Molecular basis for the interaction of [Nle⁴,D-Phe⁷]melanocyte stimulating hormone with the human melanocortin-1 receptor (melanocyte α- MSH receptor)

The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G- protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and α-, β-, and γ- melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent α-MSH analog [Nle⁴,D-Phe⁷]MSH (NDP-MSH) and the human MC1R we examined the effects of site-directed receptor mutagenesis on the binding affinity and potency of NDP-MSH. In addition, we also examined the effects of these same mutations on the binding affinity and potency of the structurally related agonists α- MSH, γ-MSH, and Ac-Nle⁴-cyclic-[Asp⁵,His⁶,D- Phe⁷,Arg⁸,Trp⁹,Lys¹⁰]NH² (MT-II). Mutagenesis of acidic receptor residues Glu⁹⁴ in TM2 and Asp¹¹⁷ or Asp¹²¹ in TM3 significantly altered the binding affinity and potency of all four agonists suggesting that these receptor residues are important to the ligand-receptor interactions of all. A disproportionate change in agonist potency versus affinity observed with simultaneous mutation of these acidic residues (mutant constructs D117A/D121A or E94A/D117A/D121A) or introduction of a single positive charge (mutant construct D121K) also implicates these residues in receptor activation. In addition, results from the individual mutation of aromatic receptor residues Phe¹⁷⁵, Phe¹⁹⁶, and Phe²⁵⁷, and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand- receptor interactions between these melanocortins and the hMC1R.