Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the hu man melanocortin-4, receptor (hMC4R), agonist structure-activity studies an d receptor point mutagenesis were pet-formed. Structure-activity studies of [Nle(4),D-Phe(7)]-alpha -melanocyte stimulating hormone (NDP-MSH) identifi ed D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full ago nist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R trans membrane (TM) residues were mutated (the rationale for choosing specific re ceptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these re ceptor residues as sites potentially involved in the sought after ligand-re ceptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found th at core melanocortin peptide residue Arg8 interacts at a molecular level wi th hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to ala nine decreased the binding affinity of AGRP (87-132), a C-terminal derivati ve of the endogenous melanocortin antagonist, 8-fold, and simultaneous muta tions D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4 R antagonist SHU 9119. These results provide further insight into the molec ular determinants of hMC4R ligand binding.