Extended Data Fig. 7: Impact of natural genetic variations in the orthosteric ligand-binding pocket on 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 agonist stimulation. (b-e) The response of 14 ghrelin receptor mutants to various agonists (10 μM ghrelin, 10 μM GHRP-6, 10 μM ibutamoren, and 10 μM anamorelin) was examined, focusing on the activation of Gq as a functional readout of the mutant receptors. Bar graphs represent the fold change compared to the wild-type receptor-induced response, illustrating the maximum amplitude and the speed of G protein activation induced by each agonist. The basal activity of the ghrelin receptor was also examined (f). Mean ± SEM of four independent experiments (e), three independent experiments (b-d), or thirteen independent experiments (f) are shown. Statistical significance was determined by One-Way ANOVA followed by Dunnett multiple comparison test (b-f), 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 S123G, A204E, V214L, S217P, F279L, and N305K, 0.0005 for P200S, 0.0028 for M213I, 0.0066 for I178T, 0.0121 for E124D, 0.0769 for L103F, 0.2792 for R102P, 0.7626 for D99E, and >0.9999 for V214A in panel b (amplitude); <0.0001 for D99E, R102P, S123G, E124D, I178T, A204E, S217P, F279L, and N305K, 0.0002 for V214A, 0.0014 for V214L, 0.0605 for M213I, 0.4336 for L103F, and 0.6960 for P200S in panel b (activation rate); <0.0001 for R102P, S123G, E124D, P200S, A204E, M213I, V214L, S217P, and F279L, 0.0004 for I178T, 0.0037 for L103F, 0.9437 for V214A, and 0.9999 for D99E in panel c (amplitude); <0.0001 for D99E, R102P, E124D, A204E, and F279, 0.0026 for S217P, 0.0219 for I178T, 0.0405 for P200S, 0.6703 for V214L, 0.9963 for M213I, 0.9995 for L103F, and >0.9999 for S123G and V214A in panel c (activation rate); <0.0001 for R102P, S123G, A204E, S217P, and F279L, 0.0003 for E124D and V214L, 0.0071 for P200S, 0.0085 for M213I, 0.0231 for I178T, 0.0667 for L103F, 0.9766 for V214A, 0.9945 for D99E, and >0.9999 for N305K in panel d (amplitude); <0.0001 for R102P and N305K, 0.0002 for D99E, 0.0008 for A204E, 0.0066 for E124D, 0.0197 for L103F, 0.0243 for F279L, 0.0343 for V214L, 0.0537 for S217P, 0.1246 for V214A, 0.2056 for P200S, 0.3043 for S123G, 0.9998 for I178T, and >0.9999 for M213I in panel d (activation rate); <0.0001 for R102P, S123G, E124D, I178T, P200S, A204E, M213I, V214L, S217P, F279L, and N305K, 0.0055 for L103F, 0.9053 for V214A, and 0.9773 for D99E in panel e (amplitude); <0.0001 for D99E, R102P, E124D, A204E, M213I, S217P, and F279L, 0.0046 for N305K, 0.0312 for P200S, 0.0427 for S123G, 0.0948 for I178T, 0.3042 for L103F, 0.6292 for V214L, and 0.9963 for V214A in panel e (activation rate); and <0.0001 for D99E, R102P, S123G, E124D, I178T, P200S, A204E, M213I, V214L, S217P, F279L, and N305K, 0.0007 for V214A, and 0.10172 for L103F in panel f. ‘N.D.’ indicates not detected.
