G Protein-Coupled Receptors as Cell Sensors

2008 Fiscal Year Final Research Report

• Project/Area Number: 15083207
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Gakushuin University
• Principal Investigator: HAGA Tatsuya Gakushuin University, 理学部, 教授 (30011646)
• Co-Investigator(Kenkyū-buntansha): ICHIYAMA Susumu 学習院大学, 理学部, 助教(当時) (00333336) ; HASHIMOTO Yuichi 学習院大学, 理学部, 助教 (00448953)
• Project Period (FY): 2003 – 2008
• Keywords: ムスカリン性アセチルコリン受容体 / Gタンパク質共役受容体 / 膜タンパク質 / 結晶構造 / 細胞内移行 / リサイクリング / オーファン受容体 / 高親和性コリントランスポーター

Research Abstract

ムスカリン性アセチルコリン受容体M2サブタイプ変異体の結晶化・高次構造の解明を主目的とした。約8Aの回折点を示す結晶が再現的に得られる条件を見いだしたが、原子構造の解明には至らなかった。副次的課題として、ムスカリン受容体の細胞内第3ループがフレキシブルな構造を取ること、ムスカリンM4受容体の細胞内移行には2つ以上の機構が併存すること、ムスカリン受容体のリサイクリングに関わるモチーフの同定、などの結果を得た。

Research Products

• [Journal Article] Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris. (2009)
  • Author(s): Yurugi-Kobayashi T, Asada H, Shiroishi M, Shimamura T, Funamoto S, Katsuta N, Ito K, Sugawara T, Tokuda N, Tsujimoto H, Murata T, Nomura N, Haga K, Haga T, Iwata S, Kobayashi T.,
  • Journal Title: Biochem. Biophys. Res. Commun 380 Pages: 271-276
  • Peer Reviewed
• [Journal Article] Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants (2008)
  • Author(s): Nakamura T, Sakai M, Sadakane Y, Haga T, Goto Y, Kinouchi T, Saito T, Fujii N.,
  • Journal Title: Biochim. Biophys. Acta. 1784 Pages: 1192-1199
• [Journal Article] Muscarinic M4 receptor recycling requires a motif in the third intracellular loop. (2008)
  • Author(s): Hashimoto Y, Morisawa K, Saito H, Jojima E, Yoshida N, Haga T.,
  • Journal Title: J. Pharmacol. Exp. Ther. 325 Pages: 947-953
• [Journal Article] The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype (2006)
  • Author(s): Ichiyama, S., Oka, Y., Haga, K., Kojima, S., Tateichi, Y., Shirakawa, M., and Haga, T.,
  • Journal Title: FEBS Letters 580 Pages: 23-26
• [Journal Article] 5-oxo-eicosatetraenoic acid-induced chemotaxis: identification of a responsible receptor hGPCR48 and negative regulation by G protein G12/13 (2006)
  • Author(s): Koike, D., Obinata, H., Yamamoto, A., Takeda, S., Komori, H., Nara, F., Izumi, T. and Haga, T.,
  • Journal Title: J. Biochem. 139 Pages: 543-549
• [Journal Article] Synthesis and Biological Evaluation of Boronic Acid Containing cis-Stilbenes as Apoptotic Tubulin Polymerization Inhibitors (2006)
  • Author(s): Nakamura, H. *, Kuroda, H., Saito, H., Suzuki, R., Yamori, T., Maruyama, K. and Haga, T.
