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Development of antibody stabilizing active or inactive state of GPCRs.

Research Project

Project/Area Number 22659059
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionKyoto 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 *help
¥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.

Report

(3 results)
  • 2011 Annual Research Report   Final Research Report ( PDF )
  • 2010 Annual Research Report
  • Research Products

    (23 results)

All 2012 2011 2010 Other

All Journal Article (12 results) (of which Peer Reviewed: 12 results) Presentation (7 results) Book (2 results) Remarks (1 results) Patent(Industrial Property Rights) (1 results) (of which Overseas: 1 results)

  • [Journal Article] Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist2012

    • Author(s)
      Haga K., et al
    • Journal Title

      Nature

      Volume: 482 Issue: 7386 Pages: 547-551

    • DOI

      10.1038/nature10753

    • Related Report
      2011 Annual Research Report 2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] G protein-coupled receptor inactivation by an allosteric inverse-agonist antibody2012

    • Author(s)
      Hino T., et al
    • Journal Title

      Nature

      Volume: 482 Issue: 7384 Pages: 237-40

    • DOI

      10.1038/nature10750

    • Related Report
      2011 Annual Research Report 2011 Final Research Report
    • 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

    • Related Report
      2011 Annual Research Report 2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] Cloning, expressionand purification of the anion exchanger 1homologue from basidiomycetePhanerochaete chrysosporium2011

    • 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

    • Related Report
      2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] Evaluation of the Pichiapastoris expression system for theproduction of GPCRs for structuralanalysis2011

    • 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

    • Related Report
      2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] Cysteine-to-serine shuffling using ayeast expression system improves proteinsecretion : case of a nonglycosylatedmutant of miracullin, a taste-modifyingprotein2011

    • 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/

    • Related Report
      2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] Crystallization and preliminary X-rayanalysis of a Glucansucrase from thedental caries pathogen Streptococcusmutans2010

    • 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

    • Related Report
      2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] Bulky high-mannose-type N-glycanblocks the taste-modifying activity ofmiraculin2010

    • 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

    • Related Report
      2011 Final Research Report
    • Peer Reviewed
  • [Journal Article] Characterizationof the・-D-glucopyranoside binding siteof the human bitter taste receptorhTAS2R162010

    • 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

    • Related Report
      2011 Final Research Report
    • 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

    • Related Report
      2010 Annual Research Report
    • 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

    • Related Report
      2010 Annual Research Report
    • 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

    • Related Report
      2010 Annual Research Report
    • 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
    • Related Report
      2011 Final Research Report
  • [Presentation] Ligand Recognition and Molecular Gating GRC in Ventura2012

    • Place of Presentation
      CA, USA
    • Year and Date
      2012-01-20
    • Related Report
      2011 Final Research Report
  • [Presentation] Towards structure based GPCR pharmacology2012

    • Author(s)
      小林拓也
    • Organizer
      Gordon Research Conference
    • Place of Presentation
      米国
    • Related Report
      2011 Annual Research Report
  • [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
    • Related Report
      2011 Final Research Report
  • [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
    • Related Report
      2011 Final Research Report
  • [Presentation] A GFP-basedplatform for rapid construction andevaluation of highly expressed and stable GPCR variants in Saccharomyces cerevisiae2010

    • Organizer
      GPCR Workshop 2010
    • Place of Presentation
      Hawaii
    • Year and Date
      2010-12-09
    • Related Report
      2011 Final Research Report
  • [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
    • Related Report
      2010 Annual Research Report
  • [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
    • Related Report
      2011 Final Research Report
  • [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
    • Related Report
      2010 Annual Research Report
  • [Remarks]

    • URL

      http://www.med.kyoto-u.ac.jp/J/grad_school/introduction/1502/

    • Related Report
      2011 Final Research Report
  • [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
    • Related Report
      2011 Final Research Report
    • Overseas

URL: 

Published: 2010-08-23   Modified: 2016-04-21  

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