The human platelet ADP receptor activates G(i2) proteins

We have previously shown that platelet ADP receptors are coupled to G-proteins by measuring the binding of [³⁵S]guanosine-5'-[γ-thio]triphosphate ([³⁵S]GTPγS) to human platelet membranes stimulated with ADP. In order to identify the activated G-proteins, we used an approach which combines photolabelling of receptor-activated G-proteins with 4-azidoanilido- [α-³²P]GTP and immunoprecipitation of the G-protein α-subunits with subtype-specific antibodies. Stimulation of human platelet membranes with ADP resulted in an increase in 4-azidoanilido - [α-³²P]GTP incorporation into the immunoprecipitates of Gα(i), but not of Gα(q), proteins, whereas stimulation with the thromboxane analogue U46619 resulted in an increase in 4-azidoanilido-[α-³²P]GTP incorporation into the immunoprecipitates of Gα(q), but not of Gα(i), proteins, and thrombin activated both G-proteins. This effect of ADP was concentration dependent and inhibited by the class P₂ purinoceptor (P(2T)) antagonist ATP. Using specific antisera against subtypes of G(i) proteins, we found that ADP stimulated labelling of the Gα(i2) immunoprecipitate, but not of the Gα(i3) precipitate. Gα(i1) was not detectable by immunoblotting of platelet membrane proteins. These data suggest that ADP inhibits cAMP formation by activation of Gα(i2) proteins and add evidence in support of the hypothesis that human platelet ADP receptors do not activate PLC through G(q) activation.