  • Journal Title: Chem. Med. Chem. 1 Pages: 729-740
• [Journal Article] Clathrin requirement for phosphorylation and internalization of beta2-adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2), (2006)
  • Author(s): Mangmool, S., Haga, T., Kobayashi, H., Kim, K.-M., Nakatya, H., Nishida, M. m and Kurose,H. *,
  • Journal Title: J. Biol. Chem 281 Pages: 31940-31949
• [Journal Article] Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis (2005)
  • Author(s): Terashima, Y., Onai, N., Murai, M., Enomoto, M., Poonpiriya, V., Hamada, T., Motomura, K., Suwa, M., Ezaki, T., Haga, T., Kanegasaki, S., and Matsushima, K.,
  • Journal Title: Nature Immunology 6 Pages: 827-835
• [Journal Article] Klavier (klv), a novel hypernodulation mutant of Lotus japonicus affected in vascular tissue organization and floral induction (2005)
  • Author(s): Oka-Kira, E., Tateno, K., Miura, K., Haga, T., Hayashi, M., Harada, K., Sato, S., Tabata, S., Shikazono, N., Tanaka, A., Watanabe, Y., Fukuhara, I., Nagata, T., and *Kawaguchi, M.,
  • Journal Title: The Plant Journal 44 Pages: 505-515
• [Journal Article] Production of N-lauroylated G protein α-subunit in Sf9 insect cells: the type of N-acyl group of Gα influences G protein-mediated signal (2004)
  • Author(s): Hashimoto, Y., Matsuda, T., Matsuura, Y., Haga, T., and Fukada, Y.
  • Journal Title: transduction, J. Biochem 135 Pages: 319-329
• [Journal Article] Effects of partial agonists and Mg2+ ions on the interaction of M2 muscarinic acetylcholinereceptor and G protein Gαi1 subunit in the M2-Gαi1 fusion protein (2004)
  • Author(s): Zhang, Q., Okamura, M., Guo, Z-D., Niwa, S., and Haga, T.
  • Journal Title: J. Biochem 135 Pages: 589-596
• [Journal Article] The receptor-Gαfusion protein as a tool for ligand screening : a model study using nociceptin receptor-Gαi2 fusion protein. (2004)
  • Author(s): Takeda, S., Okada, T., Okamura, N., Haga, T., Isoyama-Tanaka, J., Kuwahara, H., Minamino, N.
  • Journal Title: J. Biochem., 135 Pages: 597-604
• [Journal Article] Stimulation of increases in intracellular calcium and prostaglandin E2 generation in Chinese hamster ovary cells expressing receptor-Gα16 fusion proteins. (2004)
  • Author(s): Suga, H., Takeda, S., Haga, T., Okamura, M., Takao, K., and Tatemoto, K.
  • Journal Title: J. Biochem., 135 Pages: 605-613
• [Journal Article] Inhibitory role of endophilin 3 in receptor-mediated endocytosis. (2004)
  • Author(s): Sugiura, H., Iwata, K., Matsuoka, M., Hayashi, H., Takemiya, T., Yasuda, S., Ichikawa, M., Yamauchi, T., Mehlen, P., Haga, T., and Yamagata, K.,
  • Journal Title: J. Biol. Chem., 279 Pages: 23343-23348
• [Journal Article] Low pH facilitates uptake of proteins by cells through a non-endocytic pathway. (2004)
  • Author(s): Motizuki, M., Takei, T., Tasaka, K., Yokota, S., Kojima,S., Haga, T., and Tsurugi, K.,
  • Journal Title: J. Biochem. 135 Pages: 713-719
• [Presentation] The high affinity choline transporter: topology and choline binding site as studied by site- directed mutagenesis. (2008)
  • Author(s): Urashima, K., Kubo, Y., Nishikawa, S., Nakamura, T., Hayashi, K., Yamada, H., Osawa, C., Okuda, T., Haga, T.
  • Organizer: Experimental Biology
  • Place of Presentation: San Diego California, USA
  • Year and Date: 20080404-09
• [Presentation] Regulation of the high affinity choline transporter (CHT1) by ubiquitin ligase Nedd4-2 (2008)
  • Author(s): Yamada, H., Imajoh-Ohmi, S.and Haga, T.
  • Organizer: Experimental Biology
  • Place of Presentation: San Diego California, USA
  • Year and Date: 20080404-09
• [Presentation] Regulator of G-protein Signaling (RGS) 2 may bind to the third intracellular loop of the M2 muscarinic acetylcholine receptor. (2008)
  • Author(s): Watanabae, T., Ichiyama, S., Haga, T.
