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

Abstract

Although it is well established that cAMP inhibits platelet activation induced by all agonists, the thromboxane A2 signal transduction pathway was found to be particularly sensitive to such inhibition. Therefore, we examined whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A2 receptor-G-protein complex. It was found that cAMP induces protein kinase A- dependent [γ-32P]ATP labeling of solubilized membrane proteins in the region of Gα subunits, i.e. 38-45 kDa. Moreover, ligand affinity chromatography purification of thromboxane A2 receptor-G-protein complexes from these membranes revealed that 38-45-kDa phosphoproteins co-purify with thromboxane A2 receptors. Immunoprecipitation of the affinity column eluate with a Gα13 antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A2 receptor-associated Gα13 by 87 ± 27%. In separate experiments, immunopurification of Gα13 on microtiter wells coated with a different Gα13 antibody revealed that 8-Br-cAMP increased Gα13 phosphorylation by 53 ± 19%. Finally, treatment of 32P-labeled whole platelets with prostacyclin resulted in a 90 ± 14% increase in phosphorylated Gα13 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 Gα13 both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A2- mediated signaling in platelets by cAMP.