Manipulation of G12/13 signaling and and drug development strategy

• Project/Area Number: 21H04791
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 47:Pharmaceutical sciences and related fields
• Research Institution: Tohoku University
• Principal Investigator: 井上 飛鳥 東北大学, 薬学研究科, 教授 (50525813)
• Co-Investigator(Kenkyū-buntansha): 加藤 英明 東京大学, 先端科学技術研究センター, 教授 (80805961)
• Project Period (FY): 2021-04-05 – 2025-03-31
• Project Status: Granted (Fiscal Year 2024)
• Budget Amount: ¥41,730,000 (Direct Cost: ¥32,100,000、Indirect Cost: ¥9,630,000); Fiscal Year 2024: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2023: ¥7,540,000 (Direct Cost: ¥5,800,000、Indirect Cost: ¥1,740,000); Fiscal Year 2022: ¥16,120,000 (Direct Cost: ¥12,400,000、Indirect Cost: ¥3,720,000); Fiscal Year 2021: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
• Keywords: GPCR / G12 / DREADD

Outline of Research at the Start

Gタンパク質共役型受容体（GPCR）は既存薬の約3割の作用標的となる重要な膜タンパク質である。このうち、G12とG13へとシグナル伝達を行うGPCRは解析が進んでおらずこれを標的とした薬は開発されていない。本研究では、G12やG13を自在にシグナルを誘導可能な人工GPCRを開発し、これを疾患モデルにマウスに発現させることで、G12シグナルやG13シグナルが疾患治療効果を有するかを明らかにする。

Outline of Annual Research Achievements

G12共役型デザイナーGPCR(G12D)を肝細胞特異的に発現させたHep-G12Dマウスの個体レベルの代謝変化について、代謝ケージを用いて酸素消費量と二酸化炭素排出量を測定した。デザイナーリガンドを1週間前処置したHep-G12Dマウスにおいて呼吸商が低下していたことから、エネルギー源として脂質が使われることがわかった。ミトコンドリア活性が上昇していることを想定し、デザイナーリガンドを投与したHep-G12Dマウスの肝臓からミトコンドリアを単離し、電子伝達系複合体の活性を測定した。その結果、複合体IVの活性上昇が確認された。さらに、肝臓のRNA-seq解析を行ったところ、酸化的リン酸化が有意に変動する経路として見出された。G12Dを脂肪細胞に特異的に発現させたAdipo-G12Dマウスについて、初代培養脂肪細胞のベージュ誘導系を試したところ、個体での表現型と同様に、デザイナーリガンド処置により脱分化因子Ucp1の発現上昇が見られた。シグナル阻害剤を試したところ、G12経路下流のROCK阻害剤でUcp1発現が抑制された。
アセチルコリンM1受容体とGq複合体の構造情報から、M1骨格デザイナーGPCR(M1D-G12)とGタンパク質のC末端ヘリックスの複合体の分子動力学計算を行ったところ、G12とG13のC末端の結合様式に差異が見られた。Gタンパク質の点置換コンストラクトによる変異体解析により、G12とG13の選択性に関わるGタンパク質の残基を絞り込んだ。さらに、受容体側の変異体解析を行い、G12とG13の活性バランスが変化する変異体を見出した。GPCRの構造解析については、性状のよいFFAR2コンストラクトを構築することができ、Giとの安定的な複合体形成が観察された。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

当初計画に沿って研究を実施し、計画通りの研究成果を得ることができた。当初計画外の成果として、GPCRとGタンパク質の共役選択性のインフォマティクス解析（Matic et al., Nucleic Acids Research 2022; Hauser et al., eLife 2022）、アレスチンの機能制御（Janetzko et al., Cell 2022）、Gs/G13共役型受容体GPR174の構造解析、Giサブタイプ選択性の解析手法の確立（Ono et al., Commun Biol 2023）などの成果を得た。

Strategy for Future Research Activity

Hep-G12Dマウスについて全身でのエネルギー変化が観察されたことから、肝臓におけるG12D活性化により、肝臓由来の代謝制御ホルモンが産生されるという仮説を立て、ヘパトカインに着目した解析を行う。また、肝臓からのリポタンパク質分泌についての解析を進め、初代培養細胞でG12D活性化によるリポタンパク質分泌を評価する実験系を構築する。Adipo-G12Dマウスにおける脂肪細胞のベージュ化について、個体レベルの熱産生に与える役割を寒冷刺激や高脂肪負荷時の体重増減を指標に解析を進める。また、表皮G12D発現マウスを作出し、表皮におけるG12シグナルの機能を探索する。
G13共役型DREADDの開発については、前年度に得られた構造情報を元にした変異体解析を進める。最近報告されたM3D-Gq構造を元にしたG12Dの分子動力学計算を実施する。FFAR2及び他の脂質受容体の構造解析を進め、G12/G13を含めたGタンパク質選択性の構造基盤の解析に取り組む。

