Molecular Determinants of Ligand Binding to the Human Melanocortin-4 Receptor

Abstract

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor 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 receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.

High-Resolution NMR Structure of the Chemically-Synthesized Melanocortin Receptor Binding Domain AGRP(87−132) of the Agouti-Related Protein,

“…Substantial evidence suggests that this cluster serves as a docking point for arginine found in both the HFRW message sequence of the agonist peptides and the RFF triplet of AGRP and the agouti protein (42,43). Thus, the protrusion of AGRP's active loop ( Figure 5) may allow for the RFF triplet to extend into the receptor and reach this essential negatively charged cluster.…”

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

“…Binding Assays-Binding experiments were performed using the conditions described previously (25). Briefly, after removal of the medium, cells were incubated with non-radioligand ACTH from 10 Ϫ10 to 10 Ϫ6 M in 0.5 ml of minimum Eagle's medium containing 0.2% BSA and 2 ϫ 10 5 cpm of 125 I-ACTH(1-24) for 1 h. The binding reactions were terminated by removing the medium and washing the cells twice with minimum Eagle's medium containing 0.2% BSA.…”

Interactions of Human Melanocortin 4 Receptor with Nonpeptide and Peptide Agonists,

“…Alanine-scanning mutagenesis was done in thirteen positions in the area of the putative ligand binding pocket (16,33,37,38,46), whose mutation has been shown to affect binding of linear peptide agonists to MC4R and MC1R (37,38,46 The changes in IC 50 and EC 50 values for NDP-MSH were also parallel in the majority of tested mutants, with the notable exception of H264A and D126A mutations, which led to significant reduction of NDP-MSH binding affinity such that no specific binding was detected by the filtration method.…”

“…Alanine-scanning mutagenesis was done in thirteen positions in the area of the putative ligand binding pocket (16,33,37,38,46), whose mutation has been shown to affect binding of linear peptide agonists to MC4R and MC1R (37,38,46). The mutated residues include Trp 258 , which is highly conserved in rhodopsin-like GPCRs and is presumed to be involved in activation mechanism (31) 50 values are similar for each of the three agonists at the wild type receptor.…”