The α, but not the β, isoform of the human thromboxane A2 receptor is a target for prostacyclin-mediated desensitization

Abstract

In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A2 receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TPα or TPβ isoforms. Consistent with previous studies, cicaprost abrogated U46619- mediated platelet aggregation and mobilization of intracellular calcium ([Ca2+](i)). In HEK 293 cells, signaling by TPα, but not TPβ, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TPα signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of G(q) protein. Signaling by TPΔ328, a truncated variant of TP devoid of the divergent residues of the TPs, or by TPα(S329A), site- directed mutant of TPα, were insensitive to IP agonist activation. Whole cell phosphorylations established that TPα, but not TPβ or TPα(S329A), is subject to IP-mediated phosphorylation and that TPα phosphorylation is inhibited by H-89. Thus, we conclude that TPα, but not TPβ, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser329 and propose that TPα may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.