Contribution of the Conserved Amino Acids of the Melanocortin-4 Receptor in d-[Nle4,Phe7]-α-Melanocyte-stimulating Hormone Binding and Signaling

Abstract Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for α-melanocortin-stimulating hormone (α-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for α-MSH binding and signaling. α-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and α-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter α-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for α-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu100 in TM2, Asp122, Asp126 in TM3 and Trp258, Phe261, His264 in TM6 are involved in α-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for α-MSH binding and signaling.