Prostaglandin D2 receptor-mediated desensitization of the α isoform of the human thromboxane A2 receptor

Thromboxane (TX) A(2) and prostaglandin (PG) D-2 mediate opposing actions in platelets and in vascular and non-vascular smooth muscle. Here, we investigated the effects of stimulation of the PGD(2) receptor (DP) on signaling by the TXA(2) receptor (TP) expressed in human platelets and in human embryonic kidney (HEK) 293 cells over-expressing the individual TP alpha and TP beta isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca2+](i)) and inhibited platelet aggregation in response to the TXA(2) mimetic U46619. DP-mediated desensitization of TP signaling in platelets was prevented by pretreatment with the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inhibitor GF 109203X. In HEK 293 cells, signaling by TP alpha, but not TP beta, was subject to DP-mediated desensitization in a PKA-dependent, PKC-independent manner. U46619-induced signaling by TPDelta 328, a truncated variant of TP containing only those residues common to TP alpha and TP beta, was insensitive to prior DP stimulation, indicating that the carboxyl terminal tail of TP alpha contains the target site(s) for DP-mediated desensitization. Mutation of Ser(329) to Ala(329) within a consensus PKA site in TP alpha rendered the mutant TP alpha (S329A) insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TP alpha, but not TP beta or TP alpha (S329A), was subject to DP-mediated phosphorylation and thar TP alpha phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TP alpha, but not TP beta, is subject to DP-mediated cross desensitization, which occurs through direct PKA-mediated phosphorylation of TP alpha at Ser(329). (C) 2001 Elsevier Science Inc. All rights reserved.