Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin p eptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these struc turally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essenti al for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further muta tional analysis of this region of MC4R demonstrated that Tyr(268) was requi red for the selective interaction with Agouti protein, because a profound l oss of the ability of Agouti protein to inhibit I-125-labeled [Nle(4),D-Phe (7)] alpha -melanocyte-stimulating hormone (MSH) binding was observed by th e single mutation of Tyr(268) to Ile, This same residue conferred selectivi ty for the MC4R selective agonist, [D-Tyr(4)]MT-II, whereas it inhibited in teraction with the MC3R-selective agonist, [Nle(4)]Lys-gamma (2)-MSH. Conve rsely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [D-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Me-4]Lys-gamma (2)-MSH as compared wi th wild-type MC3R. This single amino acid mutation thus confers the selecti vity of MC3R toward a pharmacological profile like that observed for MC4R a gonists but not for the antagonist, Agouti protein. Thus, selectivity for s tructurally unrelated ligands with opposite activities is achieved in a sim ilar manner for MC4R but not for MC3R.