A Setmelanotide-like Effect at MC4R Is Achieved by MC4R Dimer Separation.

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R has become a target of interest in the search of treatment options. We have previously reported that the MC4R forms homodimers, affecting receptor Gs signaling properties. Recent studies introducing setmelanotide, a novel synthetic MC4R agonist, suggest a predominant role of the Gq/11 pathway regarding weight regulation. In this study, we analyzed effects of inhibiting homodimerization on Gq/11 signaling using previously reported MC4R/CB1R chimeras. NanoBRETTM studies to determine protein-protein interaction were conducted, confirming decreased homodimerization capacities of chimeric receptors in HEK293 cells. Gq/11 signaling of chimeric receptors was analyzed using luciferase-based reporter gene (NFAT) assays. Results demonstrate an improvement of alpha-MSH-induced NFAT signaling of chimeras, reaching the level of setmelanotide signaling at wild-type MC4R (MC4R-WT). In summary, our study shows that inhibiting homodimerization has a setmelanotide-like effect on Gq/11 signaling, with chimeric receptors presenting increased potency compared to MC4R-WT. These findings indicate the potential of inhibiting MC4R homodimerization as a therapeutic target to treat obesity.

Relevance of polymorphisms in MC4R and BDNF in short normal stature.

BACKGROUND: Variation in genes of the leptinergic-melanocortinergic system influence both body weight and height. Because short normal stature (SNS) is characterized by reduced body height, delayed maturation and leanness, allelic variation of genes in this pathway are hypothesized to affect this common condition. METHODS: We analyzed the coding regions of LEP, MC4R, MRAP2 and BDNF in 185 children with SNS (height < 5th percentile) to search for non-synonymous and frameshift variants. For association studies (two-sided χ2-tests) population-based data sets (ExAC, EVS and KORA) were used. Cyclic AMP accumulation, cell surface expression, central expression and MAP kinase activation were assayed in vitro to determine the functional implications of identified variants. RESULTS: We detected eleven variants predicted to be protein-altering, four in MC4R, four in BDNF, and three in MRAP2. No variants were found in LEP. In vitro analysis implied reduced function for the MC4R variant p.Met215Ile. Loss-of-function is contrary to expectations based on obesity studies, and thus does not support that this variant is relevant for SNS. The minor SNP alleles at MC4R p.Val103Ile and BDNF p.Val66Met were nominally associated with SNS. CONCLUSION: Taken together, although genes of the leptinergic-melanocortinergic system are important for normal growth, our data do not support the involvement of rare mutations in LEP, MC4R, MRAP2 or BDNF in short normal stature.

Decreased melanocortin-4 receptor function conferred by an infrequent variant at the human melanocortin receptor accessory protein 2 gene.

OBJECTIVE: The melanocortin receptor accessory protein 2 (MRAP2) is relevant for weight regulation in mice and humans. This function is likely mediated by regulation of the melanocortin-4 receptor (MC4R). Functional implications of human MRAP2 mutations have not been described yet. METHODS: A mutation screen was conducted in MRAP2 in 184 children and adolescents with (extreme) obesity and in 184 lean controls. Detected nonsynonymous variants were genotyped in larger independent study groups (300 people with obesity and 436 individuals with normal weight). The influence of mutant MRAP2 on MC4R signaling was analyzed in vitro. RESULTS: (1) Three (two novel) nonsynonymous MRAP2 variants were detected: p.Ala137Thr, p.Gln174Arg, p.Arg125His (rs115655382), two synonymous variants, and three intronic variants. (2) The impact of MRAP2 on MC4R function was dependent on the ratio between the two co-expressed proteins. Increased MC4R signaling was detected at MRAP2/MC4R ratios of 2 + 1 and above. (3) The function of MC4R was reduced with the infrequent allele at the MRAP2 p.Gln174Arg variant. (4) The three nonsynonymous mutations were each only detected once among the 484 people with obesity and not among 620 individuals with normal weight. CONCLUSIONS: This was the first study describing an effect of a MRAP2 mutation on MC4R function.

The spectrum of phenotypes associated with mutations in steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) includes severe penoscrotal hypospadias in 46,XY males without adrenal insufficiency.

OBJECTIVE: Hypospadias is a frequent congenital anomaly but in most cases an underlying cause is not found. Steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) is a key regulator of human sex development and an increasing number of SF-1 (NR5A1) mutations are reported in 46,XY disorders of sex development (DSD). We hypothesized that NR5A1 mutations could be identified in boys with hypospadias. DESIGN AND METHODS: Mutational analysis of NR5A1 in 60 individuals with varying degrees of hypospadias from the German DSD network. RESULTS: Heterozygous NR5A1 mutations were found in three out of 60 cases. These three individuals represented the most severe end of the spectrum studied as they presented with penoscrotal hypospadias, variable androgenization of the phallus and undescended testes (three out of 20 cases (15%) with this phenotype). Testosterone was low in all three patients and inhibin B/anti-Müllerian hormone (AMH) were low in two patients. Two patients had a clear male gender assignment. Gender re-assignment to male occurred in the third case. Two patients harbored heterozygous nonsense mutations (p.Q107X/WT, p.E11X/WT). One patient had a heterozygous splice site mutation in intron 2 (c.103-3A/WT) predicted to disrupt the main DNA-binding motif. Functional studies of the nonsense mutants showed impaired transcriptional activation of an SF-1-responsive promoter (Cyp11a). To date, adrenal insufficiency has not occurred in any of the patients. CONCLUSIONS: SF-1 (NR5A1) mutations should be considered in 46,XY individuals with severe (penoscrotal) hypospadias, especially if undescended testes, low testosterone, or low inhibin B/AMH levels are present. SF-1 mutations in milder forms of idiopathic hypospadias are unlikely to be common.