G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.
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We acknowledge support from National Institutes of Health, grants NS028471 (B.K.K.) and GM56169 (W.I.W.), the Stanford Medical Scientist Training Program (M.P.B.), the Lundbeck Foundation (S.G.F.R.), the University of Copenhagen and 7TM Pharma (R.N.), and the Instituto de Salud Carlos III (L.P.).
Author Contributions M.P.B. designed experiments, purified, labelled and functionally characterized β2AR, collected and analysed NMR data and wrote the paper. Y.Z. made, expressed and purified β2AR lysine mutants and collected NMR data. S.G.F.R. expressed and purified β2AR for NMR and crystallized the 13C-methylated β2AR–Fab complex. C.W.L. designed, optimized and supervised NMR experiments and collected NMR data. D.M.R., H.-J.C. and W.I.W. collected diffraction data and refined the structure of the 13C-methylated β2AR–Fab complex. R.N. collected and analysed NMR data and optimized data processing. J.J.F. performed G-protein-coupling assays on labelled β2AR. F.S.T. prepared insect cell cultures and purified β2AR. T.S.K. purified β2AR. J.D.P. advised on NMR spectroscopy experiments. L.P. performed molecular modelling and molecular dynamics simulations. R.S.P. designed and optimized NMR experiments, wrote NMR pulse sequences and collected data. L.M. conceived of lysine methylation of the β2AR, wrote NMR pulse sequences and designed NMR experiments. B.K.K. supervised the overall project, designed experiments, collected diffraction data and wrote the paper.
The authors declare no competing financial interests.
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