Fig. 6: β-arrestin2 recruitment to MC4R in the presence or absence of MRAP2.

A Bioluminescence-complementation based readout of SmBiT-β-arrestin2 recruitment to MC4R-LgBiT under basal conditions, with and without co-expression of MRAP2 (1 + 1). Data are expressed as percentage of the basal β-arrestin recruitment to the MC4R-WT in absence of MRAP2 (mock). Results are represented in a bar chart as mean ± SEM, in which individual points represent average values from the nine independent experiments, are shown as overlaid dot plot. Statistical analysis was performed with two-tailed unpaired t-test (****: adjusted p-value < 0.0001). B Average concentration-response curve of SmBiT-β-arrestin2 recruitment to MC4R-LgBiT upon α-MSH stimulation with and without co-expression of MRAP2 (1 + 1). Data represent mean ± SEM from three independent experiments with four replicates each. EC₅₀ values and normalized maximal recruitment derived from curve fitting were 100% and 60 nM ± 8 nM for control condition and 67% and 50 nM ± 8 nM with overexpression of MRAP2 (1 + 1). C Mean concentration-response curves of β-arrestin2-RlucII recruitment to the plasma membrane localization sensor rGFP-CAAX upon MC4R stimulation with α-MSH for 2 min, 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 SNAP-MC4R plasmid DNA (140 ng), with total amount of DNA being constant. EC₅₀ values were 58 ± 10 nM, 21 ± 4 nM, and 32 ± 8 nM, respectively in the presence or absence of 1 + 1 or 1 + 4 MRAP2, and were not significantly different. Normalized data are expressed as mean ± SEM of four independent experiments. D E_max values from the β-arrestin2 recruitment BRET experiments. Results are represented in a bar chart as mean ± SEM, in which individual Emax data point from the four 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 = 0.0006; ****: adjusted p-value = <0.0001). Non-significant pairwise comparisons are not all shown.