👤 Kathleen Wyatt

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

Osteoporosis diagnoses are increasing in the ageing population, and although some treatments exist, these have several disadvantages, highlighting the need to identify new drug targets. G protein-coupled receptors (GPCRs) are transmembrane proteins whose surface expression and extracellular activation make them desirable drug targets. Our previous studies have identified 144 GPCR genes to be expressed in primary human osteoclasts, which could provide novel drug targets. The development of high-throughput assays to assess osteoclast activity would improve the efficiency at which we could assess the effect of GPCR activation on human bone cells and could be utilised for future compound screening. Here, we assessed the utility of a high-content imaging (HCI) assay that measured cytoplasmic-to-nuclear translocation of the nuclear factor of activated T cells-1 (NFATc1), a transcription factor that is essential for osteoclast differentiation, and resorptive activity. We first demonstrated that the HCI assay detected changes in NFATc1 nuclear translocation in human primary osteoclasts using GIPR as a positive control, and then developed an automated analysis platform to assess NFATc1 in nuclei in an efficient and unbiased manner. We assessed six GPCRs simultaneously and identified four receptors (FFAR2, FFAR4, FPR1 and GPR35) that reduced osteoclast activity. Bone resorption assays and measurements of TRAP activity verified that activation of these GPCRs reduced osteoclast activity, and that receptor-specific antagonists prevented these effects. These studies demonstrate that HCI of NFATc1 can accurately assess osteoclast activity in human cells, reducing observer bias and increasing efficiency of target detection for future osteoclast-targeted osteoporosis therapies. 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 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. 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

Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models. Show less

Drugs targeting the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) are emerging as treatments for type-2 diabetes and obesity. GIP acutely decreases serum markers of bone resorption and transiently increases bone formation markers in short-term clinical investigations. However, it is unknown whether GIP acts directly on bone cells to mediate these effects. Using a GIPR-specific antagonist, we aimed to assess whether GIP acts directly on primary human osteoclasts and osteoblasts. Osteoclasts were differentiated from human CD14+ monocytes and osteoblasts from human bone. GIPR expression was determined using RNA-seq in primary human osteoclasts and in situ hybridization in human femoral bone. Osteoclastic resorptive activity was assessed using microscopy. GIPR signaling pathways in osteoclasts and osteoblasts were assessed using LANCE cAMP and AlphaLISA phosphorylation assays, intracellular calcium imaging and confocal microscopy. The bioenergetic profile of osteoclasts was evaluated using Seahorse XF-96. GIPR is robustly expressed in mature human osteoclasts. GIP inhibits osteoclastogenesis, delays bone resorption, and increases osteoclast apoptosis by acting upon multiple signaling pathways (Src, cAMP, Akt, p38, Akt, NFκB) to impair nuclear translocation of nuclear factor of activated T cells-1 (NFATc1) and nuclear factor-κB (NFκB). Osteoblasts also expressed GIPR, and GIP improved osteoblast survival. Decreased bone resorption and improved osteoblast survival were also observed after GIP treatment of osteoclast-osteoblast co-cultures. Antagonizing GIPR with GIP(3-30)NH2 abolished the effects of GIP on osteoclasts and osteoblasts. GIP inhibits bone resorption and improves survival of human osteoblasts, indicating that drugs targeting GIPR may impair bone resorption, whilst preserving bone formation. Show less

Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (P Show less