Melanocortin 2 receptor accessory protein 2 (MRAP2) is a membrane-bound protein expressed on hypothalamic neurons that modulate the function of appetite-regulating receptors, including melanocortin-4 receptor (MC4R). While MRAP2 variants are associated with obesity, the mechanism by which MRAP2 modulates MC4R is still unclear. The aim of this study is to investigate the functional impact of wildtype (WT) MRAP2 and MRAP2 variants on MC4R signaling.

Four MRAP2 variants (R125C, S80F, P167A and Q174X) in patients with obesity were identified by obesity gene panel analysis. The functional effects of these variants were analyzed by co-transfecting HEK293 cells with expression plasmids encoding WT or variant MRAP2 and WT MC4R. Endpoints analyses include MC4R cell surface expression, and α-MSH-induced cAMP response, β-arrestin-2 recruitment, and internalization.

WT MRAP2 significantly decreased basal MC4R cell surface expression by 68.1±4.3% (p<0.001). MC4R internalization is induced by α-MSH in the absence of MRAP2. However, in the presence of WT MRAP2, MC4R cell surface expression was increased by 89.4±6.9% upon α-MSH stimulation (p<0.001). Furthermore, in the cells expressing MC4R with WT MRAP2, the maximal α-MSH-induced cAMP and β-arrestin-2 recruitment responses were increased by 29.8±5.2% and 81.3±4.2% respectively (p<0.001), compared with those lacking MRAP2. Analyzing the MRAP2 variants for these endpoints showed that they did not significantly differ from the WT MRAP2 in modulating MC4R signaling.

Our results indicate that WT MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon α-MSH stimulation. The MRAP2 variants assessed in this study behaved as WT MRAP2 in α-MSH-induced MC4R signaling, suggesting that these may be benign variants. However, since MRAP2 can modulate multiple receptors, we cannot rule out that these MRAP2 variants affect other appetite-regulating receptors and thereby influence body weight regulation via other pathways.