Obesity-associated MRAP2 variants impair multiple MC4R-mediated signaling pathways

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed at hypothalamic neurons that has an important role in appetite suppression and food intake. Mutations in MC4R are the most common cause of monogenic obesity and can affect multiple signaling pathways including Gs-cAMP, Gq, ERK1/2, β-arrestin recruitment, internalization and cell surface expression. The melanocortin-2 receptor accessory protein 2 (MRAP2), is a single-pass transmembrane protein that interacts with and regulates signaling by MC4R. Variants in MRAP2 have also been identified in overweight and obese individuals. However, functional studies that have only measured the effect of MRAP2 variants on MC4R-mediated cAMP signaling have produced inconsistent findings and most do not reduce MC4R function. Here we investigated the effect of twelve of these previously reported MRAP2 variants and showed that all variants that have been identified in overweight or obese individuals impair MC4R function. When expressed at equal concentrations, seven MRAP2 variants impaired MC4R-mediated cAMP signaling, while nine variants impaired IP3 signaling. Four mutations in the MRAP2 C-terminus affected internalization. MRAP2 variants had no effect on total or cell surface expression of either the MRAP2 or MC4R proteins. Structural models predicted that MRAP2 interacts with MC4R transmembrane helices 5 and 6, and mutations in two MRAP2 residues in putative contact sites impaired the ability of MRAP2 to facilitate MC4R signaling. In summary, our studies demonstrate that human MRAP2 variants associated with obesity impair multiple MC4R signaling pathways and that both Gs-cAMP and Gq-IP3 pathways should be assessed to determine variant pathogenicity.