Development of antibody stabilizing active or inactive state of GPCRs.

• Project/Area Number: 22659059
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: General medical chemistry
• Research Institution: Kyoto University
• Principal Investigator: KOBAYASHI Takuya 京都大学, 医学研究科, 講師 (20311730)
• Co-Investigator(Kenkyū-buntansha): YURUGI Takami 京都大学, 医学研究科, 研究員 (90467473) ; NOMURA Norimichi 京都大学, 医学研究科, 助教 (10314246)
• Co-Investigator(Renkei-kenkyūsha): IWATA So 京都大学, 医学研究科, 教授 (60452330)
• Project Period (FY): 2010 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥3,040,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥240,000); Fiscal Year 2011: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2010: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: モノクローナル抗体 / GPCR / X線結晶構造解析 / 立体構造認識抗体 / アデノシンA2a受容体 / 結晶構造解析

Research Abstract

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The adenosine A2a receptor(A2AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A2AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AR in complex with the antibody Fab fragment(Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A2AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the activeβ2-adrenergic receptor structure, but locks A2AR in an inactive conformation.

Research Products

• [Journal Article] Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist (2012)
  • Author(s): Haga K., et al
  • Journal Title: Nature Volume: 482 Issue: 7386 Pages: 547-551
  • DOI: 10.1038/nature10753
  • Peer Reviewed
• [Journal Article] G protein-coupled receptor inactivation by an allosteric inverse-agonist antibody (2012)
  • Author(s): Hino T., et al
  • Journal Title: Nature Volume: 482 Issue: 7384 Pages: 237-40
  • DOI: 10.1038/nature10750
  • Peer Reviewed
• [Journal Article] Structure ofthe human histamine H1 receptor complexwith doxepin. (2011)
  • Author(s): Shimamura, T., Shiroishi, M, Weyand, S., Tsujimoto, H., Winter, G., Katritch, V., Abagyan, R., Cherezov, V., Liu, W., Han, G. W., Kobayashi, T., Stevens, R. C. and Iwata, S
  • Journal Title: Nature Volume: 475 Issue: 7354 Pages: 65-70
  • DOI: 10.1038/nature10236
  • Peer Reviewed
• [Journal Article] Cloning, expressionand purification of the anion exchanger 1homologue from basidiomycetePhanerochaete chrysosporium (2011)
  • Author(s): Tokuda, N., Igarashi, K., Shimamura, T., Yurugi-Kobayashi, T., Shiroishi, M., Ito, K., Sugawara, T., Asada, H., Murata, T., Nomura, N., Iwata, S. and Kobayashi, T.
  • Journal Title: Protein Expr. Purif Volume: 79 Pages: 81-87
  • URL: http://www.sciencedirect.com/science/article/pii/S1046592811000866
  • Peer Reviewed
• [Journal Article] Evaluation of the Pichiapastoris expression system for theproduction of GPCRs for structuralanalysis (2011)
  • Author(s): Asada, H., Uemura, T., Yurugi-Kobayashi, T., Shiroishi, M., Shimamura, T., Tsujimoto, H., Ito, K., Sugawara, T., Nakane, T., Nomura, N., Murata, T., Haga, T. Iwata, S. and Kobayashi, T.
  • Journal Title: Microb. Cell Fact Volume: 10 Pages: 24-24
  • URL: http://www.microbialcellfactories.com/content/10/1/24
  • Peer Reviewed
• [Journal Article] Cysteine-to-serine shuffling using ayeast expression system improves proteinsecretion : case of a nonglycosylatedmutant of miracullin, a taste-modifyingprotein (2011)
  • Author(s): Ito, K., Sugawara, T., Koizumi, A., Nakajima, K., Shimizu-Ibuka, A., Shiroishi, M., Asada, H., Yurugi-Kobayashi, T., Shimamura, T., Asada, T., Misaka, T., Iwata, S., Kobayashi, T. and Abe, K.
  • Journal Title: Biotech. Lett Volume: 33 Pages: 103-107
  • URL: http://www.springerlink.com/content/j624126g77180652/
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-rayanalysis of a Glucansucrase from thedental caries pathogen Streptococcusmutans (2010)
