Identification of ligand binding residues in extracellular loops of the melanocortin 1 receptor

To investigate whether residues in the extracellular domains of melanocortin 1 receptor (MC1R) are required for ligand binding, a number of mutants were constructed where charged residues were converted to alanine. The residues targeted for mutagenesis were Ser(6), Glu(102), Arg(109), Asp(184), Glu(269) and Thr(272). The mutant receptor DNAs were transiently expressed in COS-1 cells and their ability to bind [N1e(4), D-Phe(7)]-alpha-MSH (NDP-MSH) was evaluated. Substitution of Asp(184) by alanine completely abolished the binding of radiolabelled NDP-MSH as well as ACTH, even though the mutated receptor could be detected on cell surface using anti MC1R specific polyclonal antiserum. Mutations of Ser(6), Glu(269) and Thr(272) resulted in a considerable loss of affinity for radiolabelled NDP-MSH as well as the ability of alpha-MSH to displace the bound radiolabelled NDP-MSH. The results demonstrate that the extracellular loops of human MC1R contain important ligand binding epitopes. (C) 1996 Academic Press, Inc.