Prostaglandin D-2 receptor-mediated desensitization of the alpha isoform of the human thromboxane A(2) receptor

Thromboxane (TX) A(2) and prostaglandin (PG) D-2 mediate opposing actions i n platelets and in vascular and non-vascular smooth muscle. Here, we invest igated 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 embry onic kidney (HEK) 293 cells over-expressing the individual TP alpha and TP beta isoforms. In platelets, the selective DP agonist BW245C abolished TP-m ediated mobilization of intracellular calcium ([Ca2+](i)) and inhibited pla telet aggregation in response to the TXA(2) mimetic U46619. DP-mediated des ensitization of TP signaling in platelets was prevented by pretreatment wit h the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inh ibitor 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-indepe ndent manner. U46619-induced signaling by TPDelta 328, a truncated variant of TP containing only those residues common to TP alpha and TP beta, was in sensitive to prior DP stimulation, indicating that the carboxyl terminal ta il 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 r endered the mutant TP alpha (S329A) insensitive to BW245C-mediated desensit ization. Whole cell phosphorylation assays established that TP alpha, but n ot 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. Th ese data establish that TP alpha, but not TP beta, is subject to DP-mediate d cross desensitization, which occurs through direct PKA-mediated phosphory lation of TP alpha at Ser(329). (C) 2001 Elsevier Science Inc. All rights r eserved.