MUTATIONS IN THE CARBOXYL-TERMINUS OF THE AGOUTI PROTEIN DECREASE AGOUTI INHIBITION OF LIGAND-BINDING TO THE MELANOCORTIN RECEPTORS

Several mutations that cause ectopic expression of the agouti gene res ult in obesity, hyperinsulinemia, and yellow coat color. A candidate p athway for agouti induced obesity and hyperinsulinemia is through alte red signaling by melanocortin receptors, as agouti normally regulates coat coloration through antagonism of melanocortin receptor 1. Further more, melanocortin peptides mediate functions including steroidogenesi s, lipolysis, and thermoregulation. We report apparent inhibition diss ociation constants for mouse and human agouti protein inhibition of Li gand binding to the melanocortin receptors, to determine which of thes e receptors might be involved in agouti induced diabetes. The similari ty in the apparent K-I values for agouti inhibition of ligand binding to the brain melanocortin receptors 3 and 4 (mouse: K-I app = 190 +/- 74 and 54 +/- 18 nM; human: Kr-I app = 140 +/- 56 and 70 +/- 18 nM, re spectively) suggests that the MC3-R is a potential candidate for a rec eptor mediating the effects of agouti protein overexpression. Agouti r esidues important for melanocortin receptor inhibition were identified through the analysis of deletion constructs and site-specific variant s. Val83 is important for inhibition of binding to MC1-R (K-I app for Val83Ala agouti increased 13-fold relative to wild-type protein). Arg8 5, Pro86, and Pro89 are important for selective inhibition of binding between MC1-R and MC3-R and MC4-R as their apparent K-I values are ess entially unchanged at MC1-R, while they have increased 6-10-fold relat ive to wild-type protein at MC3-R and MC4-R.