Homologous desensitization of signalling by the alpha (α) isoform of the human thromboxane A2 receptor: A specific role for nitric oxide signalling

Abstract

Thromboxane (TX) A2 plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TPβ isoform of the human TXA2 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 TXA2 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 Ser145 within intracellular domain (IC)2 represents the key phospho-target. TPα also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr337 and Ser331, 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 Ser145 partially and transiently impairs TPα signalling while PKG- and PKC-phosphorylation at both Ser331 and Thr337, 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.

Identification of an Interaction between the TPα and TPβ Isoforms of the Human Thromboxane A2 Receptor with Protein Kinase C-related Kinase (PRK) 1

“…TP␣ and TP␤ also undergo entirely distinct mechanisms of both agonist-induced (homologous) desensitization (24,25) and heterologous desensitization or cross-talk/regulation by other signaling systems such as prostacyclin and nitric oxide (23, 26 -29). Such differences in desensitization of TP␣ and TP␤ signaling occur because of differences in their phosphorylation, occurring mainly within their unique C-tail domains, by the second messenger-regulated protein kinases, including by protein kinase (PK) C, PKA, PKG, and/or by GPCR-regulated kinases 1/2 (23)(24)(25)(26)(27)29).…”

“…The cellular responses to TXA 2 are subject to regulation by both agonist-dependent homologous desensitization (24,25) and by cross-talk with other signaling systems (23,27,29), which mainly occur through direct phosphorylation of the TP␣ and/or TP␤ isoforms themselves. For example, both TP␣ and TP␤ undergo PKC phosphorylation within their IC (e.g.…”

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

“…The calcium in turn increases eNOS activity and signaling through the NO/cGMP/PKG pathway. The subsequent PKGI phosphorylation of TP␣ receptor at Ser-331 desensitizes the receptor, thereby decreasing intracellular calcium (Reid and Kinsella, 2003;Kelley-Hickie et al, 2007). Likewise, PKGI phosphorylates the transient receptor potential canonical (TRPC)-6 channel at Thr-70 and Ser-322, which inactivates the associated inward calcium current (Koitabashi et al, 2010).…”

Transcriptional Regulation of the Human Thromboxane A2 Receptor Gene by Wilms’ Tumour (WT)

“…homologous (15,16) and heterologous (17)(18)(19) desensitization to differentially regulate their intracellular signalling. Most notably, signalling by TPα, but not by TPβ, is completely desensitized/inhibited by the counter-regulatory anti-platelet and vasodilatory agents prostacyclin/prostaglandin I 2 and nitric oxide, which is mediated by direct protein kinase (PK) A and PKG phosphorylation of TPα at Ser 329 and Ser 331 , respectively, the very first residues within its unique carboxyl-terminal tail domain of TPα and divergent from those of TPβ (13,18).…”