Cyclic AMP-dependent phosphorylation of thromboxane A(2) receptor-associated G alpha(13)

Although it is well established that cAMP inhibits platelet activation indu ced by all agonists, the thromboxane A(2) signal transduction pathway was f ound to be particularly sensitive to such inhibition. Therefore, we examine d whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A(2) receptor-G-protein complex. It was found that cAMP induces protein ki nase 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 affinit y chromatography purification of thromboxane A(2) receptor-G-protein comple xes 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 p hosphorylation of thromboxane A(2) receptor-associated G alpha(13) by 87 +/ - 27%. In separate experiments, immunopurification of G alpha(13) On microt iter wells coated with a different G alpha(13) antibody revealed that 8-Br- cAMP increased G alpha(13) phosphorylation by 53 +/- 19%. Finally, treatmen t 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 evi dence 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.