AGOUTI ANTAGONISM OF MELANOCORTIN BINDING AND ACTION IN THE B16F10 MURINE MELANOMA CELL-LINE

Several dominant mutations at the murine agouti locus result in the ex pression of a number of phenotypic changes, including a predominantly yellow coat color, obesity, and hyperinsulinemia. The mutants exhibit ectopic overexpression of normal agouti protein, suggesting that agout i regulates coat coloration by direct antagonism of the alpha-melanocy te-stimulating hormone receptor. We have tested this hypothesis by exa mining agouti inhibition of both melanocortin-stimulated cyclic adenos ine monophosphate production and the binding of a radioactive melanoco rtin analog in the murine B16F10 melanoma cell line. Inhibition of mel anocortin-induced cyclic nucleotide accumulation did not require prein cubation of the cells with agouti and was independent of the agonist u sed. Furthermore, inhibition of both agonist binding to and activation of melanocortin receptor could be described by a simple competitive m odel with similar inhibition constants of 1.9 and 0.9 nM, respectively . The mutually exclusive binding of agouti and melanocortin was verifi ed by cross-linking experiments using a radiolabeled alpha-melanocyte- stimulating hormone analog. Competitive inhibition of tr-melanocyte-st imulating hormone binding can account for the effects of agouti on coa t coloration and suggests the possibility that the other phenotypic ch anges observed on agouti overexpression may be due to direct action of agouti at a novel melanocortin receptor(s).