Abstract Disclosure: A.V. Rodriguez Rondon: None. F. Volker: None. M.S. Welling: None. C. de Groot: None. M.M. van Haelst: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. 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) and growth hormone secretagogue receptor (GHSR). While pathogenic variants in the MRAP2 gene are associated with obesity, the mechanism for this remains unclear. The aim of this study is to investigate the functional impact of wildtype (WT) MRAP2 and MRAP2 variants on MC4R and GHSR signaling. Four MRAP2 variants (S80F, R125C, P167A and Q174X) were identified in patients with obesity 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 or WT GHSR. Endpoint analyses included cell surface expression, and ligand-induced second messenger responses (α-MSH-induced cAMP response for MC4R and ghrelin-induced Ca2+ mobilization for GHSR), β-arrestin-2 recruitment, and internalization. WT MRAP2 significantly decreased basal MC4R cell surface expression by 68.1±4.3% (p<0.001). In the absence of MRAP2, α-MSH induced MC4R internalization. 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). GHSR cell surface expression and ghrelin-induced internalization were not affected by WT MRAP2. 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. We also investigated GHSR ghrelin-induced Ca2+ mobilization and found that, in the presence of WT MRAP2, this increased significantly by 95.37±21.9%, (p<0.0003). However, ghrelin-induced β-arrestin-2 recruitment was suppressed by 69.8±7.6%, (p<0.001). Using these endpoints we found no effect of the variants on the ability of MRAP2 to modulate ligand-induced signaling by MC4R and GHSR. Our results indicate that WT MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon α-MSH stimulation, and enhances GHSR Ca2+ mobilization by decreasing β-arrestin-2 recruitment upon ghrelin stimulation. The MRAP2 variants assessed in this study behaved as WT MRAP2 in α-MSH-induced MC4R and ghrelin-induced GHSR 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. Presentation: 6/3/2024