Role of GHSR1a ICL3 residues Ser252 and Thr261 for recruitment of β-arrestin.A, ghrelin stimulated β-arrestin2 recruitment to GHSR1a and GHSR1a-S252A with and without MRAP2. Arrow indicates ghrelin injection time. B, ghrelin stimulated β-arrestin1 recruitment to GHSR1a and GHSR1a-S252A with and without MRAP2. C, ghrelin-stimulated IP1 accumulation assay in cells transfected with GHSR1a and GHSR1a-S252A with and without MRAP2. D, detection of GHSR1a and GHSR1a-S252A with and without MRAP2 by ELISA in nonpermeabilized cells. E, ghrelin stimulated β-arrestin2 recruitment to GHSR1a and GHSR1a-T261A with and without MRAP2. F, ghrelin stimulated β-arrestin1 recruitment to GHSR1a and GHSR1a-T261A with and without MRAP2. G, ghrelin-stimulated IP1 accumulation assay in cells transfected with GHSR1a and GHSR1a-S252A with and without MRAP2. H, detection of GHSR1a and GHSR1a-T261A with and without MRAP2 by ELISA in nonpermeabilized cells. Data are the mean ± SEM of three independent experiments performed in triplicate. I and J, ghrelin-stimulated β-arrestin1 (I) and b-arrestin2 (J) in function of surface GHSR1a expression measured by ELISA. ∗∗∗p < 0.001. GHSR1a, growth hormone secretagogue receptor 1a; IP, inositol triphosphate.