The α, but not the β, isoform of the human thromboxane A2 receptor is a target for nitric oxide-mediated desensitization -: Independent modulation of TPα signaling by nitric oxide and prostacyclin

In humans, thromboxane A(2) signals through two thromboxane A(2) receptor (TP) isoforms termed TPalpha and TPbeta. Signaling by TPalpha, but not TPbeta, is subject to prostacyclin-induced desensitization mediated by direct protein kinase (PK) A phosphorylation where Ser(329) represents the phosphotarget (Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412-20423). In the current study, the effect of the vasodilator nitric oxide (NO) on intracellular signaling by the TP isoforms was investigated. The NO donor 3-morpholinosydnonimine, HCl (SIN-1) and 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) functionally desensitized U46619-mediated calcium mobilization and inositol 1,4,5-trisphosphate generation by TPalpha whereas signaling by TPbeta was unaffected by either agent. NO-mediated desensitization of TPalpha signaling occurred through a PKG-dependent, PKA- and PKC-independent mechanism. TPalpha, but not TPbeta, was efficiently phosphorylated by PKG in vitro and underwent NO/PKG-mediated phosphorylation in whole cells. Deletion/site-directed mutagenesis and metabolic labeling studies identified Ser(331) as the target residue of NO-induced PKG phosphorylation of TPalpha. Although TPalpha(S331A) was insensitive to NO/PKG-desensitization, similar to wild type TPalpha its signaling was fully desensitized by the prostacyclin receptor agonist cicaprost occurring through a PKA- dependent mechanism. Conversely, signaling by TPalpha(S329A) was insensitive to cicaprost stimulation whereas it was fully desensitized by NO/PKG signaling. In conclusion, TPalpha undergoes both NO- and prostacyclin-mediated desensitization that occur through entirely independent mechanisms involving direct PKG phosphorylation of Ser331, in response to NO, and PKA phosphorylation of Ser329, in response to prostacyclin, within the unique carboxyl-terminal tail domain of TPalpha. On the other hand, signaling by TPbeta is unaffected by either NO or prostacyclin.