Substituted NDP-MSH Peptides Paired with Mutant Melanocortin-4 Receptors Demonstrate the Role of Transmembrane 6 in Receptor Activation

Abstract

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis and is a potential therapeutic target for obesity and cachexia. Molecular interactions between peptide ligands and MC4R have been studied in detail. Less is known regarding the role of these interactions in the mechanism of MC4R activation. The aim of this study was to investigate the molecular mechanism of human MC4R activation by [Nle4, d-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH), by first defining the role of the His6-d-Phe7-Arg8-Trp9 residues in receptor activation (Emax for stimulation of cAMP accumulation) using modified peptides, then understanding how their interaction with the receptor modulates activation using site-directed mutagenesis and a molecular model of NDP-MSH bound to the active state of the receptor. Alanine substitution indicated that the d-Phe7, Arg8, and Trp9 side chains contribute binding energy but are not essential for the receptor activation event. Conversely, His6 to Ala6 substitution reduced receptor activation but did not affect affinity. Chlorine substitutions on the d-Phe7 side chain also inhibited receptor activation. F261(6.51)A and F284(7.35)A receptor mutations acted as gain-of-function mutations, restoring efficacy to the His6 and d-Phe7 substituted peptides that had lost efficacy at the wild-type receptor. Based on a model of NDP-MSH and MC4R interaction, the antagonist behavior of these peptides is consistent with the prevention of transmembrane 6 (TM6) rotation. This data supports the hypothesis that increasing the size of d-Phe7 directly interferes with TM6 rotation, preventing receptor activation. We further propose that removing the interaction with the His6 side chain reorients the peptide within the binding pocket, indirectly impeding TM6 rotation by strengthening peptide interaction with F261(6.51) and F284(7.35). These findings refine the molecular basis for the mechanism of ligand-stimulated hMC4R activation and will be useful for the development of hMC4R agonists and antagonists.