Foley J F, Kelley L P, Kinsella B T
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.
Biochem Pharmacol. 2001 Jul 15;62(2):229-39. doi: 10.1016/s0006-2952(01)00661-x.
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 (Ca(2+)) 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 TP(Delta 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 TPalpha 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 that 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).
血栓素（TX）A2和前列腺素（PG）D2在血小板以及血管和非血管平滑肌中发挥相反作用。在此，我们研究了刺激前列腺素D2受体（DP）对人血小板和过表达单个TPα和TPβ亚型的人胚肾（HEK）293细胞中血栓素A2受体（TP）信号传导的影响。在血小板中，选择性DP激动剂BW245C消除了TP介导的细胞内钙（[Ca2+]i）动员，并抑制了对血栓素A2模拟物U46619的血小板聚集反应。用cAMP依赖性蛋白激酶A（PKA）抑制剂H-89预处理可防止DP介导的血小板中TP信号脱敏，但不受蛋白激酶C（PKC）抑制剂GF 109203X的影响。在HEK 293细胞中，TPα而非TPβ的信号传导以PKA依赖性、PKC非依赖性方式受到DP介导的脱敏作用。U46619诱导的TP（Δ328）信号传导，TP的一种截短变体，仅包含TPα和TPβ共有的那些残基，对先前的DP刺激不敏感，表明TPα的羧基末端尾巴包含DP介导脱敏的靶位点。将TPα中一个保守PKA位点内的丝氨酸（Ser）329突变为丙氨酸（Ala）329，使突变型TPα（S329A）对BW245C介导的脱敏不敏感。全细胞磷酸化分析表明，TPα而非TPβ或TPα（S329A）受到DP介导的磷酸化，并且TPα磷酸化被PKA抑制剂H-89阻断。这些数据表明，TPα而非TPβ受到DP介导的交叉脱敏，这是通过PKA直接介导的TPα在丝氨酸329处的磷酸化发生的。
