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

We identified MRAP2 variants (R125C, S80F, P167A and Q174*) in patients with obesity using an obesity gene panel. These variants were introduced in a MRAP2 expression vector and functionally analyzed by co-transfecting HEK293 cells with expression plasmids encoding wild type (WT) MC4R with WT or variant MRAP2. We analyzed the effects of WT MRAP2 and the variants on MC4R cell surface expression and α-MSH-induced cAMP response, β-arrestin-2 recruitment, and internalization.

First, we analyzed the impact of WT MRAP2 on MC4R signaling. We observed that in the presence of MRAP2, the studied endpoints of MC4R signaling were significantly affected. 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 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 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. However, the function of the MRAP2 variants did not significantly differ from the WT MRAP2 in their modulation of MC4R signaling.

Our results indicate that MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon stimulation. However, the identified MRAP2 variants do not impact functionality of MRAP2-MC4R signaling. Since MRAP2 can modulate multiple receptors, we cannot rule out that MRAP2 variants affect other appetite-regulating receptors and thereby influence body weight regulation.