Cyclic AMP-dependent phosphorylation of thromboxane A2 receptor-associated Gα13

Although it is well established that cAMP inhibits platelet activation induced by all agonists, the thromboxane A(2) signal transduction pathway was found to be particularly sensitive to such inhibition. Therefore, we examined whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A(2) receptor-G-protein complex. It was found that cAMP induces protein kinase A-dependent [gamma-P-32]ATP labeling of solubilized membrane proteins in the region of G alpha subunits, i.e. 38-45 kDa. Moreover, ligand affinity chromatography purification of thromboxane A(2) receptor-G-protein complexes from these membranes revealed that 38-45-kDa phosphoproteins co-purify with thromboxane A(2) receptors. Immunoprecipitation of the affinity column eluate with a G alpha(13) antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A(2) receptor-associated G alpha(13) by 87 +/- 27%. In separate experiments, immunopurification of G alpha(13) On microtiter wells coated with a different G alpha(13) antibody revealed that 8-Br-cAMP increased G alpha(13) phosphorylation by 53 +/- 19%. Finally, treatment of P-32-labeled whole platelets with prostacyclin resulted in a 90 +/- 14% increase in phosphorylated Ga-13 that was abolished by pretreatment with the adenylate cyclase inhibitor MDL-12. These results provide the first evidence that protein kinase A mediates phosphorylation of G alpha(13) both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A(2)-mediated signaling in platelets by cAMP.