Abstract
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-(32)P]ATP labeling of solubilized membrane proteins in the region of Galpha 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 Galpha(13) antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A(2) receptor-associated Galpha(13) by 87 +/- 27%. In separate experiments, immunopurification of Galpha(13) on microtiter wells coated with a different Galpha(13) antibody revealed that 8-Br-cAMP increased Galpha(13) phosphorylation by 53 +/- 19%. Finally, treatment of (32)P-labeled whole platelets with prostacyclin resulted in a 90 +/- 14% increase in phosphorylated Galpha(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 Galpha(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.
Highlights
Thromboxane A2 (TXA2)1 is a potent platelet agonist and vasoconstrictor (1, 2) that modulates the hemostatic process and potentiates thrombotic events such as myocar
Treatment of 32P-labeled whole platelets with prostacyclin resulted in a 90 ؎ 14% increase in phosphorylated G␣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␣13 both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A2-mediated signaling in platelets by cAMP
It is well known that platelet activity is inhibited by elevated cAMP levels, and it is generally accepted that this inhibitory effect is mediated by protein kinase A (PKA)-dependent phosphorylation of different protein substrates
Summary
Materials—Outdated platelet units were obtained from Heartland Blood Center (Aurora, IL) and Life Source Blood Services (Chicago, IL); BM13.177 was generously supplied by Dr K. The anti-peptide G-13 antibody, directed against an internal G␣13 sequence, (Table I) was raised in rabbits according to standard protocols, and the IgG fraction was purified by protein A-Sepharose chromatography as described (17). Solubilized platelet membranes were prepared as described (52) and typically resulted in a final protein concentration of 4.5 mg/ml. The platelet-rich plasma was supplemented with 40 nM PGI2, and platelets were pelleted by centrifugation at 1600 ϫ g for 20 min. The resulting platelet pellet was resuspended in buffer A, was sonicated on ice (45-s bursts with 15-s rests) for 4 min, and was centrifuged at 1600 ϫ g for 5 min. The resulting membrane pellet was resuspended in buffer A to yield a final protein concentration of 2– 4 mg/ml. Silver-stained gels and autoradiographic films were analyzed by using a Protein Databases, Inc. densitometer (Huntington Station, NY)
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