Reid Helen M, Kinsella B Therese
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
J Biol Chem. 2003 Dec 19;278(51):51190-202. doi: 10.1074/jbc.M309314200. Epub 2003 Oct 6.
In humans, thromboxane A2 signals through two thromboxane A2 receptor (TP) isoforms termed TP alpha and TP beta. Signaling by TP alpha, but not TP beta, is subject to prostacyclin-induced desensitization mediated by direct protein kinase (PK) A phosphorylation where Ser329 represents the phosphotarget (Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412-20423). In the current study, the effect of the vasodilator nitric oxide (NO) on intracellular signaling by the TP isoforms was investigated. The NO donor 3-morpholinosydnonimine, HCl (SIN-1) and 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) functionally desensitized U46619-mediated calcium mobilization and inositol 1,4,5-trisphosphate generation by TP alpha whereas signaling by TP beta was unaffected by either agent. NO-mediated desensitization of TP alpha signaling occurred through a PKG-dependent, PKA- and PKC-independent mechanism. TP alpha, but not TP beta, was efficiently phosphorylated by PKG in vitro and underwent NO/PKG-mediated phosphorylation in whole cells. Deletion/site-directed mutagenesis and metabolic labeling studies identified Ser331 as the target residue of NO-induced PKG phosphorylation of TP alpha. Although TP alpha S331A was insensitive to NO/PKG-desensitization, similar to wild type TP alpha its signaling was fully desensitized by the prostacyclin receptor agonist cicaprost occurring through a PKA-dependent mechanism. Conversely, signaling by TP alpha S329A was insensitive to cicaprost stimulation whereas it was fully desensitized by NO/PKG signaling. In conclusion, TP alpha undergoes both NO- and prostacyclin-mediated desensitization that occur through entirely independent mechanisms involving direct PKG phosphorylation of Ser331, in response to NO, and PKA phosphorylation of Ser329, in response to prostacyclin, within the unique carboxyl-terminal tail domain of TP alpha. On the other hand, signaling by TP beta is unaffected by either NO or prostacyclin.
在人类中，血栓素A2通过两种名为TPα和TPβ的血栓素A2受体（TP）亚型进行信号传导。TPα而非TPβ的信号传导会受到前列环素诱导的脱敏作用影响，该脱敏作用由直接蛋白激酶（PK）A磷酸化介导，其中Ser329为磷酸化靶点（Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412 - 20423）。在本研究中，研究了血管舒张剂一氧化氮（NO）对TP亚型细胞内信号传导的影响。NO供体3 - 吗啉代 sydnonimine，HCl（SIN - 1）和8 - 溴 - 鸟苷3',5' - 环一磷酸（8 - Br - cGMP）在功能上使TPα介导的U46619诱导的钙动员和肌醇1,4,5 - 三磷酸生成脱敏，而TPβ的信号传导不受这两种试剂的影响。NO介导的TPα信号脱敏通过一种依赖PKG、不依赖PKA和PKC的机制发生。TPα而非TPβ在体外能被PKG有效磷酸化，并在全细胞中经历NO/PKG介导的磷酸化。缺失/定点诱变和代谢标记研究确定Ser331是NO诱导的TPα的PKG磷酸化的靶点残基。尽管TPα S331A对NO/PKG脱敏不敏感，但与野生型TPα类似，其信号传导通过依赖PKA的机制被前列环素受体激动剂西卡前列素完全脱敏。相反，TPα S329A的信号传导对西卡前列素刺激不敏感，而它被NO/PKG信号完全脱敏。总之，TPα经历NO和前列环素介导的脱敏，这两种脱敏通过完全独立的机制发生，即响应NO时，Ser331发生直接PKG磷酸化；响应前列环素时，TPα独特的羧基末端尾域内的Ser329发生PKA磷酸化。另一方面，TPβ的信号传导不受NO或前列环素的影响。
