Homologous desensitization of signalling by the beta (β) isoform of the human thromboxane A2 receptor

Thromboxane (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 TPβ 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 TPβ. It was established that TPβ 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: TPβ 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, TPβ 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 β-arrestin association, thereby blocking TPβ internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs. TPβ 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 β-arrestin association and, in turn, facilitates TPβ internalization. Thromboxane (TX) A2 is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA2 mediated through the TPβ isoform of the human TXA2 receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPβ 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: TPβ undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 was identified as the key phospho-target. In addition, TPβ 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 β-arrestin association, thereby blocking TPβ internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPβ 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 β-arrestin association and, in turn, facilitates TPβ internalization.