Covalent binding of arachidonate to G protein α subunits of human platelets

The α 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 α 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 α 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 α 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 α subunit function.