Homologous desensitization of signalling by the alpha (α) isoform of the human thromboxane A ₂ receptor: A specific role for nitric oxide signalling

Abstract

Thromboxane (TX) A ₂ plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TPβ isoform of the human TXA ₂ receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TPα isoform. TPα undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA ₂ mimetic U46619 but, unlike that of TPβ, this is independent of GRKs. Similar to TPβ, TPα undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser ¹⁴⁵ within intracellular domain (IC) ₂ represents the key phospho-target. TPα also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr ³³⁷ and Ser ³³¹, respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors l-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TPα involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser ¹⁴⁵ partially and transiently impairs TPα signalling while PKG- and PKC-phosphorylation at both Ser ³³¹ and Thr ³³⁷, respectively, within its C-tail domain profoundly desensitizes TPα, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TPα.