Mutations in the Carboxyl Terminus of the Agouti Protein Decrease Agouti Inhibition of Ligand Binding to the Melanocortin Receptors

Abstract

Several mutations that cause ectopic expression of the agouti gene result in obesity, hyperinsulinemia, and yellow coat color. A candidate pathway for agouti induced obesity and hyperinsulinemia is through altered signaling by melanocortin receptors, as agouti normally regulates coat coloration through antagonism of melanocortin receptor 1. Furthermore, melanocortin peptides mediate functions including steroidogenesis, lipolysis, and thermoregulation. We report apparent inhibition dissociation constants for mouse and human agouti protein inhibition of ligand binding to the melanocortin receptors, to determine which of these receptors might be involved in agouti induced diabetes. The similarity 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: K(I) app = 140 +/- 56 and 70 +/- 18 nM, respectively) suggests that the MC3-R is a potential candidate for a receptor mediating the effects of agouti protein overexpression. Agouti residues important for melanocortin receptor inhibition were identified through the analysis of deletion constructs and site-specific variants. Val83 is important for inhibition of binding to MC1-R (K(I) app for Val83Ala agouti increased 13-fold relative to wild-type protein). Arg85, 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 essentially unchanged at MC1-R, while they have increased 6-10-fold relative to wild-type protein at MC3-R and MC4-R.

Down-regulation of Melanocortin Receptor Signaling Mediated by the Amino Terminus of Agouti Protein in XenopusMelanophores

“…Dose-response curve at different antagonist concentrations were fitted individually to a sigmoid logistic; in each case, the minima, maxima, and slopes of individual dose-response curves were identical. (36,52). None of these residues are conserved in Agrp, which suggests that they are not crucial for antagonist binding and instead may play a role in protein folding and/or stability or, as described here, a signaling role distinct from inhibition of ␣-MSH binding.…”

The hypothalamus and the regulation of energy homeostasis: lifting the lid on a black box

“…Generation of an MC4-R 'knockout' mouse produced a syndrome of obesity very similar to that demonstrated by the A VY mouse . Additionally, agouti peptide was shown to act as a highly specific functional antagonist at MC4-R, with over 100-fold greater selectivity as compared with its activity at MC3-R (Lu et al 1994;Fan et al 1997); however, subsequently, this degree of selectivity has been questioned by Kiefer et al (1997), who reported a similar affinity at MC3-R and MC4-R. However, administration of the potent α-MSH analogue, MT-II, reduces food intake, while injection of the agouti mimetic, SHU9119, stimulates feeding; both these peptides demonstrate at least 10-fold higher selectivity for MC4-R over MC3-R (Fan et al 1997).…”

Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo

“…The conclusion that Agouti protein is not a simple competitive antagonist of melanocortin receptors may help to explain the discrepancy between estimates of Agouti protein affinity based on functional compared with binding assays (Lu et al 1994;Blanchard et al 1995;Willard et al 1995;Kiefer et al 1997;Yang et al 1997). Furthermore, the observation that Agouti protein does Phenotypic classes were tallied, and genotypes were determined using molecular techniques as described in Materials and Methods only for the last cross, which represents two breeding pairs.…”