  • Organizer: 日本農芸化学会
  • Place of Presentation: 名古屋/名城大学天白キャンパス
  • Year and Date: 20080326-29
• [Presentation] The role of G protein-coupled receptor kinase 2 in internalization of muscarinic acetylcholine receptor M4 subtype (2008)
  • Author(s): Saito, K., Kakegai, R., Hashimoto, Y., Haga, T.
  • Organizer: 第81回日本薬理学会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 20080317-19
• [Presentation] Experimental evidence for topology of the high affinity choline transporter (CHT1) (2007)
  • Author(s): Urashima, K., Kubo, Y., Yamada, H., Okuda, T., Haga, T.
  • Organizer: 第30回日本分子生物学会年会、第80回日本生化学会大会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 20071211-15
• [Presentation] ヒトαB-クリスタリン変異体中のAsp, Asn 残基のラセミ化反応速度定数の解析と構造活性相関 (2007)
  • Author(s): 中村徹、酒井美世、定金豊、芳賀達也、後藤祐児、藤井紀子
  • Organizer: 第30回日本分子生物学会年会、第80回日本生化学会大会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 20071211-15
• [Presentation] 高親和性コリントランスポ-タ-の負電荷アミノ酸の変異解析 (2007)
  • Author(s): 西川静枝、大澤千恵子、松田弘子、奥田隆志、芳賀達也
  • Organizer: 第80回日本薬理学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 20070314-16
• [Presentation] Topology model for the high-affinity choline transporter by site-directed chemical labeling (2006)
  • Author(s): Kubo, Y., Yamada, H., Okuda, T., Haga, T.
  • Organizer: 第28回日本生物学的精神医学会、第36回日本神経精神薬理学会、第49回日本神経化学会大会合同年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 20060914-16
• [Presentation] Effect of G protein βγ subunits on the interaction of the muscarinic acetylcholine receptor M2 or M4 subtype with G protein Gαi1 subunit (2006)
  • Author(s): Ichiyama, S., Nemoto, R., Tanabe, H., Okamura, M., Furukawa, H., and Haga, T.
  • Organizer: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOB Congress
  • Place of Presentation: Kyoto International Conference Hall, Kyoto, Japan
  • Year and Date: 20060618-23
• [Presentation] A riegion in the third intracellular loop is involved in the recycling of internalized muscarinic acetylcholine receptor M4 subtype (2006)
  • Author(s): Hashimoto, Y., Morisawa, K., Saito, H., Jojima, E., Yoshida, N., and Haga, T.
  • Organizer: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
  • Place of Presentation: Kyoto International Conference Hall, Kyoto, Japan
  • Year and Date: 20060618-23
• [Presentation] Molecular characterization by mutagenesis of the high affinity choline transporter (CHT1) (2006)
  • Author(s): Haga, T., Okuda, T., Kaitsuka, C., Osawa , C., Nishiyama, N., Yamada, H., Nakamura, T., Hayashi, K, Ito, S., Hamano, T., Sato, M., Ushio, T., Nishikawa, S., Kubo, Y., Matsuda, H.
  • Organizer: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
  • Place of Presentation: Kyoto International Conference Hall, Kyoto, Japan
  • Year and Date: 20060618-23
• [Presentation] Kinetic study of racemization at Asp58 and Asp151 residues in recombinant human αA-crystallin (2006)
  • Author(s): Nakamura T., Sadakane Y., Haga, T., Fujii, N.
  • Organizer: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
  • Place of Presentation: Kyoto International Conference Hall, Kyoto, Japan
  • Year and Date: 20060618-23
• [Presentation] Dynamin-dependent internalization and recycling of M4 muscarinic acetylcholine receptors: the role of the third intracellular loop. (2006)
  • Author(s): Hashimoto, Y., Morisawa, K., Saito, H., Jojima, E., Yoshida, N., and Haga, T..
  • Organizer: 12th International Conference on Retinal Proteins
  • Place of Presentation: Awaji Yumebutai International Conference Center, Hyogo, Japan
  • Year and Date: 20060604-08
• [Presentation] Interaction of muscarinic M2 receptor with G protein Gai 1 subunit in their fusion protein expressed in Escherichia coli: the effect of expression level (2006)
  • Author(s): Ichiyama, S., Nemoto, R., Haga, T.
