The MRAP2 accessory protein directly interacts with melanocortin-3 receptor to enhance signalling

The central melanocortin system links nutrition to energy expenditure, with melanocortin-4 receptor (MC4R) controlling appetite and food intake, and MC3R regulating timing of sexual maturation, rate of linear growth and lean mass accumulation. Melanocortin-2 receptor accessory protein-2 (MRAP2) is a single transmembrane protein that interacts with MC4R to potentiate it’s signalling, and human mutations in MRAP2 cause obesity, with hyperglycaemia and hypertension. Previous studies have been unable to consistently show whether MRAP2 affects MC3R activity. Here we used single-molecule pull-down (SiMPull) to confirm that MC3R and MRAP2 interact. Analysis of fluorescent photobleaching steps showed that MRAP2 forms monomers or occasionally homodimers at cell surfaces, while MC3R is predominantly monomeric. When co-transfected, MC3R and MRAP2 readily form heterodimers most commonly with a 1:1 (~74%) or 1:2 (~17%) stoichiometry. snRNA-seq of human hypothalamic neurons showed MRAP2 was expressed in 57% of MC3R-positive neurons, while spatial transcriptomics revealed MRAP2 transcripts are present under the same spatially barcoded spots as MC3R transcripts in regions of the human hypothalamus with roles in energy homeostasis and appetite control, including the arcuate nucleus, ventromedial hypothalamus and periventricular region. We then showed that MRAP2 enhances MC3R cAMP signalling, impairs β-arrestin recruitment, and reduces receptor internalisation in HEK293 cells. Structural homology models revealed putative interactions between the two proteins and alanine mutagenesis of five MRAP2 and three MC3R transmembrane helix-6 residues significantly reduced MRAP2 effects on MC3R signalling. Finally, we showed genetic variants in MRAP2 that have been identified in individuals that are overweight or obese prevent MRAP2’s enhancement of MC3R-driven signalling. Thus, these studies reveal MRAP2 as an important regulator of MC3R function and provide further evidence for the crucial role of MRAP2 in energy homeostasis.