Regulation of Protease-activated Receptor-1 Signaling

描述(由申请者提供)：本研究计划的长期目标是了解凝血酶调节信号的机制。凝血酶是一种凝血酶，是在血管损伤和血栓性疾病中产生的。凝血酶推动纤维蛋白沉积和激活血小板，这对止血和血栓形成至关重要。凝血酶引起的细胞反应也与炎症、血管发育和癌症进展有关。因此，了解凝血酶信号转导机制并发展其调控能力，可能为凝血酶相关血管疾病的预防和治疗提供新的策略。蛋白水解酶激活受体(PARs)是G蛋白偶联受体(GPCRs)，介导细胞中的大部分凝血酶反应。凝血酶激活受体-1(PAR1)是该家族的原型，是凝血酶在人血小板、内皮细胞、成纤维细胞和平滑肌细胞中诱导的细胞效应的主要来源。凝血酶激活PAR1通过不可逆的蛋白分解机制发生，导致产生一种不能扩散的系链配体。因此，PAR1信号必须受到严格的调控。调节PAR1信号的幅度、持续时间和空间方面的信号终止机制仍然知之甚少。磷酸化和阻滞素是导致PAR1信号脱敏的一些重要成分，但也可能存在其他机制。PAR1与多种G蛋白亚型结合，包括GQ、GI和G12/13，而PAR1与这些不同G蛋白亚型解偶联的分子基础尚不清楚。内化和溶酶体分选也是调节蛋白水解性激活的PAR1的关键，但人们对此知之甚少。我们发现，激活的PAR1内化是通过依赖于磷酸化、笼蛋白和动力蛋白的途径发生的，该途径不依赖于拦阻蛋白。这导致了我们的假设，即我们的clathrin适配器在PAR1内化过程中起着关键的中介作用。我们最近发现泛素化调节PAR1内化。我们提供的初步证据表明，网状蛋白适配器epsin调节PAR1的内化。泛素化和Eepsin参与调节PAR1信号和运输的分子机制尚不清楚。我们将使用异位表达PAR1的HeLa和HEK 293细胞以及表达内源性PAR1的人内皮细胞来描述泛素化和epsin在PAR1信号和运输中的作用。该提案的具体目的是：1)确定泛素化和Eepsin对PAR1细胞信号转导的功能后果；2)定义通过网状蛋白适配器epsin对PAR1内化进行新的调控的分子基础；3)描述泛素化对PAR1内化进行新的调控的分子机制。公共卫生相关性：凝血酶是凝血级联反应的关键效应物，在血管损伤和血栓性疾病中产生。蛋白水解酶激活的受体-1介导细胞内的凝血酶信号，对与血管损伤相关的血液凝结和炎症反应至关重要。因此，了解蛋白水解酶激活的受体-1信号，并发展其操控能力，可能为凝血酶相关血管疾病的预防和治疗提供新的策略。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to understand the mechanisms of thrombin-regulated signaling. Thrombin, a coagulant protease, is generated in response to vascular injury and in thrombotic disease. Thrombin drives fibrin deposition and activates platelet, which are critical for hemostasis and thrombosis. Thrombin elicited cellular responses have also been implicated in inflammation, vascular development and cancer progression. Thus, understanding thrombin signaling, and developing the ability to manipulate it, may provide new strategies for the prevention and treatment of thrombin-related vascular diseases. Protease- activated receptors (PARs) are G-protein coupled receptors (GPCRs) that mediate most, if not all, thrombin responses in cells. Protease-activated receptor-1 (PAR1), the prototype for this family, accounts for the majority of the cellular effects elicited by thrombin in human platelets, endothelial cells, fibroblasts and smooth muscle cells. Thrombin activation of PAR1 occurs through an irreversible proteolytic mechanism that results in the generation of a tethered ligand that cannot diffuse away. Thus, PAR1 signaling must be tightly regulated. The signal termination mechanisms that regulate the magnitude, duration and spatial aspects of PAR1 signaling remain poorly understood. Phosphorylation and arrestins are some important components that contribute to desensitization of PAR1 signaling, but other mechanisms are likely to exist. PAR1 couples to multiple G-protein subtypes including Gq, Gi and G12/13 and the molecular basis of PAR1 uncoupling from these distinct G-protein subtypes is not known. Internalization and lysosomal sorting are also critical for the regulation of proteolytically activated PAR1 and are poorly understood. We showed that activated PAR1 internalization occurs through a phosphorylation-, clathrin- and dynamin-dependent pathway that is independent of arrestins. This led to our hypothesis that our clathrin adaptors function as critical mediators of PAR1 internalization. We recently discovered that ubiquitination regulates PAR1 internalization. We provide initial evidence that the clathrin adaptor epsin regulates internalization of PAR1. The molecular mechanisms by which ubiquitination and epsin contribute to the regulation of PAR1 signaling and trafficking are not known. We will use HeLa and HEK 293 cells ectopically expressing PAR1 and human endothelial cells expressing endogenous PAR1 to delineate the role of ubiquitination and epsin in PAR1 signaling and trafficking. The specific aims of the proposal are to: 1) define the functional consequences of ubiquitination and epsin on PAR1 cellular signaling, 2) define the molecular basis for the novel regulation of PAR1 internalization by the clathrin adaptor epsin, and 3) delineate the molecular mechanism for the underlying novel regulation of PAR1 internalization by ubiquitination. Public Health Relevance: Thrombin, the key effector protease of the coagulation cascade, is generated in response to vascular injury and in thrombotic disease. The protein protease-activated receptor-1 mediates thrombin signaling in cells and is critical for blood clotting and inflammatory responses associated with vascular injury. Thus, understanding protease-activated receptor-1 signaling, and developing the ability to manipulate it, may provide new strategies for the prevention and treatment of thrombin-related vascular diseases.