THROMBIN RECEPTOR-G PROTEIN SIGNALING MECHANISMS

描述（申请人逐字描述）： 凝血酶受体激活了一系列异常多样的生理活性， 应答这些包括血小板聚集、细胞 增殖/凋亡，细胞-细胞粘附和炎症，以及潜在的 癌症中的侵入性和组织重组过程。激活 血小板凝血酶受体可能在启动中起主要作用， 和维持病理性动脉和静脉血栓形成， 动脉粥样硬化病变的发展。四个G蛋白偶联， 已经鉴定了蛋白酶激活的受体：PAR 1、PAR 2、PAR 3和PAR 4。 原型蛋白酶激活受体PAR 1已被证明与 先生，Gi（β-γ）和G12/13。的 PAR 1如何与G蛋白相互作用的细节和相对重要性 差异G蛋白激活的时间顺序仍然未知。 目前对G蛋白的身份知之甚少，G蛋白与新的 发现了PAR 3和PAR 4凝血酶受体。这些研究的目的是：1） 研究G蛋白差异激活的机制基础， PAR 1在体内条件下，2）了解潜在的差异 PAR 4和PAR 1 G蛋白依赖性信号传导和信号终止之间的关系，以及 3)以扩大细胞内蛋白质的库，这些蛋白质可能与 蛋白酶激活受体。在第一个具体目标中， 利用我们新发现的细胞穿透肽蛋白这些 试剂快速穿透完整细胞的质膜 血小板和成纤维细胞，并引起特异性激活和/或抑制 在体内条件下的受体依赖性细胞内信号转导。 我们已经证明，这些pepducins表现出的性质， 激活的PAR 1。这些包括完全血小板聚集， 形状变化、刺激调节的Ca++通量、激活磷脂酶 C和凝血酶受体反应的脱敏。我们预计 使用pepducins研究细胞内信号转导将开辟新的 在以前不适合分子生物学的细胞中进行实验研究的途径 技术.最终，这些可能被证明是第一个治疗上有用的 靶向受体-G蛋白界面的药物。第二个目标将 探索PAR 4与PARI激活细胞内 使用各种遗传和生物化学技术的信号通路。我们 我将扩展我们实验室最近的工作，证明PAR 1和PAR 4 在血小板中具有不同的Ca++信号传导动力学。第三个目标，我们 将研究在酵母中共表达的PAR 1和哺乳动物G蛋白，并将鉴定 与PAR 1和PAR 4细胞内特异性相互作用的新蛋白质 域.

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.