  • Author(s): Ito, K., Ito, S., Shimamura, T., Kawarasaki, Y., Kobayashi, T. and Iwata, S
  • Journal Title: Acta. Crystallogr. Sect. F Volume: 66(Pt9) Pages: 1086-1088
  • URL: http://scripts.iucr.org/cgi-bin/paper?S1744309110029714
  • Peer Reviewed
• [Journal Article] Bulky high-mannose-type N-glycanblocks the taste-modifying activity ofmiraculin (2010)
  • Author(s): Ito, K., Sugawara, T., Koizumi, A., Nakajima, K. I., Shimizu-Ibuka, A., Shiroishi, M., Asada, H., Yurugi-Kobayashi, T., Shimamura, T., Asakura, T., Masuda, K., Ishiguro, M., Misaka, T., Iwata, S., Kobayashi, T. and Abe, K.
  • Journal Title: Biochim. Biophys. Acta Volume: 1800 Pages: 986-992
  • URL: http://www.sciencedirect.com/science/article/pii/S0304416510001625
  • Peer Reviewed
• [Journal Article] Characterizationof the・-D-glucopyranoside binding siteof the human bitter taste receptorhTAS2R16 (2010)
  • Author(s): Sakurai, T., Misaka, T., Ishiguro, M., Masuda, K., Sugawara, T., Ito, K., Kobayashi, T., Matsuo, S., Ishimaru, Y., Asakura, T. and Abe, K.
  • Journal Title: J. Biol. Chem Volume: 285 Pages: 28373-28378
  • URL: http://www.jbc.org/content/285/36/28373.long
  • Peer Reviewed
• [Journal Article] Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin. (2010)
  • Author(s): Ito K., et al.
  • Journal Title: Biochim.Biophys.Acta. Volume: 1800 Pages: 986-992
  • Peer Reviewed
• [Journal Article] Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTASR16. (2010)
  • Author(s): Sakurai T., et al.
  • Journal Title: J.Biol.Chem. Volume: 285 Pages: 28373-28378
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray analysis of a glucansucrase form the dental caries pathogen Streptococcus mutants. (2010)
  • Author(s): Ito K., et al
  • Journal Title: Acta.Crystallogr.Sect.F Struct.Biol.Cryst.Commun. Volume: 66 Pages: 1086-1088
  • Peer Reviewed
• [Presentation] X線結晶構造解析とシステムバイオロジーの融合によるG蛋白質共役受容体(GPCR)の新たな創薬展開 (2012)
  • Organizer: 第85回日本薬理学会年会
  • Place of Presentation: 京都
  • Year and Date: 2012-03-15
• [Presentation] Ligand Recognition and Molecular Gating GRC in Ventura (2012)
  • Place of Presentation: CA, USA
  • Year and Date: 2012-01-20
• [Presentation] Towards structure based GPCR pharmacology (2012)
  • Author(s): 小林拓也
  • Organizer: Gordon Research Conference
  • Place of Presentation: 米国
• [Presentation] 多様な生物種における膜蛋白質の機能理解を目指した新戦略「多様な生物種における膜蛋白質の機能理解を目指した新戦略」 (2011)
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都
  • Year and Date: 2011-09-24
• [Presentation] 構造生物学と薬理学「Gタンパク質共役受容体(GPCR)の三次元結晶構造解析への試み」 (2011)
  • Organizer: 第84回日本薬理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2011-03-24
• [Presentation] A GFP-basedplatform for rapid construction andevaluation of highly expressed and stable GPCR variants in Saccharomyces cerevisiae (2010)
  • Organizer: GPCR Workshop 2010
  • Place of Presentation: Hawaii
  • Year and Date: 2010-12-09
• [Presentation] Yeast expression : a GFP-based platform for rapid construction and evaluation of highly expressed and stable GPCR variants in S.cerevisiae. (2010)
  • Author(s): Takuya Kobayashi
  • Organizer: GPCR Workshop
  • Place of Presentation: Hawaii(USA)(招待講演)
  • Year and Date: 2010-12-09
• [Book] Gタンパク共役受容体(GPCR)の三次元結晶構造解析への試み (2011)
  • Author(s): 小林拓也
  • Total Pages: 10
  • Publisher: シーエムシー出版
• [Book] バイオインダストリーGタンパク質共役受容体(GPCR)の三次元結晶構造解析への試み (2011)
  • Author(s): 小林拓也
  • Publisher: シーエムシー出版
• [Remarks]
  • URL: http://www.med.kyoto-u.ac.jp/J/grad_school/introduction/1502/
• [Patent(Industrial Property Rights)] 膜蛋白質の立体構造を認識するモノクローナル抗体のスクリーニング方法 (2010)
  • Inventor(s): 岩田想・小林拓也・野村紀通
  • Industrial Property Rights Holder: 科学技術振興機構
  • Filing Date: 2010
  • Overseas