Fig. 3: Binding of NDP-α-MSH is increased in HEK293 expressing MRAP2.

A Confocal maximum intensity projections of representative HEK293 cells transfected with either SNAP-MC4R + mock or SNAP-MC4R + MRAP2 (1 + 4), seeded on the same coverslip and then labelled using SNAP-surface549. The cells were then incubated for 5 min with either 0.5 nM or 1000 nM Alexa647-NDP-α-MSH and imaged. Contrast is set to the same values in each channel. Maximum intensity projections were constructed in Fiji before running Cellpose-based cell segmentation to extract individual cells intensity values. Scale bar is 10 µm. B Chart of the normalised NDP-α-MSH ligand signal (to SNAP-MC4R receptor expression) per cell for 0.5 nM ligand (turquoise circles, n = 5 independent transfections, 226 cells) and 1000 nM ligand (green square, n = 2 independent transfections, 45 cells). Shading indicates MRAP2 positive cells, as determined by visual inspection of confocal micrographs. C Affinity and D maximal binding parameters extracted from saturation radioligand binding experiments using [I]¹²⁵-NDP-α-MSH on membrane preparations of HEK293-SL cells transfected with MC4R and MRAP2 or RAMP3 at 1 + 1 ratio. Results are represented as mean ± SEM, in which individual data point from the three individual experiments are shown. Results show an increase in B_max for MRAP2 and not from the negative control RAMP3, but no change in affinity (pK_D) for both conditions. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons post-hoc test (* = p < 0.05, adjusted p-value mock vs MRAP2 = 0.0125 and adjusted p-value MRAP2 vs RAMP3 = 0.0377). Non-significant comparison is not shown in (D).