Covalent binding of arachidonate to G protein alpha subunits of human platelets.
The alpha subunits of GTP-binding regulatory proteins (G proteins) are subject to lipid modifications required for anchorage to membrane and/or interactions with other proteins. With the knowledge that alpha subunits are palmitoylated, which we demonstrate here for human platelets, we sought to determine whether these subunits also bind arachidonate and myristate in a covalent, post-translational manner. All alpha subunits examined were found to incorporate radioactivity upon incubation of human platelets with [3H]palmitate, [3H]arachidonate, and [3H]myristate. The identity of [3H]palmitate and [3H]arachidonate as covalently bound fatty acids was confirmed by high pressure liquid chromatography following alkaline methanolysis. With [3H]myristate, however, the bound fatty acid proved to be [3H]palmitate, presumably generated by a 2-carbon chain elongation. Protein-bound [3H]palmitate and [3H]arachidonate were released by hydroxylamine at neutral pH, implying a thioester linkage between protein and fatty acid. Thus, post-translational modifications of G protein alpha subunits include palmitoylation and arachidonoylation, but not myristoylation. Given the different physical properties of saturated and unsaturated fatty acids and the large-scale release of arachidonate during platelet activation, changes in arachidonate incorporation may serve as an important regulator of alpha subunit function.
Hallak
H
H
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Muszbek
L
L
Laposata
M
M
Belmonte
E
E
Brass
L F
LF
Manning
D R
DR
D000527:Alprostadil / Q000494:pharmacology; D016718:Arachidonic Acid / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D046911:Macromolecular Substances; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000097:blood; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D014316:Tritium
D006898:Hydroxylamines; D046911:Macromolecular Substances; D009227:Myristic Acids; D010169:Palmitic Acids; D019814:Myristic Acid; D014316:Tritium; D016718:Arachidonic Acid; D019811:Hydroxylamine; D019308:Palmitic Acid; D019204:GTP-Binding Proteins; D000527:Alprostadil