Regulation of Protease-activated Receptor-1 Signaling

描述(由申请人提供)：本提案的总体目标是了解凝血酶调节信号的机制。凝血酶是一种凝血酶，可引起多种细胞效应，对止血和血栓形成以及血管损伤所产生的炎症和增殖反应是必不可少的。因此，了解凝血酶信号转导机制可能为凝血酶相关心血管疾病的防治提供新的策略。蛋白水解酶激活受体(PARs)是G蛋白偶联受体(GPCRs)，介导细胞内大部分凝血酶反应。PAR1是该家族的原型，是人类血小板、内皮细胞、成纤维细胞和平滑肌细胞中凝血酶信号的主要介体。PAR1被不可逆转的蛋白水解性激活，被内化并直接与溶酶体结合。导致PAR1信号终止的机制尚不清楚。Betaarrestins与大多数磷酸化的GPCRs结合，介导脱敏和内化。磷酸化对于终止PAR1信号非常重要(至少对Gq而言)。我们发现，Betaarr1是PAR1解偶联到Gq信号的主要介体。PAR1与GQ、GI和G12/13偶联，PAR1与这些不同的G蛋白亚型解偶联的分子基础尚不清楚。我们将使用来自Betaarrestin基因敲除的小鼠胚胎成纤维细胞(MEF)和COS-7细胞来描述？arrestins在PAR1信号转导中的作用。激活的PAR1的内化和溶酶体分选也是终止受体信号的关键。PAR1是通过依赖于笼蛋白和动力素的途径内化的，该途径不依赖于拦阻蛋白。我们提供了基于酪氨酸的基序和可能是一种新的接头蛋白调节PAR1内化的初步证据。最后，我们的研究证明了SNX1和SNX2在调节PAR1的溶酶体分类中的一个新的作用，并提出了SNXS在GPCR运输中普遍发挥作用的令人兴奋的可能性。这项建议的具体目的是：(1)描述PAR1脱敏的分子基础，(2)定义PAR1新的内化特性，以及(3)确定分类连接蛋白调节激活的PAR1溶酶体降解的分子机制。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the mechanisms of thrombin-regulated signaling. Thrombin, a coagulant protease, elicits a variety of cellular effects that are essential for hemostasis and thrombosis, as well as inflammatory and proliferative responses produced by vascular damage. Therefore, understanding the mechanisms of thrombin signaling may provide new strategies for the prevention and treatment of thrombin-related cardiovascular diseases. Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that mediate most of thrombin responses in cells. PAR1, the prototype of this family, is the predominant mediator of thrombin signaling in human platelets, as well as in endothelial cells, fibroblasts and smooth muscle cells. PAR1 is irreversibly proteolytically activated, intemalized and sorted directly to lysosomes. The mechanisms that contribute to termination of PAR1 signaling are not clearly understood. Betaarrestins bind to most phosphorylated GPCRs to mediate desensitization and internalization. Phosphorylation is important for termination of PAR1 signaling (at least to Gq). We found that Betaarr1 is the predominant mediator of PAR1 uncoupling to Gq signaling. PAR1 couples to Gq, Gi, and G12/13, and the molecular basis of PAR1 uncoupling to these distinct G protein subtypes is not known. We will use mouse embryonic fibroblasts (MEFs) derived from betaarrestin knockouts and COS-7 cells to delineate the role of ?arrestins in PAR1 signaling. Internalization and lysosomal sorting of activated PAR1 are also critical for termination of receptor signaling. PAR1 is internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. We provide initial evidence that a tyrosine-based motif and perhaps a novel adaptor protein regulate internalization of PAR1. Finally, our studies demonstrate a novel role for SNX1 and perhaps SNX2 in regulating lysosomal sorting of PAR1 and raise the exciting possibility that SNXs function generally in GPCR trafficking. The specific aims of this proposal are to: (1) delineate the molecular basis of PAR1 desensitization, (2) define the novel internalization properties of PAR1, and (3) define the molecular mechanisms by which sorting nexins regulate lysosomal degradation of activated PAR1.