Figure 5.

The effect of RAMP coexpression on MRGPRX4 β-arrestin recruitment.A and B, BRET² β-arrestin recruitment assays were carried out in the presence of increasing amounts of MRGPRX4 coexpressed with β-arrestin1 (A) or β-arrestin2 (B) upon stimulation with 100 μM nateglinide or DCA. C, time course of β-arrestin2 recruitment in cells expressing MRGPRX4 upon stimulation with nateglinide (red) and DCA (blue) as compared to basal levels (open black). Cells coexpressing CALCRL with RAMP2 and stimulated with 200 nM adrenomedullin served as the positive control (gray star). Red and gray smooth curves are fits to a two-phase decay model (see Table S3). D, comparison of normalized net BRET² for β-arrestin1 or β-arrestin2 recruitment to MRGPRX4 upon nateglinide stimulation. Cells coexpressing CALCRL with RAMP2 and stimulated with adrenomedullin served as the positive control. Data are normalized to nateglinide-dependent β-arrestin2 recruitment to MRGPRX4, and individual data points for each independent experiment are overlaid. The statistical significance was determined by unpaired two-tailed t test (see Table S2). E, time course of β-arrestin2 recruitment to MRGPRX4 in cells expressing MRGPRX4 alone or with RAMP2 upon stimulation with nateglinide (red) and DCA (blue). The red smooth curve is the fit to a two-phase decay model (see Table S3). F, comparison of normalized net BRET² for β-arrestin2 recruitment to MRGPRX4 under basal conditions and nateglinide or DCA stimulation. MRGPRX4 was expressed alone or co-expressed with each RAMP. Data are normalized to nateglinide-dependent β-arrestin2 recruitment to MRGPRX4, and data points representing the mean of each independent experiment are overlaid. The statistical significance was determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test to nateglinide-stimulated MRGPRX4 (see Table S2). (∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05, if not marked then not significant). Error bars signify the mean ± SEM. A, B, D, and F, data are from three independent experiments with three replicates each, except for mock (D and F), which had two replicates per experiment. C, and E, data are from three independent experiments with two replicates each, except the CALCRL-RAMP2 dataset in (C) for which the data are from two independent experiments with two replicates each. BRET2, bioluminescence resonance energy transfer; CALCRL, calcitonin receptor-like receptor; DCA, deoxycholic acid; RAMP, receptor activity–modifying protein.