Research Products

• [Int'l Joint Research] Stanford University/Thomas Jefferson University/Indiana University(米国)
• [Int'l Joint Research] Chinese University of Hong Kong/Harbin Institute of Technology(中国)
• [Int'l Joint Research] Indian Institute of Technology, Kanpur(インド)
• [Int'l Joint Research] Scuola Normale Superiore(イタリア)
• [Int'l Joint Research] University of Copenhagen(デンマーク)
• [Int'l Joint Research] Stanford University/UCSD(米国)
• [Int'l Joint Research] Scuola Normale Superiore(イタリア)
• [Journal Article] Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels (2023)
  • Author(s): Tatsumi Manae、Kishi Takayuki、Ishida Satoru、Kawana Hiroki、Uwamizu Akiharu、Ono Yuki、Kawakami Kouki、Aoki Junken、Inoue Asuka
  • Journal Title: PLOS ONE Volume: 18 Issue: 1 Pages: e0280448-e0280448
  • DOI: 10.1371/journal.pone.0280448
  • Peer Reviewed / Open Access
• [Journal Article] Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor (2023)
  • Author(s): Xu Jun、Wang Qinggong、Hubner Harald、Hu Yunfei、Niu Xiaogang、Wang Haoqing、Maeda Shoji、Inoue Asuka、Tao Yuyong、Gmeiner Peter、Du Yang、Jin Changwen、Kobilka Brian K.
  • Journal Title: Nature Communications Volume: 14 Issue: 1 Pages: 376-376
  • DOI: 10.1038/s41467-022-35726-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs (2023)
  • Author(s): Ono Yuki、Kawakami Kouki、Nakamura Gaku、Ishida Satoru、Aoki Junken、Inoue Asuka
  • Journal Title: Communications Biology Volume: 6 Issue: 1 Pages: 112-112
  • DOI: 10.1038/s42003-023-04465-2
  • Peer Reviewed / Open Access
• [Journal Article] Structural basis of lysophosphatidylserine receptor GPR174 ligand recognition and activation (2023)
  • Author(s): Liang Jiale、Inoue Asuka、Ikuta Tatsuya、Xia Ruixue、Wang Na、Kawakami Kouki、Xu Zhenmei、Qian Yu、Zhu Xinyan、Zhang Anqi、Guo Changyou、Huang Zhiwei、He Yuanzheng
  • Journal Title: Nature Communications Volume: 14 Issue: 1 Pages: 1012-1012
  • DOI: 10.1038/s41467-023-36575-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Identification and Functional Characterization of Fungal Chalcone Synthase and Chalcone Isomerase (2023)
  • Author(s): Furumura Sho、Ozaki Taro、Sugawara Akihiro、Morishita Yohei、Tsukada Kento、Ikuta Tatsuya、Inoue Asuka、Asai Teigo
  • Journal Title: Journal of Natural Products Volume: 86 Issue: 2 Pages: 398-405
  • DOI: 10.1021/acs.jnatprod.2c01027
  • Peer Reviewed / Open Access
• [Journal Article] Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae (2022)
  • Author(s): Shibata Takeaki、Kawakami Kouki、Kawana Hiroki、Aoki Junken、Inoue Asuka
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 602 Pages: 70-76
  • DOI: 10.1016/j.bbrc.2022.02.098
  • Peer Reviewed
• [Journal Article] Common coupling map advances GPCR-G protein selectivity (2022)
  • Author(s): Hauser Alexander S、Avet Charlotte、Normand Claire、Mancini Arturo、Inoue Asuka、Bouvier Michel、Gloriam David E
  • Journal Title: eLife Volume: 11
  • DOI: 10.7554/elife.74107
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Activation and allosteric regulation of the orphan GPR88-Gi1 signaling complex (2022)
  • Author(s): Chen Geng、Xu Jun、Inoue Asuka、Schmidt Maximilian F.、Bai Chen、Lu Qiuyuan、Gmeiner Peter、Liu Zheng、Du Yang
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 2375-2375
  • DOI: 10.1038/s41467-022-30081-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Cryo-EM structures of the β3 adrenergic receptor bound to solabegron and isoproterenol (2022)
  • Author(s): Nureki Ikko、Kobayashi Kazuhiro、Tanaka Tatsuki、Demura Kanae、Inoue Asuka、Shihoya Wataru、Nureki Osamu
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 611 Pages: 158-164
