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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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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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.
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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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DOI: https://doi.org/10.1038/nature10750
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