👤 Alejandra V Rodríguez Rondón

Melanocortin-2 receptor accessory protein-2 (MRAP2) modulates the activity of hypothalamic melanocortin-4 (MC4R) and growth hormone-secretagogue (GHSR) receptors, which suppress and promote appetite, respectively. We investigate whether obesity-associated variants of MRAP2 alter their ability to modulate MC4R and GHSR signalling as a possible mechanistic link to the development of obesity. Functional effects of five obesity-associated MRAP2 variants were analysed in HEK293 cells by co-expressing wild-type or variant MRAP2 with MC4R or GHSR. Endpoints included cell-surface and total expression, and ligand-induced second-messenger responses, β-arrestin-2 recruitment, and alternative G-protein activation. MRAP2 decreased basal MC4R cell-surface expression while GHSR cell-surface expression was not affected. In MC4R/MRAP2 expressing cells, maximal α-MSH-induced cAMP and β-arrestin-2 recruitment responses were increased. Similarly, ghrelin-induced Ca2+-mobilization in GHSR/MRAP2 expressing cells was increased, but β-arrestin-2 recruitment was suppressed. MRAP2 did not bias G-protein activation by either receptor, although previous reports show MRAP2 biases MC4R signalling towards Gαq/11. The variants did not significantly affect the ability of MRAP2 to modulate MC4R and GHSR signalling. Our results indicate that MRAP2 potentiates the ligand responsiveness of MC4R and GHSR, but has differential effects on β-arrestin-2 recruitment. The MRAP2 variants had no significant effects on the signalling endpoints tested. This suggests that, despite their association with obesity, the variants may be functionally benign, or that the absence of effects reflects limitations inherent to our cellular model. In addition, since MRAP2 can modulate multiple receptors and differentially modulate their signalling, we cannot rule out their influence on body weight regulation via other mechanisms. Show less

The melanocortin-4 receptor (MC4R) plays an important role in body weight regulation. Pathogenic MC4R variants are the most common cause of monogenic obesity. We have identified 17 MC4R variants in adult and pediatric patients with obesity. Here we aimed to functionally characterize these variants by analyzing 4 different aspects of MC4R signaling. In addition, we aimed to analyze the effect of setmelanotide, a potent MC4R agonist, on these MC4R variants. Cell surface expression and α-melanocyte stimulating hormone (α-MSH)- or setmelanotide-induced cAMP response, β-arrestin-2 recruitment, and ERK activation were measured in cells expressing either wild type or variant MC4R. We found a large heterogeneity in the function of these variants. We identified variants with a loss of response for all studied MC4R signaling, variants with no cAMP accumulation or ERK activation but normal β-arrestin-2 recruitment, and variants with normal cAMP accumulation and ERK activation but decreased β-arrestin-2 recruitment, indicating disrupted desensitization and signaling mechanisms. Setmelanotide displayed a greater potency and similar efficacy as α-MSH and induced significantly increased maximal cAMP responses of several variants compared to α-MSH. Despite the heterogeneity in functional response, there was no apparent difference in the obesity phenotype in our patients. We show that these obesity-associated MC4R variants affect MC4R signaling differently yet lead to a comparable clinical phenotype. Our results demonstrate the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity, which may aid in improving personalized treatment. Show less