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

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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.

At a glance


  1. Effect of Fab2838 on A2AAR-ligand binding.
    Figure 1: Effect of Fab2838 on A2AAR–ligand binding.

    a, Saturation binding curves for an antagonist [3H]-ZM241385 binding to A2AAR with (open circles) or without (filled circles) Fab2838. b, c, Inhibition of [3H]-ZM241385 binding by the antagonists theophylline (b) and SCH442416 (c) with (open circles) and without (filled circles) Fab2838. The binding of [3H]-ZM241385 in the absence of a competitor was set at 100%. d, As in a, but for the agonist [3H]-NECA. e, f, As in c and d, but for the agonists adenosine (e) and NECA (f). All data are the mean±s.e.m. of three independent experiments performed in duplicate.

  2. Structure of the A2AAR-Fab2838 complex.
    Figure 2: Structure of the A2AAR–Fab2838 complex.

    a, Overall structure viewed parallel to the membrane. A2AAR and the Fab light (Fab(L)) and heavy (Fab(H)) chains are shown in blue-grey, cyan and magenta, respectively. The three disulphide bonds in the extracellular loops (ECLs) are represented by yellow sticks. The bound antagonist ZM241385 in the ligand-binding pocket is shown as a space-filling model. The CDRs of Fab2838 are coloured as follows: CDR-H1, yellow; CDR-H2, orange; CDR-H3, red; CDR-L1, green; CDR-L2, purple; CDR-L3, marine blue. EXT, extracellular; IN, intracellular. b, Surface representation of the interface between A2AAR (top) and Fab2838 (bottom). Relative to a, A2AAR has been rotated 90° around a horizontal axis, whereas Fab2838 is shown in the same orientation. c, View of the interface between A2AAR (green residues) and CDR-H3 (orange residues). The main chain of A2AAR is shown as ribbon representation as in a. Red spheres show the positions of water molecules. Red dotted lines indicate hydrogen-bond interactions. d, Schematic representation of the interface between A2AAR and CDR-H3.

  3. Comparison of the structures of the opsin-G[agr]CT, [bgr]2AR-Nb80 and A2AAR-Fab2838 complexes.
    Figure 3: Comparison of the structures of the opsin–GαCT, β2AR–Nb80 and A2AAR–Fab2838 complexes.

    Left, middle and right panels show the structures of an active form of opsin (green) in complex with GαCT (yellow), an active form of β2AR (brown) bound agonist BI-167107 in complex with Nb80 CDR-3 (blue) and an inactive form of A2AAR (blue-grey) bound antagonist ZM241385 in complex with Fab2838 CDR-H3 (red). a, Views parallel to the membrane. Bound ligands are shown as stick models in β2AR and A2AAR. The residues involved in the ionic lock formation are also shown. Nitrogen and oxygen atoms are coloured blue and red, respectively. b, Cytoplasmic views of the complexes. c, Surface representations of cytoplasmic surfaces of the receptors. Surfaces within 4Å of GαCT, CDR-3 or CDR-H3 are coloured red.

Accession codes

Primary accessions

Protein Data Bank


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Author information

  1. These authors contributed equally to this work.

    • Tomoya Hino,
    • Takatoshi Arakawa,
    • Hiroko Iwanari &
    • Takami Yurugi-Kobayashi


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

    • Tomoya Hino


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.

Competing financial interests

The authors declare no competing financial interests.

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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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  1. Supplementary Information (3.3M)

    This file contains Supplementary Figures 1-11 with legends, Supplementary Tables 1-2 and a Supplementary Discussion.

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