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-opiomelanocortin peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), to the murine melanocortin-1 receptor (MC1-R). We report the purification of a secreted murine agouti to homogeneity by a two-step procedure from baculovirus-infected Trichoplusia ni (T. ni). The protein is glycosylated and exhibits competitive, high-affinity antagonism (Ki = 0.8 nM) versus alpha-MSH in cell-based assays employing B16F10 cells. Association state analysis by analytical ultracentrifugation reveals that agouti exists in a monomer--dimer plus aggregate equilibrium at low micromolar concentrations. Data from secondary structure studies indicate that the protein is highly stable to thermal denaturation. Enzymatic 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 agouti. This bioactive domain contains all 10 cysteines which exhibit sequence homology when aligned with several conotoxins.
Willard
D H
DH
Department of Biochemistry, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
Bodnar
W
W
Harris
C
C
Kiefer
L
L
Nichols
J S
JS
Blanchard
S
S
Hoffman
C
C
Moyer
M
M
Burkhart
W
W
Weiel
J
J
D054366:Agouti Signaling Protein; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D018680:Cholinergic Antagonists; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000235:genetics; D009036:Moths / Q000166:cytology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D011979:Receptors, Pituitary Hormone / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D000521:alpha-MSH / Q000378:metabolism*
D054366:Agouti Signaling Protein; D018680:Cholinergic Antagonists; D036341:Intercellular Signaling Peptides and Proteins; D008978:Mollusk Venoms; D010446:Peptide Fragments; D011506:Proteins; D011979:Receptors, Pituitary Hormone; D011994:Recombinant Proteins; D000521:alpha-MSH; C029446:MSH receptor; D003545:Cysteine