GPCR Cytoprotective Signaling Mechanisms

描述(申请人提供)：这项建议的长期目标是确定新的G蛋白偶联受体(GPCRs)细胞保护信号通路，以恢复或增强内皮屏障的完整性，以防止与脓毒症相关的血管渗漏。GPCRs是哺乳动物细胞中表达的最大的信号受体家族，也是被批准的治疗药物中最大的一类药物靶点。已有两种GPCRs介导内皮细胞保护反应，即蛋白酶激活受体-1(PAR1)和鞘氨醇1-磷酸受体-1(S1PR1)。PAR1是凝血酶的GPCR，但可以被激活的蛋白C(APC)切割和激活。APC与它的辅助因子EPCR结合，在一个独特的位置切割PAR1的N端，产生一个独特的系链配体，促进细胞保护信号的传递。S1PR1信号还促进内皮屏障的维持和抗凋亡反应，并参与APC/PAR1诱导的细胞保护。因此，PAR1和S1PR1在内皮细胞保护中都起着重要作用。然而，APC/PAR1和S1PR1在体外和体内协调细胞保护信号的机制尚不清楚，了解这些受体作为药物靶点的地位对于开发预防和治疗脓毒症的新疗法至关重要。我们假设b-arrestins在小窝中协调APC/PAR1和S1/S1PR1信号，以促进内皮细胞保护反应。我们发现，APC/PAR1诱导的内皮细胞保护需要PAR1在小窝中定位。在最近的工作中，我们发现APC/PAR1启动的细胞保护信号是由ç-arrestin-2和disheveled-2支架介导的，而不是由异源三聚体G蛋白介导的。在初步研究中，我们发现PAR1和S1PR1在质膜上共结合，并在小窝中共存。此外，APC/PAR1还通过一种依赖于arrestin-2的途径诱导S1P生成和S1PR1激活的重要中介--鞘氨醇激酶-1(SK1)的激活。阻滞素在APC诱导的抗细胞凋亡反应中也起着关键作用。我们进一步使用斑马鱼证明了PAR1、S1PR1和B-arrestin-2在脓毒症模型中调节血管通透性。我们将采用一种多学科的方法，在体外使用培养的人内皮细胞识别内皮细胞保护信号通路，并使用斑马鱼血管通透性和脓毒症的模型进行体内鉴定。这些研究将促进我们对PAR1和S1PR1如何在脓毒症模型中正常调节血管内皮细胞屏障完整性和细胞凋亡的功能的理解。该提案的具体目的是：1)确定PAR1和S1PR1如何协调内皮细胞保护信号；2)确定APC/PAR1与S1P/S1PR1整合促进细胞保护信号传递的机制(S)；3)研究PAR1、S1PR1和B-arrestin-2如何在体内调节内皮屏障的完整性。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify novel G protein-coupled receptor (GPCR) cytoprotective signaling pathways that can restore or enhance endothelial barrier integrity to prevent vascular leakage associated with sepsis. GPCRs represent the largest family of signaling receptors expressed in mammalian cells and the largest class of drug targets for approved therapeutics. Two GPCRs have been shown to mediate endothelial cytoprotective responses, protease-activated receptor-1 (PAR1) and the sphingosine 1-phosphate receptor-1 (S1PR1). PAR1 is a GPCR for thrombin, but can be cleaved and activated by activated Protein C (APC). APC bound to its co-factor EPCR cleaves the N-terminus of PAR1 at a unique site generating a distinct tethered ligand that promotes cytoprotective signaling. S1PR1 signaling also promotes endothelial barrier maintenance and anti-apoptotic responses and contributes to APC/PAR1-induced cytoprotection. Thus, both PAR1 and S1PR1 make important contributions to endothelial cytoprotection. However, the mechanism by which APC/PAR1 and S1PR1 coordinate cytoprotective signaling in vitro and in vivo is not known and critical to understand to advance the status of these receptors as drug targets for the development of new therapeutics for the prevention and treatment of sepsis. We hypothesize that b-arrestins coordinate APC/PAR1 and S1/S1PR1 signaling in caveolae to promote endothelial cytoprotective responses. We found that APC/PAR1- induced endothelial cytoprotection requires PAR1 localization in caveolae. In recent work, we discovered that APC/PAR1-promoted cytoprotective signaling is mediated by ß-arrestin-2 and dishevelled-2 scaffolds rather than by heterotrimeric G proteins. In preliminary studies we show that PAR1 and S1PR1 co-associate at the plasma membrane and co-exist in caveolae. Moreover, APC/PAR1 induces activation of sphingosine kinase-1 (SK1), an important mediator of S1P generation and S1PR1 activation, through a ß-arrestin-2-dependent pathway. ß-arrestins are also critical for APC-induced anti-apoptotic responses. We further demonstrate using zebrafish that PAR1, S1PR1 and ß-arrestin-2 regulate vascular permeability in a sepsis model. We will pursue a multidisciplinary approach to identify endothelial GPCR cytoprotective protective signaling pathways in vitro using cultured human endothelial cells and in vivo using a zebrafish model of vascular permeability and sepsis. The proposed studies will advance our understanding of how PAR1 and S1PR1 function to regulate vascular endothelial barrier integrity and apoptosis normally and in a sepsis model. The specific aims of the proposal are to: 1) determine how PAR1 and S1PR1 coordinate endothelial cytoprotective signaling, 2) identify the mechanism(s) by which APC/PAR1 integrates with S1P/S1PR1 to promote cytoprotective signaling, and 3) investigate how PAR1, S1PR1 and ß-arrestin-2 regulate endothelial barrier integrity in vivo.