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GPCR Cytoprotective Signaling Mechanisms

GPCR 细胞保护信号机制

基本信息

批准号：
9514364
负责人：
Neil C Chi
金额：
$ 10.01万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9514364
关键词：
Adherens Junction Apoptosis Apoptotic Arrestins Bacterial Infections Biochemical Blood Vessels Caveolae Cell membrane Cleaved cell Clinical Clinical Trials Cytoprotection Cytoskeleton Development Drug Targeting Edema Endothelial Cells Experimental Models Extravasation FDA approved Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs Generations Goals Heterotrimeric GTP-Binding Proteins Human In Vitro Infection Inflammatory Response Ligands Link Maintenance Mammalian Cell Mediating Mediator of activation protein Microscopy Modeling Morbidity - disease rate PAR-1 Receptor Pathway interactions Pharmaceutical Preparations Prevention Receptor Signaling Regulation Reporting Role SPHK1 enzyme Sepsis Signal Pathway Signal Transduction Site Small Interfering RNA Sphingosine-1-Phosphate Receptor Testing Therapeutic Therapeutic Uses Thrombin Receptor Tissues Transactivation Transgenic Animals Vascular Permeabilities Work Zebrafish activated Protein C activated protein C receptor arrestin 2 base beta-arrestin endothelial dysfunction experimental study genetic manipulation in vivo insight interdisciplinary approach microbial mortality mouse model novel novel therapeutic intervention novel therapeutics prevent public health relevance receptor response scaffold

项目摘要

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.

描述（由申请方提供）：本提案的长期目标是确定新型G蛋白偶联受体（GPCR）细胞保护信号通路，该通路可恢复或增强内皮屏障完整性，以预防脓毒症相关的血管渗漏。GPCR代表了哺乳动物细胞中表达的最大的信号传导受体家族，也是获批治疗药物的最大一类药物靶标。两种GPCR已被证明介导内皮细胞保护反应，蛋白酶激活受体-1（PAR 1）和1-磷酸鞘氨醇受体-1（S1 PR 1）。PAR 1是凝血酶的GPCR，但可以被活化的蛋白C（APC）切割和活化。APC与其辅因子EPCR结合，在独特的位点切割PAR 1的N-末端，产生促进细胞保护信号传导的独特的拴系配体。S1 PR 1信号还促进内皮屏障维持和抗凋亡反应，并有助于APC/PAR 1诱导的细胞保护。因此，PAR 1和S1 PR 1都对内皮细胞保护做出重要贡献。然而，APC/PAR 1和S1 PR 1在体外和体内协调细胞保护信号传导的机制尚不清楚，并且对于理解这些受体作为药物靶点的地位以开发用于预防和治疗脓毒症的新疗法至关重要。我们假设b-抑制蛋白协调APC/PAR 1和S1/S1 PR 1信号在小窝，以促进内皮细胞保护反应。我们发现APC/PAR 1诱导的内皮细胞保护作用需要PAR 1定位于细胞膜小窝。在最近的工作中，我们发现APC/PAR 1促进的细胞保护信号是由β-arrestin-2和dishevelled-2支架介导的，而不是由异源三聚体G蛋白介导的。在初步的研究中，我们表明，PAR 1和S1 PR 1在质膜上共同缔合，并共存于小窝中。此外，APC/PAR 1通过β-arrestin-2依赖性途径诱导鞘氨醇激酶-1（SK 1）的活化，所述SK 1是S1 P产生和S1 PR 1活化的重要介质。β-抑制蛋白对于APC诱导的抗凋亡应答也是关键的。我们使用斑马鱼进一步证明了PAR 1、S1 PR 1和β-arrestin-2在脓毒症模型中调节血管通透性。我们将采用多学科方法，使用培养的人类内皮细胞在体外以及使用血管渗透性和败血症的斑马鱼模型在体内鉴定内皮GPCR细胞保护信号通路。拟议的研究将推进我们的理解PAR 1和S1 PR 1功能如何调节血管内皮屏障的完整性和细胞凋亡正常和脓毒症模型。该提案的具体目的是：1）确定PAR 1和S1 PR 1如何协调内皮细胞保护信号，2）鉴定APC/PAR 1与S1 P/S1 PR 1整合以促进细胞保护信号的机制，以及3）研究PAR 1、S1 PR 1和β-arrestin-2如何在体内调节内皮屏障完整性。