Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter (Culter alburnus).

Melanocortin-4 receptor (MC4R) plays important roles in regulation of multiple physiological processes, and interaction of MC4R and melanocortin receptor accessory protein 2 (MRAP2) is suggested to play pivotal role in energy balance of vertebrates. Topmouth culter (Culter alburnus) is an economically important freshwater fish in China. Herein we cloned culter mc4r, mrap2a, and mrap2b. Culter mc4r consisted of a 981 bp open reading frame encoding a protein of 326 amino acids. qRT-PCR revealed that mc4r, mrap2a, and mrap2b were primarily expressed in the central nervous system. In the periphery, mc4r and mrap2b were expressed more widely in the male, while mrap2a was expressed more widely in the female. Culter MC4R could bind to four peptide agonists and increase intracellular cAMP production dose dependently. Culter MC4R was constitutively active in both cAMP and ERK1/2 pathways, which was differentially regulated by culter MRAP2a and MRAP2b. Culter MRAP2a significantly increased Bmax and decreased agonist-stimulated cAMP, while MRAP2b increased cell surface and total expression but did not affect Bmax and agonist-stimulated cAMP. These results will aid the investigation of the potential physiological processes that MC4R might be involved in topmouth culter.