THIOPHOSPHORYLATION OF THE G-PROTEIN BETA-SUBUNIT IN HUMAN PLATELET MEMBRANES - EVIDENCE AGAINST A DIRECT PHOSPHATE TRANSFER-REACTION TO G(ALPHA)SUBUNITS

A direct phosphate transfer reaction from the G protein beta subunits to either G(s alpha) or G(i alpha) has been proposed to account for th e ability of thiophosphorylated transducin beta gamma-dimers to bidire ctionally regulate adenylyl cyclase activity in human platelet membran es. We searched for experimental evidence for this reaction. Incubatio n of human platelet membranes with [S-35]guanosine-5'-(3-O-thio)tripho sphate ([S-35]GTP gamma S) results in the predominant incorporation of [S-35]thiophosphate into a 36-kDa protein, which comigrates with the G protein beta subunit and is immunoprecipitated by a beta subunit-spe cific antiserum. Thiophosphorylation of the beta subunit is specific f or guanine nucleotides and abolished by the histidine-modifying agent diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of [S-35]thiophosphorylated beta subunits is accelerated in the presence of GDP, but not ADP, UDP, or guanosine-5'-(2-O-thio)diphosphate. Neit her the thiophosphorylation nor the dephosphorylation is sensitive to receptor agonists (alpha(2)-adrenergic, A(2) adenosine, thrombin, or i nsulin), and purified G protein alpha subunits do not act as thiophosp hate donors. An approach was designed to demonstrate direct thiophosph ate transfer to protein-bound nucleotides; platelet membranes were seq uentially exposed to NaIO4, NaCNBH3, and NaBH4, an oxidation-reduction step that covalently incorporates prebound nucleotides into proteins. Under these conditions, multiple radiolabeled proteins are visualized on subsequent addition of [S-35]GTP gamma S. This reaction is specifi c because both oxidation and reduction are required and pretreatment o f platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyroc arbonate blocks the subsequent labeling in oxidized and reduced membra nes. The G protein beta subunit may participate in this thiophosphate transfer reaction. Most important, however, no labeled G protein alpha subunits (G(s alpha)) and G(i alpha) were recovered by immunoprecipit ation from oxidized and reduced membranes subsequent to the addition o f [S-35]GTP gamma S. Thus,our results clearly rule out the existence o f a postulated G protein activation by phosphate transfer reactions, w hich lead to the formation of GTP from GDP prebound to the or subunit.