MOLECULAR-CLONING, FUNCTIONAL EXPRESSION AND PHARMACOLOGICAL CHARACTERIZATION OF A MOUSE MELANOCORTIN RECEPTOR GENE

We describe the cloning of the mouse HGMP01A gene that encodes a melan ocortin receptor functionally distinct from the adrenal cortex cortico tropin (adrenocorticotrophic hormone; ACTH) receptor and the melanocyt e-stimulating hormone (MSH) receptor expressed in melanoma. The gene e ncodes a protein of 323 amino acids with a calculated molecular mass o f 35800 Da, displaying potential sites for N-linked glycosylation and phosphorylation by protein kinase C. An RNAase protection assay detect ed weak expression in the brain, but not in adrenal gland, skin, or an y of the other tissues tested. Stable CHO cell lines expressing over 1 00000 receptors per cell were generated. The recombinant receptor bind s iodinated [Nle(4),D-Phe(7)]alpha-MSH (NDP-MSH) with an apparent K-d of 700 pM. Displacement of she ligand by a variety of pro-opiomelanoco rtin-derived peptides revealed a pharmacological profile distinct from that of the classical ACTH and MSH receptors. NDP-MSH was the most po werful competitor (IC50 1.4 nM), followed by gamma-MSH (IC50 7 nM). al pha-MSH, beta-MSH and ACTH-(1-39) were significantly less potent, with IC50 values of 30, 19 and 21 nM respectively. ACTH-(4-10) was poorly active (IC50 2.4 mu M), while corticotropin-like intermediate lobe pep tide (CLIP) and beta-endorphin were totally ineffective. The recombina nt receptor was found to stimulate adenylate cyclase. The potency orde r of the agonists in this assay was consistent with that of the bindin g displacement assays. This receptor represents the orthologue of the human melanocortin 3 receptor reported recently. The growing family of melanocortin receptors constitute the molecular basis for the variety of actions of melanocortins that have been described over the years. The availability of functionally expressed receptors from the melanoco rtin family will allow the development of a specific pharmacology, and a better understanding of the function of the pro-opiomelanocortin-de rived peptides.