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 Gs alpha or Gi alpha has been proposed to account for the ability of thiophosphorylated transducin beta gamma-dimers to bidirectionally regulate adenylyl cyclase activity in human platelet membranes. We searched for experimental evidence for this reaction. Incubation of human platelet membranes with [35S]guanosine-5'-(3-O-thio)triphosphate ([35S]GTP gamma S) results in the predominant incorporation of [35S]thiophosphate into a 36-kDa protein, which comigrates with the G protein beta subunit and is immunoprecipitated by a beta subunit-specific antiserum. Thiophosphorylation of the beta subunit is specific for guanine nucleotides and abolished by the histidine-modifying agent diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of [35S]thiophosphorylated beta subunits is accelerated in the presence of GDP, but not ADP, UDP, or guanosine-5'-(2-O-thio)diphosphate. Neither the thiophosphorylation nor the dephosphorylation is sensitive to receptor agonists (alpha 2-adrenergic, A2 adenosine, thrombin, or insulin), and purified G protein alpha subunits do not act as thiophosphate donors. An approach was designed to demonstrate direct thiophosphate transfer to protein-bound nucleotides; platelet membranes were sequentially 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 [35S]GTP gamma S. This reaction is specific because both oxidation and reduction are required and pretreatment of platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyrocarbonate blocks the subsequent labeling in oxidized and reduced membranes. The G protein beta subunit may participate in this thiophosphate transfer reaction. Most important, however, no labeled G protein alpha subunits (Gs alpha and Gi alpha) were recovered by immunoprecipitation from oxidized and reduced membranes subsequent to the addition of [35S]GTP gamma S. Thus, our results clearly rule out the existence of a postulated G protein activation by phosphate transfer reactions, which lead to the formation of GTP from GDP prebound to the alpha subunit.