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素调节信号的机制。凝血酶是一种凝血蛋白酶，可引起多种细胞效应，对止血和血栓形成以及血管损伤产生的炎症和增殖反应至关重要。因此，了解凝血酶信号传导的机制可能为预防和治疗凝血酶相关的心血管疾病提供新的策略。蛋白酶激活受体（PARs）是介导细胞中大部分凝血酶反应的G蛋白偶联受体（gpcr）。PAR1是该家族的原型，是人血小板、内皮细胞、成纤维细胞和平滑肌细胞中凝血酶信号传导的主要介质。PAR1是不可逆的蛋白水解激活，内化和直接分类到溶酶体。导致PAR1信号终止的机制尚不清楚。β -拘捕素结合到大多数磷酸化的gpcr介导脱敏和内化。磷酸化对PAR1信号的终止很重要（至少对Gq）。我们发现Betaarr1是PAR1解耦到Gq信号的主要介质。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。我们将使用源自β捕雷素敲除和COS-7细胞的小鼠胚胎成纤维细胞（mef）来描述？PAR1信号中的阻滞。活化PAR1的内化和溶酶体分选也对受体信号的终止至关重要。PAR1通过一个独立于抑制因子的网格蛋白和动力蛋白依赖途径内化。我们提供的初步证据表明，基于酪氨酸的基序和可能是一种新的接头蛋白调节PAR1的内化。最后，我们的研究证明了SNX1和SNX2在调节PAR1溶酶体分选中的新作用，并提出了SNXs在GPCR运输中普遍起作用的令人兴奋的可能性。本提案的具体目标是：(1)描述PAR1脱敏的分子基础，(2)定义PAR1新的内化特性，(3)定义分选连接蛋白调节活化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.