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

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. 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 in HEK293 cells. Analysis of fluorescent photobleaching steps showed that MC3R and MRAP2 readily form heterodimers most commonly with a 1:1 stoichiometry. Human single-nucleus and spatial transcriptomics show MRAP2 is co-expressed with MC3R in hypothalamic neurons with important roles in energy homeostasis and appetite control. Functional analyses showed MRAP2 enhances MC3R cAMP signalling, impairs β-arrestin recruitment, and reduces internalization in HEK293 cells. Structural homology models revealed putative interactions between the two proteins and alanine mutagenesis of five MRAP2 and three MC3R transmembrane 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.