳川正隆, 平塚寿々音, 佐甲靖志, 井上飛鳥

GPCR研究会プログラム・抄録集　17th　2022
第三の結合様式におけるβアレスチンの機能の解明

桑原莉来, 川上耕季, 倉本律輝, 生田達也, 井上飛鳥

GPCR研究会プログラム・抄録集　17th　2022
Discovering GPCR genomic variants unique to Japanese people

鈴木璃子, 生田達也, 川上耕季, 井上飛鳥

日本分子生物学会年会プログラム・要旨集(Web)　45th　2022
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.
分子から迫る神経薬理学 Gタンパク質共役型受容体のシグナル伝達-バイアスシグナル創薬の黎明

川上耕季, 井上飛鳥

Clinical Neuroscience　40　(4)　401-403　2022
Publisher: (株)中外医学社
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)　557-559　2022
Publisher: (株)中外医学社
ISSN： 0289-0585
Toward understanding the role of G-protein signaling

Ryoji Kise, Yuki Ono, Kouki Kawakami, Asuka Inoue

Current Opinion in Endocrine and Metabolic Research　16　51-55　2021/02

DOI： 10.1016/j.coemr.2020.08.006 　

eISSN： 2451-9650
Molecular mechanisms underlying μ-opioid receptor agonist-induced adverse effects

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

日本薬学会年会要旨集(Web)　141st　27V09-pm19S　2021
Publisher: (公社)日本薬学会
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　[1T13e-680)]　2021
Publisher: (公社)日本生化学会
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　[P-566]　2021
Publisher: (公社)日本生化学会
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　[P-541]　2021
Publisher: (公社)日本生化学会
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　[1T15m-194)]　2021
Publisher: (公社)日本生化学会
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, 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, 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

日本生化学会大会プログラム・講演要旨集　94th　[P-546]　2021
Publisher: (公社)日本生化学会
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　[P-599]　2021
Publisher: (公社)日本生化学会
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
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　[P-422]　2020
Publisher: (公社)日本生化学会
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　[1Z10-420)]　2020
Publisher: (公社)日本生化学会
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　[P-424]　2020
Publisher: (公社)日本生化学会
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　[2Z11-434)]　2020
Publisher: (公社)日本生化学会
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, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢, 青木淳賢

日本薬学会年会要旨集(CD-ROM)　139th　(3)　64-64　2019
Publisher: (公社)日本薬学会
ISSN： 0918-9823
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　[2P-207]　2019
Publisher: (公社)日本生化学会
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　[3T07m-03]　2019
Publisher: (公社)日本生化学会
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
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, 井上飛鳥, 川上耕季, 木瀬亮次, 木瀬亮次, 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　2018
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, 井上飛鳥, 青木淳賢, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　91st　[1P-216]　2018
Publisher: (公社)日本生化学会
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
Gαi欠損HEK293細胞の作製と評価

小野雄基, 井上飛鳥, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　91st　[1P-219]　2018
Publisher: (公社)日本生化学会
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, 井上飛鳥, 木瀬亮次, 川上耕季, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　90th　2017
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
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, 井上飛鳥, 青木淳賢, 青木淳賢

日本細胞生物学会大会(Web)　69th　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

日本薬学会年会要旨集(CD-ROM)　137th　2017
三量体Gタンパク質の活性化センサーの開発

井上飛鳥, 井上飛鳥, KADJI Francois Marie, 川上耕季, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　90th　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
Gαi欠損HEK293細胞の作成

小野雄基, 井上飛鳥, 井上飛鳥, 井上飛鳥, 石田覚, 川上耕季, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　90th　2017
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, 井上飛鳥, 井上飛鳥, 柴田剛明, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　90th　2017
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, 井上飛鳥, 井上飛鳥, 柴田剛明, 金藤奨, 青木淳賢, 青木淳賢

日本薬学会年会要旨集(CD-ROM)　137th　2017
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, 井上飛鳥, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　89th　2016
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
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
GPCRシグナル解析ツールの開発

