Pharmacological Characterization of 40 Human Melanocortin-4 Receptor Polymorphisms with the Endogenous Proopiomelanocortin-Derived Agonists and the Agouti-Related Protein (AGRP) Antagonist^,

Abstract

The melanocortin-4 receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating energy homeostasis and obesity. Up to a remarkable 6% of morbidly obese adults and children studied possess single nucleotide polymorphisms (SNPs) of the MC4R. Upon stimulation by agonist, the MC4R signals through the intracellular adenylate cyclase signal transduction pathway. Posttranslational modification of the pro-opiomelanocortin (POMC) gene transcript results in the generation of several endogenous melanocortin receptor agonists including alpha-, beta-, gamma-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH) ligands. The endogenous MC4R antagonist, agouti-related protein (AGRP), is expressed in the brain and is only one of two naturally occurring antagonists of GPCRs identified to date. Herein, we have generated 40 hMC4 polymorphic receptors and evaluated their cell surface expression by flow cytometry as well as pharmacologically characterized their functionality using the endogenous agonists alpha-MSH, beta-MSH, gamma2-MSH, ACTH(1-24), the antagonist hAGRP(87-132), and the synthetic agonists NDP-MSH and MTII. This is the first study in which polymorphic hMC4Rs have been pharmacologically characterized simultaneously with multiple endogenous ligands. Interestingly, at the N97D, L106P, and C271Y hMC4Rs beta-MSH was more potent than the other endogenous agonists alpha-MSH, gamma2-MSH, ACTH(1-24). The S58C and R165Q/W hMC4Rs possessed significantly reduced endogenous agonist potency (15- to 90-fold), but the synthetic ligands NDP-MSH and MTII possessed only 2-9-fold reduced potency as compared to the wild-type receptor, suggesting their potential as therapeutic ligands to treat individuals with these polymorphisms.

Mouse models for the central melanocortin system

“…About 6% of severe obese subjects carry point mutations in or near the MC4R gene [15,38,50]. Investigation of signaling activities of these mutant MC4Rs in assays based on cell culture demonstrated that the characteristic of obesity correlates with dysfunctions in receptor properties [14,25,39,52]. The most common MC4R polymorphism V103I is negatively associated with obesity [18], a finding meanwhile replicated in several independent studies [18,23,47,53].…”

“…Despite of a negative association of V103I with obesity, a strong pharmacological phenotype of this mutant receptor expressed in cell culture was not identified. Only one report demonstrated a lower potency of orexigenic AGRP on V103I corresponding to the distribution of this allele in population screens [52]. Several point mutations in the Mc4r gene introduced by chemical mutagenesis have been shown to cause different severity of obesity in mice [40].…”

Increased constraints on MC4R during primate and human evolution

“…Only one of the 11 non-synonymous mutations (N62S) exhibited a reduced function, as indicated by a strongly reduced cell surface expression, and by signal transduction Table 2 MC4R functional assays MC4R cAMP accumulation and cell surface expression assays. Each amino acid replacement observed in the HGDP-CEPH panel is found in Table. Three sites (R7C, V103I, I251L) were not functionally tested in this study as these sites have been identiWed and assayed previously (Srinivasan et al 2004;Stutzmann et al 2007;Xiang et al 2006). Construct properties that did not result in an enhancement of intracellular cAMP levels (Table 2).…”

Controversial association results for INSIG2 on body mass index may be explained by interactions with age and with MC4R

“…INSIG2 rs7566605 and MC4R rs2229616 were the first SNPs with a replicated polygenic effect on body mass in the general population 3,4 and known functional implications. 5,6 The INSIG2 region has been connected with obesity in human linkage studies 7 as well as in one of the first genome-wide association studies (GWAS) 3 to address obesity. This GWAS identified common variant rs7566605, located 10-kb upstream of the INSIG2 transcription start site.…”

“…1,27 MC4R harbours several susceptibility loci for obesity. 6,28,29 Known functional SNPs in MC4R are all rare, have been found mostly in obesity studies and mostly associate with elevated BMI. The missense variant V103I (rs2229616), however, is relatively frequent and well-studied in population-based samples.…”