CHARACTERIZATION OF MELANOCORTIN RECEPTOR SUBTYPES BY RADIOLIGAND BINDING ANALYSIS

The DNAs encoding three melanocortin receptor subtypes (melanocortin MC(1) receptor, melanocortin MC(3) receptor and melanocortin MC(5) receptor) were expressed individually in COS (CV-1 Origin, SV40) cells to characterise their ligand binding properties. The results indicated that [I-125][Nle(4), D-Phe(7)]alpha-MSH (melanocyte stimulating hormone) bound to a single saturable site with K-d values of 85.1 +/- 8.0 pmol/l (mean +/- S.E.M), 396 +/- 65 pmol/l and 5.05 +/- 1.00 nmol/l for melanocortin MC(1) receptor, melanocortin MC(3) receptor and melanocortin MC(3) receptor, respectively. The melanocortin MC(1) receptor and the melanocortin MC(5) receptor showed a similar potency order to the melanocortic peptides examined which was markedly different from the potency order of the melanocortin MC(3) receptor. The melanocortin MC(1) receptor and melanocortin MC(5) receptor had a relatively higher affinity for alpha-MSH than gamma-MSH and beta-MSH, whereas the melanocortin MC(3) receptor had higher affinity for desacetyl-alpha-MSH, gamma-MSH and beta-MSH compared to alpha-MSH. The inclusion of the endopeptidase inhibitor phosphoramidon to prevent the breakdown of ACTH-(1-39) (adrenocorticotrophic hormone) to alpha-MSH, decreased ACTH-(1-39) binding affinity showing that ACTH-(1-39) had a much lower affinity for melanocortin MC(1) receptor than reported earlier.