石田覚, 井上飛鳥, 井上飛鳥, 新上雄司, 川上耕季, 青木淳賢, 青木淳賢

日本生化学会大会(Web)　88th　2015

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Industrial Property Rights 4

TSH受容体に対する自己抗体活性の測定方法及びキット

川嵜淳史, 保科元気, 青木淳賢, 川上耕季, 井上飛鳥

特許第7211596号

Property Type: Patent
TSH受容体に対する自己抗体活性の測定方法及びキット

川嵜淳史, 保科元気, 青木淳賢, 川上耕季, 井上飛鳥

Property Type: Patent
改変型バソプレシン受容体を発現する培養細胞

土居耕介, 井上飛鳥, 生田達也, 川上耕季

Property Type: Patent
AVP受容体及びその利用

土居耕介, 井上飛鳥, 生田達也, 川上耕季

Property Type: Patent

Research Projects 6

局所pHシグナルの人為的操作・計測技術の開発

加藤英明, 川上耕季

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(A)

Institution: 東京大学

2025/04 - 2030/03
Gタンパク質シグナル阻害ツールセットの創出

川上耕季

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(B)

Institution: 東京大学

2026/04/01 - 2029/03/31
GPCR低分子創薬を加速する革新的DELスクリーニングプラットフォーム開発研究

加藤英明, 川上耕季

Offer Organization: 日本医療研究開発機構

System: 創薬基盤推進研究事業

2026/04 - 2029/03
コア型βアレスチンの機能とその分子基盤の解明

川上耕季

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 若手研究

Institution: 東京大学

2024/04 - 2026/03
GPCR網羅的な内在化経路の同定と内在化経路選択の構造基盤解明

川上耕季

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特別研究員奨励費

Category: 特別研究員奨励費

Institution: 東京大学

2023/04 - 2026/03
易活性化型変異を導入した人工Ｇタンパク質共役型受容体の創製と応用

川上耕季

Offer Organization: 日本学術振興会

System: 科学研究費助成事業特別研究員奨励費

Category: 特別研究員奨励費

Institution: 東北大学

2019/04 - 2021/03

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本研究においては、これまでに同定している多数のGPCRの共通に活性化させる変異(BW:3.43Q)を、GPCR解析ツールとして応用し、未解明のGPCR研究の課題に取り組む。その前段階として、より多くのGPCRを活性化させる変異の適用拡大を図った。まず、我々がこれまでに解析してきた多数のGPCRのシグナル解析の結果に加え、ClassAに属するGPCRの配列アライメント、既報のGPCR三次元構造の情報を組み合わせることで、GPCR活性化、Gタンパク質共役に関わるアミノ酸位置とアミノ酸の性質の網羅的同定と、活性化、Gタンパク質共役を予測する配列アルゴリズムの構築(Inoue et al., Cell 177, 1-15 (2019))を行なった。この解析から、多くのGPCR活性化に関わると想定されるアミノ酸位置や、特定のシグナルを伝達する上で重要と推測されるアミノ酸位置を抽出した。その結果、3.43Q以外にもGPCRを活性化、さらには、特定のシグナルのみを活性化しうる変異の候補が得られた。その候補変異を導入したDNAコンストラクトを作成し、HEK293細胞に発現させ、シグナルアッセイを行い、実際に特定のシグナルを高選択的に伝達する変異の同定に至っている。さらに、前述の3.43Qが与えるGPCR活性化に与える影響を解析を行なった。特に既報の構造情報から、不活性化状態、活性化状態における構造変化から、相互作用するアミノ酸位置と性質に着目した。3.43Q変異に加え、着目した3箇所の変異をさらに加えることで、NTS1(ニューロテンシン受容体)やEP4(プロスタグランジンE2受容体)など、リガンド既知GPCRにおいて、より多くのGPCRに適用可能な、より強力に活性化を誘導可能な変異の同定に至った。

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