Cyclic AMP-dependent phosphorylation of thromboxane A(2) receptor-associated Galpha(13).
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.
Manganello
J M
JM
Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA.
Djellas
Y
Y
Borg
C
C
Antonakis
K
K
Le Breton
G C
GC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D017482:Receptors, Thromboxane / Q000097:blood* / Q000302:isolation & purification
D008565:Membrane Proteins; D010446:Peptide Fragments; D017482:Receptors, Thromboxane; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins