THROMBIN RECEPTOR-G PROTEIN SIGNALING MECHANISMS

描述(申请人的逐字描述)：凝血酶裂解 凝血酶受体激活一系列异常多样的生理学 回应。这些包括血小板聚集，细胞 增殖/凋亡、细胞间黏附和炎症，以及潜在的 与癌症有关的侵袭和组织重组过程。激活 可能在血小板凝血酶受体的启动中起主要作用 和维持病理性动脉和静脉血栓以及 动脉粥样硬化病变的发展。四个G蛋白偶联， 已发现的蛋白水解酶激活受体有：PAR1、PAR2、PAR3和PAR4。 典型的蛋白水解酶激活的受体，PAR1，已被证明与 GQ、GI(Betagamma)和G12/13在各种体外条件下。这个 PAR1如何与G蛋白相互作用的细节及其相对重要性 差异G蛋白激活的时间顺序仍然未知。 关于与新的G蛋白偶联的G蛋白的身份，人们知之甚少 发现了PAR3和PAR4凝血酶受体。这些研究的目标是：1) 探讨差异G蛋白激活的机制基础 活体条件下的PAR1，2)了解潜在的差异 在PAR4和PAR1G蛋白依赖的信号和信号终止之间，以及 3)扩大可能与之相互作用的细胞内蛋白质库 蛋白水解酶激活受体的类别。在第一个具体目标中，我们将 利用我们新发现的一类可穿透细胞的多肽。这些 试剂迅速穿透完整细胞的质膜，如 血小板和成纤维细胞，并引起特异性激活和/或抑制 活体条件下受体依赖的细胞内信号转导。 我们已经证明了这些缩氨酸蛋白具有正常情况下 可归因于激活的PAR1。这些包括完全的血小板聚集和 形状变化、调节的钙离子通量的刺激、磷脂酶的激活 C，以及凝血酶受体反应的脱敏。我们预计， 利用肽类化合物研究细胞内信号转导将开启新的 以前对分子不敏感的细胞的实验研究途径 技巧。最终，这些药物可能被证明是第一个在治疗上有用的药物。 针对受体-G蛋白界面的药物。第二个目标是 探讨PAR4与细胞内准激活的机制 使用各种遗传和生化技术的信号通路。我们 我将详细介绍我们实验室最近的工作，这些工作证明了PAR1和PAR4 在血小板内有明显的钙信号动力学。在第三个目标中，我们 将研究在酵母中共表达的PAR1和哺乳动物G蛋白，并将鉴定 细胞内与PAR1和PAR4特异性相互作用的新蛋白 域名。

DESCRIPTION (Applicant's Description Verbatim): Thrombin cleavage of the thrombin receptors activates an extraordinarily diverse array of physiologic responses. These include platelet aggregation, cellular proliferation/apoptosis, cell-cell adhesion and inflammation, and potentially the invasive and tissue-reorganizing processes involved in cancer. Activation of platelet thrombin receptors is likely to play a major role in the initiation and maintenance of pathological arterial and venous thromboses and the development of atherosclerotic lesions. Four G protein-coupled, protease-activated receptors have been identified: PAR1, PAR2, PAR3, and PAR4. The prototypical protease-activated receptor, PAR1, has been shown to couple to Gq., Gi(betagamma), and G12/13 under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation is still unknown. Far less is known about the identity of the G proteins that couple to the newly discovered PAR3 and PAR4 thrombin receptors. The goals of these studies are: 1) to investigate the mechanistic basis of differential G protein activation by PAR1 under in vivo conditions, 2) to understand the underlying differences between PAR4 and PAR1 G-protein dependent signaling and signal termination, and 3) to expand the repertoire of intracellular proteins that might interact with the class of protease-activated receptors. In the first specific aim we will exploit our newly discovered class of cell-penetrating pepducins. These reagents rapidly penetrate the plasma membrane of intact cells such as platelets and fibroblasts and cause specific activation and/or inhibition of receptor-dependent intracellular signal transduction under in vivo conditions. We have demonstrated that these pepducins exhibit properties that are normally attributable to activated PAR1. These include full platelet aggregation and shape change, stimulation of regulated Ca++ fluxes, activation of phospholipase C, and desensitization of thrombin receptor responses. We anticipate that the use of the pepducins to study intracellular signal transduction will open new avenues of experimental research in cells previously not amenable to molecular techniques. Ultimately, these may prove to be the first therapeutically useful agents that are targeted at receptor-G protein interfaces. The second aim will explore the mechanistic basis of PAR4 versus PARI-activation of intracellular signaling pathways using a variety of genetic and biochemical techniques. We will expand on recent work done in our lab that demonstrated that PAR1 and PAR4 have distinct kinetics of Ca++ signaling in platelets. In the third aim, we will study coexpressed PAR1 and mammalian G proteins in yeast and will identify novel proteins which specifically interact with PAR1 and PAR4 intracellular domains.