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传导的机制。凝血酶是一种促凝蛋白酶，可激发止血和血栓形成所必需的多种细胞效应，以及血管损伤产生的炎症和增殖反应。因此，了解凝血酶信号转导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G蛋白偶联受体（GPCR），介导细胞内大多数凝血酶反应。PAR 1是该家族的原型，是人血小板以及内皮细胞、成纤维细胞和平滑肌细胞中凝血酶信号传导的主要介质。PAR 1被不可逆地蛋白水解激活、内化并直接分选至溶酶体。导致PAR 1信号传导终止的机制尚不清楚。β抑制蛋白与大多数磷酸化GPCR结合以介导脱敏和内化。磷酸化对于终止PAR 1信号传导（至少终止至Gq）是重要的。我们发现Betaarr 1是PAR 1解偶联至Gq信号传导的主要介质。PAR 1与Gq、Gi和G12/13偶联，PAR 1与这些不同的G蛋白亚型解偶联的分子基础尚不清楚。我们将使用小鼠胚胎成纤维细胞（MEFs）来自β抑制蛋白基因敲除和COS-7细胞来描绘的作用？PAR 1信号传导抑制蛋白。活化的PAR 1的内化和溶酶体分选对于受体信号传导的终止也是至关重要的。PAR 1通过不依赖于抑制蛋白的网格蛋白和动力蛋白依赖性途径内化。我们提供的初步证据表明，酪氨酸基序，也许是一种新的衔接蛋白调节PAR 1的内化。最后，我们的研究证明了SNX 1和SNX 2在调节PAR 1的溶酶体分选中的新作用，并提出了SNXs通常在GPCR运输中起作用的令人兴奋的可能性。该提案的具体目标是：（1）描绘PAR 1脱敏的分子基础，（2）定义PAR 1的新内化特性，（3）定义分选连接蛋白调节激活PAR 1的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.