AGOUTI STRUCTURE AND FUNCTION - CHARACTERIZATION OF A POTENT ALPHA-MELANOCYTE-STIMULATING HORMONE-RECEPTOR ANTAGONIST

The murine agouti gene encodes for a novel 131 amino acid protein. The sequence includes a 22 residue putative secretion signal, an internal basic region, and a C-terminal domain containing 10 cysteines. Agouti has been found to antagonize the binding of certain pro-opiomelanocor tin peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH) , to the murine melanocortin-1 receptor (MC1-R). We report the purific ation of a secreted murine agouti to homogeneity by a two-step procedu re from baculovirus-infected Trichoplusia ni (T. ni). The protein is g lycosylated and exhibits competitive, high-affinity antagonism (K-i = 0.8 nM) versus alpha-MSH in cell-based assays employing B16F10 cells. Association state analysis by analytical ultracentrifugation reveals t hat agouti exists in a monomer-dimer plus aggregate equilibrium at low micromolar concentrations. Data from secondary structure studies indi cate that the protein is highly stable to thermal denaturation. Enzyma tic digestion to probe disulfide bond arrangement yielded a discrete C -terminal (Val 83-Cys 131) domain. The isolated highly cysteine-rich C -terminal domain retains alpha-MSH antagonism equipotent with mature a gouti. This bioactive domain contains all 10 cysteines which exhibit s equence homology when aligned with several conotoxins.