Involvement of N-myristoylation in monoclonal antibody recognition sites on chimeric G protein alpha subunits.
Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (Gt alpha), inhibited Gt beta gamma-dependent, pertussis toxin-catalyzed ADP ribosylation of Gt alpha and was specific for Gt alpha. Immunoblotting studies on proteolytic fragments of Gt alpha were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant Gt alpha was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyl-transferase. Amino-terminal fatty acylation of Gt alpha, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of Gt alpha and the remainder Go alpha, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of Gt alpha were present in a Go alpha chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both Gt alpha-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it "folds back" on and stabilizes the amino-terminal structure of Gt alpha as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor.
Justice
J M
JM
Pulmonary Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Bliziotes
M M
MM
Stevens
L A
LA
Moss
J
J
Vaughan
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002417:Cattle; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009227:Myristic Acids; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D019204:GTP-Binding Proteins