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G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

Nature volume 482, pages 237–240 (2012)Cite this article

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Abstract

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states1,2,3,4,5,6,7,8,9,10,11,12,13. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain14. Here we report the raising of a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AAR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A2AAR 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 structure1 and to CDR-3 of the nanobody in the active β2-adrenergic receptor structure2, but locks A2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

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Figure 1: Effect of Fab2838 on A 2A AR–ligand binding.
Figure 2: Structure of the A 2A AR–Fab2838 complex.
Figure 3: Comparison of the structures of the opsin–GαCT, β 2 AR–Nb80 and A 2A AR–Fab2838 complexes.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the A2AAR–Fab structure have been deposited in the Protein Data Bank under the accession codes 3VG9 (2.7Å) and 3VGA (3.1Å).

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Acknowledgements

This work was supported by the ERATO Human Receptor Crystallography Project of the Japan Science and Technology Agency (S.I.), by the Targeted Proteins Research Program of MEXT, Japan (S.I.), and by Development of New Functional Antibody Technologies (New Energy and Industrial Technology Development Organization, Japan) (T. Hamakubo). It was also partly funded by the Biotechnology and Biological Sciences Research Council (BB/G023425/1) (S.I.). The work was partly performed in the Membrane Protein Laboratory funded by the Wellcome Trust (grant 062164/Z/00/Z) (S.I.) at the Diamond Light Source, UK. Data were collected at Diamond Light Source beamline I24 with the assistance of G. Evans, R. Owen, D. Axford and J. Aishima.

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Author notes

  1. Tomoya Hino

    Present address: Present address: Department of Biotechnology, Graduate School of Engineering, Tottori University, 4-101, Koyama-cho minami, Tottori, 680-8552, Japan.,

  2. Tomoya Hino, Takatoshi Arakawa, Hiroko Iwanari and Takami Yurugi-Kobayashi: These authors contributed equally to this work.

Authors and Affiliations

  1. Iwata Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan,

    Tomoya Hino, Takatoshi Arakawa, Takami Yurugi-Kobayashi, Chiyo Ikeda-Suno, Simone Weyand, Tatsuro Shimamura, Norimichi Nomura, Alexander D. Cameron, Takuya Kobayashi, So Iwata & Takeshi Murata

  2. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan,

    Tomoya Hino, Takatoshi Arakawa, Takami Yurugi-Kobayashi, Chiyo Ikeda-Suno, Tatsuro Shimamura, Norimichi Nomura, Takuya Kobayashi, So Iwata & Takeshi Murata

  3. Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan,

    Hiroko Iwanari, Yoshiko Nakada-Nakura, Osamu Kusano-Arai & Takao Hamakubo

  4. Perseus Proteomics Inc, 4-7-6 Komaba, Meguro, Tokyo 153-0041, Japan,

    Yoshiko Nakada-Nakura

  5. Institute of Immunology Co. Ltd, 1-1-10 Koraku, Bunkyo, Tokyo 112-0004, Japan,

    Osamu Kusano-Arai

  6. Division of Molecular Bioscience, Membrane Protein Crystallography Group, Imperial College London, Exhibition Road, London SW7 2AZ, UK,

    Simone Weyand, Alexander D. Cameron & So Iwata

  7. Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot OX11 0DE, UK,

    Simone Weyand, Alexander D. Cameron & So Iwata

  8. Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot OX11 0FA, UK,

    Simone Weyand, Alexander D. Cameron & So Iwata

  9. Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan,

    Takuya Kobayashi

  10. Systems and Structural Biology Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan,

    So Iwata & Takeshi Murata

  11. Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan,

    Takeshi Murata

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Contributions

S.I. and T.M. designed the original research project. T.Y.-K. and T.K. established the A2AAR expression and purification protocols. T. Hino and C.I.-S. expressed, purified and characterized the receptor. H.I., Y.N.-N., O.K.-A. and T. Hamakubo performed the immunization, selection and isolation of antibodies. T. Hino, T.A. and C.I.-S. purified and characterized antibodies. N.N. sequenced antibodies. T. Hino, T.A. and T.S. purified and crystallized the receptor/Fab-fragment complex. S.W., A.D.C. and S.I. performed data collection. T. Hino solved and refined the structure. T. Hino, S.I. and T.M. wrote the manuscript and all authors provide editorial input. The project was managed by T.K., T. Hamakubo, S.I. and T.M.

Corresponding authors

Correspondence to So Iwata or Takeshi Murata.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-11 with legends, Supplementary Tables 1-2 and a Supplementary Discussion. (PDF 3397 kb)

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Hino, T., Arakawa, T., Iwanari, H. et al. G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature 482, 237–240 (2012). https://doi.org/10.1038/nature10750

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