Prostaglandin D 2 receptor-mediated desensitization of the α isoform of the human thromboxane A 2 receptor

Abstract

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 TPα and TPβ isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca 2+] 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 TPα, but not TPβ, was subject to DP-mediated desensitization in a PKA-dependent, PKC-independent manner. U46619-induced signaling by TP Δ328, a truncated variant of TP containing only those residues common to TPα and TPβ, was insensitive to prior DP stimulation, indicating that the carboxyl terminal tail of TPα contains the target site(s) for DP-mediated desensitization. Mutation of Ser 329 to Ala 329 within a consensus PKA site in TPα rendered the mutant TPα S329A insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TPα, but not TPβ or TPα S329A, was subject to DP-mediated phosphorylation and that TPα phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TPα, but not TPβ, is subject to DP-mediated cross desensitization, which occurs through direct PKA-mediated phosphorylation of TPα at Ser 329.