👤 Joshua Levitz

The melanocortin-2 receptor accessory protein (MRAP) family interacts with and regulates the signaling of diverse G protein-coupled receptors (GPCRs). MRAP2 modifies the signaling of three distinct GPCRs, melanocortin-4 receptor (MC4R), MC3R, and ghrelin receptor (GHSR), all essential for appetite regulation. The nature of MRAP2/GPCR complexes and whether there are shared mechanisms for complex assembly, critical structural regions, or consistent effects on receptor signaling remains unknown. Here, we show that all three GPCRs preferentially interact with MRAP2 as 1:1 complexes and MRAP2 binding disrupts GPCR homodimerization. MRAP2 interacts with shared receptor transmembrane regions to promote GPCR signaling and impairs β-arrestin-2 recruitment to prolong signaling and delay internalization. Deletion of the MRAP2 cytoplasmic region impairs GPCR signaling by modulating constitutive activity. Human MRAP2 variants associated with overweight/obesity modify the constitutive activity of all three GPCRs. Thus, MRAP2 regulates GPCR function using shared molecular mechanisms, and we provide further evidence for the importance of GHSR constitutive activity. Show less

Post-labelling cleavable substrates for self-labelling protein tags, such as SNAP- and Halo-tags, can be used to study cell surface receptor trafficking events by stripping dyes from non-internalized protein pools. Since the complexity of receptor biology requires the use of multiple and orthogonal approaches to simultaneously probe multiple receptor pools, we report the development of four membrane impermeable probes that covalently bind to either the SNAP- or the Halo-tag in the red to far-red range. These molecules bear a disulfide bond to release the non-internalized probe using the reducing agent sodium 2-mercaptoethane sulfonate (MESNA). As such, our approach allows the simultaneous visualization of multiple internalized cell surface proteins in two colors which we showcase using G protein-coupled receptors. We use this approach to detect internalized group II metabotropic glutamate receptor (mGluRs), homo- and heterodimers, and to reveal unidirectional crosstalk between co-expressed glucagon-like peptide 1 (GLP1R) and glucose-dependent insulinotropic polypeptide receptors (GIPR). In these applications, we translate our method to both high resolution imaging and quantitative, high throughput assays, demonstrating the value of our approach for a wide range of applications. Show less

The central melanocortin system links nutrition to energy expenditure. Melanocortin-4 receptor (MC4R) controls appetite and food intake, and its signaling is potentiated by melanocortin-2 receptor accessory protein 2 (MRAP2). Human mutations in Show less

Accessory proteins such as members of the melanocortin-2 receptor accessory protein family (MRAP) have been described to interact with and regulate the signaling of diverse G protein-coupled receptors (GPCRs), however, surprisingly little is known about the mechanisms by which they mediate these effects. MRAP2 modifies signaling of three distinct GPCRs, melanocortin receptor 4 (MC4R), MC3R and the ghrelin receptor (GHSR), which each play essential roles in appetite regulation. Human mutations in MRAP2 cause obesity with hyperglycaemia and hypertension, suggesting that its regulation of GPCRs is critical for maintaining metabolic homeostasis. However, the nature of MRAP2/GPCR complexes and whether there are shared mechanisms for complex assembly, critical structural regions or consistent effects on receptor signaling and trafficking remains unknown. Here we showed all three GPCRs preferentially interact with MRAP2 as 1:1 complexes and that MRAP2 binding disrupts GPCR homodimerization. MRAP2 interacts with the same receptor transmembrane regions to promote GPCR signaling, and the accessory protein impairs β-arrestin-2 recruitment to prolong signaling and delay internalization. Deletion of the cytoplasmic region of MRAP2 impairs GPCR signaling by modulating receptor constitutive activity. Genetic variants in MRAP2 associated with overweight or obesity modulate the constitutive activity of all three GPCRs. Thus, MRAP2 regulates GPCR function using shared molecular mechanisms and these studies provide further evidence of the importance of GHSR constitutive activity. Show less

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. Show less