  • Organizer: 12th International Conference on Retinal Proteins第12回レチナ-ルタンパク質国際会議
  • Place of Presentation: Awaji Yumebutai International Conference Center, Hyogo, Japan
  • Year and Date: 20060604-08
• [Presentation] クラスリンはGタンパク質共役受容体キナ-ゼ2(GRK)の活性化に必須である (2006)
  • Author(s): Mangmool, S., 芳賀達也、西田基宏、黒瀬等
  • Organizer: 第79回日本薬理学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 20060308-10
• [Presentation] ヒト-クリスタリンにおけるラセミ化部位の役割 (2005)
  • Author(s): 中村徹、酒井美世、定金豊、芳賀達也、後藤祐児、木野内忠稔、藤井紀子
  • Organizer: 第78回日本生化学会大会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 20051019-22
• [Presentation] ムスカリン性アセチルコリン受容体M4サブタイプのダイナミン依存的細胞内移行 (2005)
  • Author(s): 森澤加乃子、橋本祐一、斉藤宏幸、城島依里、吉田典弘、芳賀達也
  • Organizer: 第78回日本生化学会大会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 20051019-22
• [Presentation] Does the third intracellular loop of the muscarinic acetylcholine receptor have a rigid tertiary structure? (2005)
  • Author(s): Ichiyama, S., Oka, Y., Haga, K., Kojima, S., Tateishi, Y., Shirakawa, M., and Haga, T.
  • Organizer: Gordon Research Conference (Second Messengers & Protein Phosphorylation)
  • Place of Presentation: Biddeford, U.S. A.
  • Year and Date: 20050612-19
• [Presentation] Internalization and Recycling of Muscarinic Acetylcholine Receptor M2 and M4 Subtypes. (2005)
  • Author(s): Hashimoto, Y., Yoshida, N., Jojima, E., Saito, H., Morisawa, K., and Haga, T.
  • Organizer: Gordon Research Conference (Second Messengers & Protein Phosphorylation)
  • Place of Presentation: Biddeford, U.S. A.
  • Year and Date: 20050612-19
• [Presentation] 大腸菌で発現させたムスカリン性アセチルコリン受容体細胞内第3ル-プの精製と構造機能解析 (2005)
  • Author(s): 市山進、岡良彰、小島修一、芳賀達也
  • Organizer: 2005年度日本農芸化学会大会
  • Place of Presentation: 札幌コンベンションセンタ-
  • Year and Date: 20050328-30
• [Book] Springer Publishers (2008)
  • Author(s): Ichiyama, S., and Haga, T.,
  • Publisher: Muscarinic acetylcholine receptor, in Handbook of Neurochemistry and Molecular Neurobiology, 3rd ed., edited by Mikoshiba, K.,
• [Book] 生物物理学ハンドブック IV (2007)
  • Author(s): 佐藤元康,芳賀達也,石渡信一,桂勲,桐野豊,美宅成樹編
  • Total Pages: 236-239
  • Publisher: 朝倉書店
• [Book] Ligand screening system using fusion proteins of G protein-coupled receptors with G protein a subunits (2007)
  • Author(s): Suga, H., and Haga, T.,
  • Total Pages: 140-164
  • Publisher: Neurochemistry International
• [Book] Screening of ligands for human GPCRs by the use of receptor-Gα fusion proteins, in "G protein-coupled receptors: structure, function, and ligand screening" (2006)
  • Author(s): Haga, T. and Takeda, S., Haga, T. and Takeda, S.,
  • Total Pages: 37-66
  • Publisher: Haga, T. and Takeda, S., CRC Press, Taylor & Francis Group
• [Book] Haga, T. and Takeda, S.
  • Author(s): Furukawa, H., Hamada, T., Hirota, H., Ishigur, M., and Haga, T.,
  • Publisher: Determination of steric structure of muscarinic ligands bound to muscarinic acetylcholine receptors: approaches by TRNOE (transferred nuclear overhauser effect), in "G protein-coupled receptors: structure, function, and ligand screening"