Colours represent cLoF (purple), pLoF (light brown), WT-like (green) and GoF (black). β-arrestin-2 coupling activity of MC4R mutations were characterised using a protein-protein interaction assay. (a) Dose response curves of MC4R mutants upon activation by NDP-MSH grouped by cLoF, pLoF, WT-like and GoF compared wild-type MC4R. Means +/- S.E.M. are shown (N=3-8 independent experiments); and (b) The relative EC50 (fold WT response, left panel) and Emax (% WT response right panel) were determined for each mutants and are presented in mean and 95% CI. * indicates p<0.05 by paired t-test (p-values were two-sided and uncorrected for multiple comparisons). CI = confidence interval; cLoF = complete loss of function; GoF = gain of function; N.D. = not determined; NDP-αMSH = [Nle4,D-Phe7]-α-melanocyte-stimulating hormone; pLoF = partial loss of function; WT-like = wild-type like.