Extended Data Fig. 8: Impact of natural genetic variations in the orthosteric ligand-binding pocket on antagonist/inverse agonist actions.
From: The structure and function of the ghrelin receptor coding for drug actions
(a) Schematic representation of the BRET-based kinetic assay used for real-time monitoring of the ghrelin receptor’s activity on Gq following antagonist/inverse agonist stimulation. (b-e) The response of 14 ghrelin receptor mutants to various ligands (100 μM YIL 781, 32 μM CPD21, 100 μM JMV 2959, and 10 μM PF-05190457) was examined, focusing on the activation of Gq as a functional readout of the mutant receptors. Bar graphs illustrate the maximum amplitude and the speed of G protein activation induced by each ligand. Gq deactivation induced by an inverse agonist was quantified (e). Mean ± SEM of four independent experiments (b) or three independent experiments (c-e) are shown. Statistical significance was determined by One-Way ANOVA with Dunnett multiple comparison test (b, c, d, and e), with significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Adjusted P values in comparison with the wild-type receptor are as follows: <0.0001 for I178T, P200S, and S217P, 0.0005 for D99E, 0.0023 for S123G, 0.0024 for V214A, 0.0245 for M213I, and 0.9996 for A204E in panel b (amplitude); <0.0001 for D99E, I178T, P200S, and V214A, 0.0011 for S217P, 0.0020 for A204E, 0.7644 for S123G, and >0.9999 for M213I in panel b (activation rate); <0.0001 for I178T and S217P, 0.0002 for M213I, 0.0173 for D99E, 0.0330 for E124D, 0.5048 for V214A, 0.7065 for P200S, and 0.9998 for S123G in panel c (amplitude); <0.0001 for D99E, S123G, E124D, I178T, P200S, and S217P, 0.0038 for V214A, and 0.0080 for M213I in panel c (activation rate); 0.0002 for E124D, 0.0003 for R102P and I178T, and 0.0007 for V214A in panel d (amplitude); <0.0001 for R102P, E124D, I178T, and V214A in panel d (activation rate); <0.0001 for no GHSR, S213G, E124D, I178T, A204E, M213I, V214L, S217P, F279L, and N305K, 0.0002 for P200S, 0.6001 for L103F, 0.9942 for D99E, and 0.9994 for V214A in panel e. ‘N.D.’ indicates not detected.
