1 Heterologous desensitization or intermolecular cross-talk plays a critical role in regulating intracellular signalling by diverse members of the G-protein-coupled receptor superfamily. We have previously established that the a and b isoforms of the human thromboxane A 2 receptor (TP) undergo differential desensitization of signalling in response to 17 phenyl trinor prostaglandin (PG)E 2 , an agonist of the EP 1 subtype of the PGE 2 receptor (EP) family. 2 Herein, we investigated the molecular basis of TPa and TPb desensitization in human embryonic kidney (HEK) 293 cells and in renal mesangial cells in response to 17 phenyl trinor PGE 2 and in response to the PGF 2a receptor (FP) agonist PGF 2a , and sought to identify the target site(s) of those desensitizations.3 Our results demonstrated that TPa and TPb receptors are subject to desensitization in response to both EP 1 and FP receptor activation and that these effects are mediated by direct protein kinase (PK)C phosphorylation of the individual TP isoforms within their unique carboxyl-terminal (C)-tail domains. 4 Moreover, deletion/site-directed mutagenesis and metabolic labelling studies identified Thr 337 , within TPa, and Thr 399 , within TPb, as the specific target residues for PKC phosphorylation and EP 1 -and FP-mediated desensitization of TPa and TPb signalling, respectively. 5 Hence, in conclusion, while the TPa and TPb diverge within their C-tail domains, they have evolved to share a similar mechanism of PKC-induced phosphorylation and desensitization in response to EP 1 and FP receptor activation, though it occurs at sites unique to the individual TP isoforms.
Publisher ElsevierLink to online version http://dx.doi.org/10.1016/j.bbalip.2006.07.012Item record/more information http://hdl.handle.net/10197/3146
Publisher's statement þÿ T h i s i s t h e a u t h o r s v e r s i o n o f a w o r k t h a t w a s a c c e p t e d f o r p u b l i c a t i o n i n B i o c h i m i c a e tBiophysica Acta. Changes resulting from the publishing process, such as peer review,
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.