Comparative Functional Alanine Positional Scanning of the α-Melanocyte Stimulating Hormone and NDP-Melanocyte Stimulating Hormone Demonstrates Differential Structure-Activity Relationships at the Mouse Melanocortin Receptors

Abstract

The melanocortin system has been implicated in the regulation of various physiological functions including melanogenesis, steroidogenesis, energy homeostasis, and feeding behavior. Five melanocortin receptors have been identified to date and belong to the family of G protein-coupled receptors (GPCR). Post-translational modification of the proopiomelanocortin (POMC) prohormone leads to the biosynthesis of the endogenous melanocortin agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, γ-MSH, and adrenocorticotropic hormone (ACTH). All the melanocortin agonists derived from the POMC prohormone contain a His-Phe-Arg-Trp tetrapeptide sequence that has been implicated in eliciting the pharmacological responses at the melanocortin receptors. Herein, an alanine (Ala) positional scan is reported for the endogenous α-MSH ligand and the synthetic, more potent, NDP-MSH peptide (Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH2) at the cloned mouse melanocortin receptors to test the assumption that the structure-activity relationships of one ligand would apply to the other. Several residues outside of the postulated pharmacophore altered potency at the melanocortin receptors, most notably the 1560-, 37-, and 15-fold potency loss when the Glu(5) position of α-MSH was substituted with Ala at the mMC1R, mMC3R, and mMC4R, respectively. Importantly, the altered potencies due to Ala substitutions in α-MSH did not necessarily correlate with equivalent Ala substitutions in NDP-MSH, indicating that structural modifications and corresponding biological activities in one of these melanocortin ligands may not be predictive for the other agonist.

Keywords: GPCR; Melanotropin; NDP-MSH; alanine positional scan; obesity; α-MSH.

Figure 1

Amino acid sequence of the endogenous POMC melanocortin agonists and NDP-MSH synthetic analog. The numbering of α-MSH is indicated. The tetrapeptide sequence His-Phe-Arg-Trp, common to all naturally occurring MCR agonists, is indicated in bold. The more potent NDP-MSH ligand is modified from α-MSH by replacing Met⁴ with Nle and inverting the stereochemistry at the Phe⁷ position.

Figure 2

(A) Graphical illustration summarizing the ligand potency of an Ala positional scan of α-MSH at the mouse melanocortin receptors. (B) Graphical illustration summarizing the ligand potency of an Ala positional scan of NDP-MSH at the mouse melanocortin receptors.

Figure 3

(A) Amino acid sequence and numbering of α-MSH and NDP-MSH. (B) Graphical illustration comparing the fold difference of Ala substitutions in α-MSH and NDP-MSH at the mMC1R, mMC3R, mMC4R, and mMC5R. Fold difference is defined as the EC₅₀ value of a particular substitution divided by the EC₅₀ value of the native sequence of the parent ligand.