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pubmed:abstractText |
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 Ser6, Glu102, Arg109, Asp184, Glu269, and Thr272. The mutant receptor DNAs were transiently expressed in COS-1 cells and their ability to bind [N1e4,D-Phe7]-alpha-MSH (NDP-MSH) was evaluated. Substitution of Asp184 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 Ser6, Glu269 and Thr272 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.
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