Functional characterization and pharmacological rescue of the human and porcine neural melanocortin receptors

Abstract

During the past decade, numerous studies showed that both melanocortin-3 and -4 receptors (MC3R and MC4R) are critically involved in regulation of mammalian energy balance. The MC4R plays a vital role in regulation of food intake and energy expenditure, and the MC3R is critical in regulation of fat storage/adiposity. In this study, porcine (p) MC3R was cloned, expressed and functionally characterized. Sequence analysis revealed that pMC3R was highly homologous (>80%) at nucleotide and amino acid sequences to human, rat, and mouse MC3Rs. Pharmacological analysis showed that pMC3R bound NDP-MSH with the highest affinity followed by D-Trp8-?-MSH, ?-MSH and a-MSH. Both pMC3R and hMC3R bound Agouti-related protein (AgRP) with high affinity. D-Trp8-?-MSH was the most potent agonist to stimulate cAMP generation followed by NDP-MSH, ?-MSH, and a-MSH. These results suggest that D-Trp8-?-MSH is the best tool for in vivo studies. Two naturally occurring pMC4R mutants, D298N and R236H, were functionally characterized. Both mutant pMC4Rs had similar binding capacities and affinities for the natural agonist a-MSH and the natural antagonist AgRP as wild-type (WT) pMC4R. In signaling assays, both mutants had normal EC50 and maximal signaling to a-MSH. Thus, both mutants do not have any overt functional defects. Therefore we urge caution using these variants as genetic markers for selection in breeding programs. In addition, ten human (h) MC4R mutants were functionally characterized. The results showed that R7C had decreased cell surface expression but had similar binding capacity and signaling potency as WT MC4R. C84R, S127L, W174C and F261S were defective in either total or cell surface expression or both resulting in mutants with impaired function. A219V had normal total and cell surface expressions; however, its ability for binding and signaling were significantly reduced. P230L had decreased cell surface expression but had similar signaling potency as the WT hMC4R. S136F had normal total and cell surface expression and ligand binding but was defective in signaling. I317V and L325F might be polymorphisms of hMC4R. We also tested the effect of an MC4R antagonist, ML00253764, on the cell surface expression of C84R and W174C. The results showed that the cell surface expression and signaling of the mutants were rescued. These results suggested a novel way to treat obese patients carrying intracellularly retained MC4R mutations.