  • DOI: 10.1016/j.bbrc.2022.04.065
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Effect of Ligands and Transducers on the Neurotensin Receptor 1 Conformational Ensemble (2022)
  • Author(s): Dixon Austin D.、Inoue Asuka、Robson Scott A.、Culhane Kelly J.、Trinidad Jonathan C.、Sivaramakrishnan Sivaraj、Bumbak Fabian、Ziarek Joshua J.
  • Journal Title: Journal of the American Chemical Society Volume: 144 Issue: 23 Pages: 10241-10250
  • DOI: 10.1021/jacs.2c00828
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] PRECOGx: exploring GPCR signaling mechanisms with deep protein representations (2022)
  • Author(s): Matic Marin、Singh Gurdeep、Carli Francesco、De Oliveira Rosa Natalia、Miglionico Pasquale、Magni Lorenzo、Gutkind J Silvio、Russell Robert B、Inoue Asuka、Raimondi Francesco
  • Journal Title: Nucleic Acids Research Volume: 50 Issue: W1 Pages: W598-W610
  • DOI: 10.1093/nar/gkac426
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] 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 (2022)
  • Author(s): Heo Yunseok、Ishimoto Naito、Jeon Ye-Eun、Yun Ji-Hye、Ohki Mio、Anraku Yuki、Sasaki Mina、Kita Shunsuke、Fukuhara Hideo、Ikuta Tatsuya、Kawakami Kouki、Inoue Asuka、Maenaka Katsumi、Tame Jeremy R. H.、Lee Weontae、Park Sam-Yong
  • Journal Title: PLOS Biology Volume: 20 Issue: 8 Pages: e3001714-e3001714
  • DOI: 10.1371/journal.pbio.3001714
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Endogenous ligand recognition and structural transition of a human PTH receptor (2022)
  • Author(s): Kobayashi Kazuhiro、Kawakami Kouki、Kusakizako Tsukasa、Miyauchi Hirotake、Tomita Atsuhiro、Kobayashi Kan、Shihoya Wataru、Yamashita Keitaro、Nishizawa Tomohiro、Kato Hideaki E.、Inoue Asuka、Nureki Osamu
  • Journal Title: Molecular Cell Volume: 82 Issue: 18 Pages: 3468-3483
  • DOI: 10.1016/j.molcel.2022.07.003
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody (2022)
  • Author(s): Baidya Mithu、Chaturvedi Madhu、Dwivedi-Agnihotri Hemlata、Ranjan Ashutosh、Devost Dominic、Namkung Yoon、Stepniewski Tomasz Maciej、Pandey Shubhi、Baruah Minakshi、Panigrahi Bhanupriya、Sarma Parishmita、Yadav Manish K.、Maharana Jagannath、Banerjee Ramanuj、Kawakami Kouki、Inoue Asuka、et al
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 4634-4634
  • DOI: 10.1038/s41467-022-32386-x
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex (2022)
  • Author(s): 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
  • Journal Title: Cell Rep. Volume: 40(11) Issue: 11 Pages: 111323-111323
  • DOI: 10.1016/j.celrep.2022.111323
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Enhanced membrane binding of oncogenic G protein αqQ209L confers resistance to inhibitor YM-254890 (2022)
  • Author(s): Randolph Clinita E.、Dwyer Morgan B.、Aumiller Jenna L.、Dixon Alethia J.、Inoue Asuka、Osei-Owusu Patrick、Wedegaertner Philip B.
  • Journal Title: Journal of Biological Chemistry Volume: 298 Issue: 11 Pages: 102538-102538
  • DOI: 10.1016/j.jbc.2022.102538
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics (2022)
  • Author(s): Janetzko John、Kise Ryoji、Barsi-Rhyne Benjamin、Siepe Dirk H.、Heydenreich Franziska M.、Kawakami Kouki、Masureel Matthieu、Maeda Shoji、Garcia K. Christopher、von Zastrow Mark、Inoue Asuka、Kobilka Brian K.
  • Journal Title: Cell Volume: 185 Issue: 24 Pages: 4560-4573
  • DOI: 10.1016/j.cell.2022.10.018
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Synthesis of unnatural morphinan compounds to induce itch-like behaviors in mice: Towards the development of MRGPRX2 selective ligands (2022)
