Abstract
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.
Highlights
The ability of f3"1 to stimulate pertussis toxin-catalyzed ADPribosylation of Gta [6] as did amino-terminal deletions in a recombinant Gia 3 [7]
We describe here an anti-Gto< monoclonal antibody, LAS-2, whose epitope involves the Gt13y-binding domain of Gto< and requires amino-terminal fatty acylation as well as specific Gto< amino-terminal sequence
PAGE, t ransferred to nit rocellulose, and reacted wit h antibodies LAS-2 and AS/7
Summary
Vol 270, No 12, Issue of March 24, pp. 6436-6439, 1995 Printed in U.S.A. Involvement ofN-Myristoylation in Monoclonal Antibody Recognition Sites on Chimeric G Protein a Subunits*. Were present in a Goa chimera and the protein was aminoterminally myristoylated; there was no reactivity without myristoylation It appears that the LAS-2 epitope requires both Gtu-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 Gtu' as opposed to protruding from an amino-terminal a-helix and serving as an amino-terminal membrane anchor. We describe here an anti-Gto< monoclonal antibody, LAS-2, whose epitope involves the Gt13y-binding domain of Gto< and requires amino-terminal fatty acylation as well as specific Gto< amino-terminal sequence
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