RAPID, AGONIST-DEPENDENT PHOSPHORYLATION IN-VIVO OF HUMAN THROMBOXANERECEPTOR ISOFORMS - MINIMAL INVOLVEMENT OF PROTEIN-KINASE-C

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet ago nist. Its biological function is tightly regulated, G protein-coupled membrane receptors transduce the effects of TxA(2). However, although a single thromboxane receptor (TP) gene has been identified, two splic e variants have been cloned from human placenta and megakaryocytic lin es (TP alpha) and from human endothelial cells (TP beta). These differ in the length of their carboxyl-terminal extensions (15 versus 79 res idues), which contain multiple potential sites for receptor phosphoryl ation. Given that TP agonists activate protein kinase C (PKC), it woul d seem possible that PRC-dependent phosphorylation of Tps might play a central role in homologous desensitization of these receptors, To det ermine if the TP isoforms were differentially phosphorylated in respon se to agonist in vivo, human embryonic kidney (HEK) 293 cells were sta bly transfected with TP alpha and TP beta. Isoform-specific anti-pepti de antibodies were developed and used to immunoprecipitate the phospho rylated receptors, U46619, a PGH(2)/TxA(2), mimetic, induced specific phosphorylation of both isoforms. Phosphorylation of the two isoforms was similar in dose and time dependence, reaching a plateau at around 100 nM U46619, Inhibition of PKC with either GF 109203X (5 mu M) or RO 31-8220 (5 mu M) or of protein kinase A with H-89 (50 mu M) marginall y influenced agonist-dependent phosphorylation of either isoform and f ailed to modulate homologous desensitization of agonist-induced stimul ation of inositol phosphate formation. Similar results were obtained w hen PRC was down-regulated by long term incubation with the phorbol es ter, phorbol myristate acetate, Although short term stimulation with p horbol myristate acetate caused PKC-dependent phosphorylation of TPs i n vivo, thrombin stimulation of the TP-transfected HEK cells in vivo f ailed to phosphorylate either of the TP isoforms, Thus, despite the ca pacity of PRC to phosphorylate TPs in HER 293 cells and the likely act ivation of PRC by TP stimulation, this enzyme, like protein kinase A, contributes marginally to rapid, agonist-induced phosphorylation of ei ther TP isoform.