  • Author(s): Iio Keita、Kutsumura Noriki、Nagumo Yasuyuki、Saitoh Tsuyoshi、Tokuda Akihisa、Hashimoto Kao、Yamamoto Naoshi、Kise Ryoji、Inoue Asuka、Mizoguchi Hirokazu、Nagase Hiroshi
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 56 Pages: 128485-128485
  • DOI: 10.1016/j.bmcl.2021.128485
  • Peer Reviewed
• [Journal Article] Heterotrimeric Gq proteins?act as a switch for GRK5/6 selectivity underlying β-arrestin transducer bias (2022)
  • Author(s): Kawakami Kouki、Yanagawa Masataka、Hiratsuka Suzune、Yoshida Misaki、Ono Yuki、Hiroshima Michio、Ueda Masahiro、Aoki Junken、Sako Yasushi、Inoue Asuka
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 870-870
  • DOI: 10.1038/s41467-022-28056-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Biased agonists of the chemokine receptor CXCR3 differentially signal through Gα _i :β-arrestin complexes (2022)
  • Author(s): Zheng Kevin、Smith Jeffrey S.、Eiger Dylan S.、Warman Anmol、Choi Issac、Honeycutt Christopher C.、Boldizsar Noelia、Gundry Jaimee N.、Pack Thomas F.、Inoue Asuka、Caron Marc G.、Rajagopal Sudarshan
  • Journal Title: Science Signaling Volume: 15 Issue: 726
  • DOI: 10.1126/scisignal.abg5203
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors (2021)
  • Author(s): Pandey Shubhi、Kumari Punita、Baidya Mithu、Kise Ryoji、Cao Yubo、Dwivedi-Agnihotri Hemlata、Banerjee Ramanuj、Li Xaria X.、Cui Cedric S.、Lee John D.、Kawakami Kouki、Maharana Jagannath、Ranjan Ashutosh、Chaturvedi Madhu、Jhingan Gagan Deep、Laporte St?phane A.、Woodruff Trent M.、Inoue Asuka、Shukla Arun K.
  • Journal Title: Molecular Cell Volume: 81 Issue: 22 Pages: 4605-4621.e11
  • DOI: 10.1016/j.molcel.2021.09.007
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors (2021)
  • Author(s): Seibel-Ehlert Ulla、Plank Nicole、Inoue Asuka、Bernhardt Guenther、Strasser Andrea
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Issue: 18 Pages: 9739-9739
  • DOI: 10.3390/ijms22189739
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism (2021)
  • Author(s): Xu Zhenmei、Ikuta Tatsuya、Kawakami Kouki、Kise Ryoji、Qian Yu、Xia Ruixue、Sun Ming-Xia、Zhang Anqi、Guo Changyou、Cai Xue-Hui、Huang Zhiwei、Inoue Asuka、He Yuanzheng
  • Journal Title: Nature Chemical Biology Volume: 18 Issue: 3 Pages: 281-288
  • DOI: 10.1038/s41589-021-00930-3
  • Peer Reviewed / Int'l Joint Research
• [Presentation] 結合様式の違いによる beta アレスチンの機能的、構造的差異 (2022)
  • Author(s): 桑原 莉来、川上 耕季、井上 飛鳥
  • Organizer: 日本生化学会東北支部第88回例会
• [Presentation] G12共役型DREADDを用いたG12シグナルの脂肪肝抑制作用の評価とその機構解析 (2022)
  • Author(s): 荒井魁斗、小野雄基、井上飛鳥
  • Organizer: 第17回 GPCR研究会
• [Presentation] デザイナーGPCRを用いたG12シグナルの表皮における機能解析 (2022)
  • Author(s): 平井夏実、古川史歩、小野雄基、井上飛鳥
  • Organizer: 第95回日本生化学会大会
• [Presentation] Biased ligands and GRK-subtype selectivity: negative regulations of GRK5/6 by Gq and its consequence for β-arrestin functionality (2021)
  • Author(s): Asuka Inoue
  • Organizer: TARGET 2035-Chemical Probes for GPCRs
  • Int'l Joint Research / Invited
• [Presentation] GPCR effector-KO HEK293 cell lines - how to dissect complexity of GPCR signaling (2021)
  • Author(s): Asuka Inoue
  • Organizer: British Pharmacological Society Pharmacology 2021
  • Int'l Joint Research / Invited
• [Presentation] Back and forth between purified and cellular systems for GPCR biology (2021)
  • Author(s): 井上飛鳥
  • Organizer: 第59回日本生物物理学会年会
  • Invited
• [Presentation] 酸化ストレス代謝物の標的膜受容体とその活性化検出 (2021)
  • Author(s): 井上飛鳥
  • Organizer: 第74回日本酸化ストレス学会
  • Invited
• [Remarks] 東北大学分子細胞生化学分野ホームページ
  • URL: http://www.pharm.tohoku.ac.jp/~seika/2020/index.html
• [Remarks] 研究室HP
  • URL: http://www.pharm.tohoku.ac.jp/~seika/2020/index.html