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

Abstract

To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopus melanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time-and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein.

Mahoganoid and Mahogany Mutations Rectify the Obesity of the Yellow Mouse by Effects on Endosomal Traffic of MC4R Protein

“…1). Pharmacological effects of ASP antagonism on the melanocortin receptor present in isolated Xenopus melanophores indicate that ASP action involves both time-and temperature-dependent inhibition of melanocortin signaling (38). Similar conclusions were reached in B16-F1 murine melanoma cells that express MC1R (42).…”

“…4, A and B). The predicted molecular mass of ASP after signal peptide cleavage is 11.8 kDa; however, Western blotting of mouse skin extracts for endogenous ASP detects a protein of ϳ21.5 kDa (38). The upper V5-positive band migrates at ϳ25 kDa, the anticipated molecular mass of the ASP-V5/His construct; the lower band is probably ASP that has not been fully processed (i.e.…”

The serine protease Corin is a novel modifier of the agouti pathway

“…In ventral skin, where agouti levels remain high and promote pheomelanin production throughout the hair cycle, the majority of agouti protein detected by western analysis migrates at the position expected of the full-length form (Ollmann and Barsh, 1999). Unfortunately, the low and variable level of agouti protein in dorsal skin, the limited sensitivity of available reagents, and the partial nature of the predicted cleavage events prevent direct assessment of these potential cleavage models in dorsal skin in vivo.…”

“…The fact that the N-terminal and C-terminal domains of agouti bind separately and independently to Atrn and Mc1r, respectively, and the failure of these two domains to rescue in trans the activity of full-length agouti (He et al, 2001;Ollmann and Barsh, 1999), suggest that binding of agouti to both Atrn and Mc1r forms a ternary complex that is required for agouti signaling. If so, a proteolytic cleavage that physically separates the N-terminal domain from the C-terminal domain would not only render the agouti protein inactive but could also produce competitive antagonists that interfere with agouti binding to both Mc1r and Atrn.…”

New ligands for melanocortin receptors

“…78 By contrast, the active form of Asip in tissues is the full-length protein. 82 A summary of the similarities and differences in mechanisms of Asip and Agrp action are summarized in Figure 2a. It is also interesting to consider these results in the context of a molecular evolutionary analysis (Figure 2c), in which protein alignments of Asip and Agrp from a diverse group of vertebrates were used to estimate regional levels of evolutionary constraint.…”