The Role of Protease-Activated Receptors in the Regulation of eNOS

描述(申请人提供)：本研究的目的是探讨蛋白酶激活受体(PARs)调节内皮细胞内皮型一氧化氮合酶(ENOS)磷酸化和激活的信号转导机制。蛋白水解酶通过激活PARs在血管内皮细胞生理和病理生理中发挥重要作用。它们的功能包括调节内皮依赖性收缩/松弛、内皮通透性/屏障功能、白细胞黏附、血管生成、内皮细胞迁移和增殖。因此，PARs很可能参与心血管疾病的发生发展。然而，PARs调控这些功能的详细分子机制在很大程度上仍不清楚。PAR1和PAR2被认为在内皮细胞功能中发挥重要作用。最近的研究发现，在内皮细胞和其他细胞中，PAR1的多个异源三聚体G蛋白偶联(GQ、GI、G12/13)和几个下游信号级联反应。相比之下，PAR2在内皮细胞中的信号转导知之甚少，它主要与GQ偶联。我们的目标是研究PARs所利用的特定信号转导途径，包括异源三聚体G蛋白偶联，偶联过程中PARs的构效关系，以及它们在调节eNOS下游信号中的作用。我们假设PAR1和PAR2通过不同的信号通路相互调节eNOS，并提出了以下具体目标来解决这一假说。我们的计划：1.为了证明不同的受体C-尾巴结构与不同的G蛋白偶联有关，需要通过PARS相互作用的eNOS磷酸化。2.确定eNOS激酶在不同的PAR反应中相互调节eNOS活性的作用。3.鉴定参与eNOS双向调控的不同PAR C-Tail相关蛋白。培养的内皮细胞将用于检测eNOS的磷酸化。将使用激酶分析、位点突变和新的蛋白质组学方法来鉴定参与eNOS相互磷酸化的其他蛋白质。这项研究的结果将对开发可用于治疗导致心血管疾病的内皮功能障碍的特效治疗药物具有价值。公共卫生相关性：该项目与公共卫生相关，因为它对了解心血管疾病的一些可能原因具有重要意义，心血管疾病是美国的头号杀手，特别是在非裔美国人社区中。它将提供有关蛋白酶激活受体在调节内皮细胞中的作用的信息，内皮细胞在血管张力中起着重要作用。内皮功能障碍是高血压的主要原因，这些研究将有助于开发减轻或预防心血管疾病的治疗策略。

DESCRIPTION (provided by applicant): The objective of this study is to explore the signal transduction mechanism by which protease-activated receptors (PARs) regulate endothelial nitric oxide synthase (eNOS) phoshorylation and activation in endothelial cells. Proteases play very important roles in endothelial physiology and pathophysiology through activation of PARs. Their functions include regulations of endothelium-dependent contraction/relaxation, endothelial permeability/barrier function, leukocyte adhesion, angiogenesis, endothelial cell migration and proliferation. Thus, PARs are very likely to be involved in the development of cardiovascular diseases. However, the detailed molecular mechanisms by which PARs regulate these functions remain largely unclear. PAR1 and PAR2 are believed to play major roles in endothelial cell function. Recent studies revealed multiple heterotrimeric G protein-coupling (Gq, Gi, G12/13) and several downstream signaling cascades of PAR1 in endothelial cells and other cells. By contrast, much less is known regarding PAR2 signal transduction in endothelial cells, which mainly couples to Gq. Our goal is to study the specific signal transduction pathways utilized by PARs including heterotrimeric G protein- coupling together with the structure-activity relationship of PARs during coupling and their roles in mediating downstream signals that regulate eNOS. We hypothesize that PAR1 and PAR2 reciprocally regulate eNOS through distinct signaling pathways and have proposed the following specific aims to address this hypothesis. We plan: 1. To demonstrate that a distinct receptor C-tail structure associated with distinct G protein-coupling is required for reciprocal eNOS phosphorylation by PARs. 2. To identify eNOS kinases that reciprocally regulate eNOS activity in response to distinct PARs. 3. To identify the distinct PAR C-tail associated proteins that are responsible for reciprocal eNOS regulation. Cultured endothelial cells will be used to detect phosphorylation of eNOS. Kinase assays, site mutations, and novel proteomic approaches will be used to identify other proteins involved in the reciprocal phosphorylation of eNOS. The results of this study will be valuable in developing specific-acting therapeutic drugs that can be used to treat endothelial dysfunctions that lead to cardiovascular diseases. PUBLIC HEALTH RELEVANCE: This project is relevant to public health because of its importance in understanding some of the possible causes of cardiovascular disease, the number one killer in America, especially among the African American community. It will provide information regarding the role of protease-activated receptors in the regulation of the endothelium, which plays an important role in vascular tone. Endothelial dysfunction is a major cause of hypertension, and these studies will contribute to the development of treatment strategies to alleviate or prevent cardiovascular disease.