Fig. 5: MRAP2 decreases constitutive and ligand-dependent endocytosis of MC4R.

A Representative confocal micrographs of the localization and trafficking of SNAP-tagged MC4R in basal conditions or after the addition of 100 nM of the inverse agonist AgRP for 30 min in HEK293T cells in absence of presence of co-expression of MRAP2 (1 + 4). More than 3 independent experiments have been done. Scale bar is 10 µm. B Internalization of SNAP-tagged MC4R after 30 min incubation using NDP-α-MSH (1 µM), co-expressed with the endosomal marker Cerulean-Rab5, with or without co-expression of eYFP-MRAP2 (3 transfections, 14 cells). Scale bar is 10 µm. C Time evolution of SNAP-MC4R intensity at the plasma membrane of cells expressing SNAP-MC4R alone or in co-expression with eYFP-MRAP2 (1 + 4) upon stimulation with NDP-α-MSH (1 µM). Mean values ± 95% CI of three independent experiments are indicated. D Quantification of the number of endosomes per cell detected using Cerulean-Rab5 staining with and without co-expression of MRAP2 (1 + 4) together with MC4R, both in basal and agonist stimulated conditions. Results are represented in a bar chart as mean ± SEM, in which individual points represent replicates from the three independent experiments are shown as overlaid dot plot. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons post-hoc test (****: adjusted p-value < 0.0001). E Mean concentration-response curves of the translocation of MC4R-RlucII to the early endosome localization sensor rGFP-FYVE upon stimulation with α-MSH for 1 h, in the presence or absence of MRAP2 or RAMP3 in transiently transfected HEK293-SL cells. MRAP2 or RAMP3 plasmid DNA is co-transfected with the same amount (1 + 1) or fourfold the amount (1 + 4) of the MC4R-RlucII plasmid DNA. Normalized data are expressed as mean ± SEM of three independent experiments. F E_max values from the translocation to early endosome BRET experiments. Results are represented in a bar chart as mean ± SEM, in which individual Emax data point from the three individual experiments are shown as overlaid dot plot. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons post-hoc test (***: adjusted p-value = 0.0005; ****: adjusted p-value < 0.0001). Non-significant pairwise comparison are not shown.