Kelley-Hickie, Leanne P.Leanne P.Kelley-HickieKinsella, B. ThereseB. ThereseKinsella2011-09-022011-09-022006 Elsev2006-09Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids1388-1981http://hdl.handle.net/10197/3146Thromboxane (TX) A2 is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA2 mediated through the TPbeta isoform of the human TXA2 receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.1329458 bytesapplication/pdfenThis is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1761 (9): 1114-1131 DOI 10.1016/j.bbalip.2006.07.012.Thromboxane receptorDesensitizationPhosphorylationPhospholipase CG protein coupled receptorG protein coupled receptor kinaseArrestinThromboxanesPhosphorylationPhospholipase CG proteinsReceptors, ThromboxanePhosphorylationType C PhospholipasesReceptors, G-Protein-CoupledArrestinJournal Article176191114113110.1016/j.bbalip.2006.07.012https://creativecommons.org/licenses/by-nc-sa/1.0/