EVIDENCE FOR ALTERNATE POINTS OF ATTACHMENT FOR ALPHA-MSH AND ITS STEREOISOMER [NLE(4),D-PHE(7)]-ALPHA-MSH AT THE MELANOCORTIN-1 RECEPTOR

The molecular basis for the alpha-melanocyte stimulating hormone (alpha-MSH) stereoselectivity was studied by examining ligand binding to site specific mutants of the melanocortin 1 receptor (MC1R). The amino acids Asp(117), phe(179), His(209) and His(260) were targeted for mutation to alanine as they are conserved in the melanocortin receptor family. Expression of the wild type and the MC1R mutants in COS-7 cells revealed that the binding affinities for the alpha-MSH (L-isomer) was reduced by 267 fold for the D-117-->A mutant and 132 fold for the H-260-->A mutant. In contrast, the binding affinity for the [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH; D-isomer) remain unchanged between the wild type and the mutants. Moreover, the mutants also displayed reduction in affinity to L-isomers of all the other melanocortic peptides examined. Thus, the mutation of single amino acids in the third and sixth transmembrane segments results in the display of ligand stereoselertivity of the MC1R. In addition, these data represent the first evidence of the different binding epitopes on a G-protein coupled receptor for a peptide ligand stereoisomers, both of which are shown to be potent agonists.(C) 1994 Academic Press, Inc.