Direct Rap1-talin interaction in platelets, leukocytes, and endothelial cells

Rap1-talin 在血小板、白细胞和内皮细胞中的直接相互作用

• 批准号: 10676892
• 负责人: Mark HOWARD Ginsberg
• 金额: $ 48.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-05 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676892
• 关键词: Achievement; Adhesions; Age; Agonist; Alleles; Binding; Binding Sites; Biological Assay; Blood; Blood Cells; Blood Platelets; Blood Vessels; Carotid Arteries; Cell Adhesion; Cell physiology; Cells; Collaborations; Compensation; Data; Development; Disabled Persons; Embryo; Endothelial Cells; Endothelium; Foundations; Hemorrhage; Hemostatic function; Homing; In Vitro; Individual; Inflammation; Integrins; Intercellular Junctions; Knockout Mice; Laser injury; Leukocytes; Ligands; Lung; Lymphoid Tissue; Mediating; Megakaryocytes; Modeling; Molecular Conformation; Mus; Mutation; Nature; Pathway interactions; Peripheral; Phenotype; Play; Role; Signal Transduction; Site; Stenosis; Structure; T-Lymphocyte; Talin; Tamoxifen; Testing; Thrombosis; Tumor Angiogenesis; Vena caval structure; Venous Thrombosis; brain endothelial cell; cell type; ferric chloride; in vivo; mutant; neutrophil; overexpression; platelet function; recruit

Abstract Rap1 is a central signaling node connecting agonist stimulation to talin recruitment to integrins, a final common step in integrin activation. In leukocytes, RIAM is a Rap1 effector that mediates talin-dependent activation; however, in platelets the identity of such Rap1 effectors is obscure and is a key gap in our understanding. Preliminary data suggest a new evolutionarily-conserved paradigm that talin contains two Rap1 binding sites that enable it to serve as a Rap1 effector. The applicant hypothesizes that talin itself is the principal, and perhaps only, Rap1 effector implicated in platelet integrin activation. Secondly, he suggests that that the talin-Rap1 interaction may contribute to integrin function even in leukocytes that contain an abundant Rap1 effector, RIAM and in endothelial cells that contain substantial lamellipodin, a RIAM paralogue. To examine these ideas: Specific Aim 1 will test the hypothesis that a direct Rap1-talin interaction plays a central role in platelet integrin activation. Mice in which the megakaryocytes and platelets express talin with either or both Rap1 binding sites disabled will be analyzed for platelet structure, function, and formation. Hemostasis and thrombosis will be tested in a variety of assays. A biomembrane force probe will characterize the role of the Rap1-talin interaction in individual IIb3 integrin-ligand bonds. Specific Aim 2 will test the hypothesis that direct Rap1-Talin interaction is important in RIAM-replete cells. In collaboration with Project 2 Integrin activation, conformation, and topology in leukocytes will be analyzed in cells bearing each of the three talin alleles described in Aim1. The effect of each mutation in combination with RIAM deletion or with RIAM over-expression will test the relative roles of these two Rap1 effectors. Specific Aim 3 will test the hypothesis that the talin-Rap1 interaction is important in endothelial cells. Mice expressing one of the three mutant talin alleles selectively in endothelium will be studied for hemorrhage and developmental phenotypes. Effects on integrin activation, spreading, and cell-cell junctions will be assessed in vitro in primary lung and brain microvascular endothelial cells. Lamellipodin deletion and over expression will enable an analysis of the relationship of lamellipodin and Rap1 binding to talin in endothelial cell functions. Together, achievement of these aims will test the paradigm-shifting hypothesis that talin itself serves as the major Rap1 effector in platelet function in hemostasis and thrombosis and evaluate the importance of the Rap1-talin interaction in leukocytes and endothelial cells.

抽象的 RAP1是一种将激动剂刺激连接到塔林募集到整合素的中心信号节点 整合素激活中的最终共同步骤。在白细胞中，Riam是介导的Rap1效应子 塔林依赖性激活；但是，在血小板中，这种Rap1效应的身份晦涩难懂 这是我们理解的关键差距。初步数据表明了一个新的进化保存的 Talin包含两个RAP1结合位点的范式，使其能够用作RAP1效应子。这 申请人假设塔林本身是主要的，也许只是实施了RAP1效应器 在血小板整合素激活中。其次，他建议talin-rap1相互作用 即使在包含丰富RAP1效应子的白细胞中有助于整联蛋白功能 在含有大量层lamelipodin的内皮细胞中，是Riam paralogue。检查 这些想法：特定目标1将检验直接Rap1-Talin互动的假设 血小板整合素激活中的核心作用。巨核细胞和血小板的小鼠 将残障的带有或两个RAP1结合位点的Express Talin分析血小板结构， 功能和形成。止血和血栓形成将在各种测定中进行测试。一个 生物膜力探针将表征Rap1-Talin相互作用在个体中的作用 IIB3整联蛋白 - 配体键。特定目标2将检验直接Rap1-Talin的假设 相互作用在RIAM复发细胞中很重要。与Project 2 Intemin合作 白细胞中的激活，构象和拓扑结构将在包含每一个的细胞中进行分析 AIM1中描述的三个塔林等位基因。每个突变与RIAM结合的效果 删除或RIAM过表达将测试这两种RAP1效应的相对作用。 具体目标3将检验以下假设：talin-rap1相互作用在 内皮细胞。在选择性地表达三个突变塔林等位基因之一的小鼠 内皮将研究出血和发育表型。对整合素的影响 将在原发性肺和大脑中评估激活，扩散和细胞 - 细胞连接 微血管内皮细胞。 lamellipodin删除和过度表达将实现分析 片状脂蛋白和RAP1与内皮细胞功能中塔林结合的关系。一起， 这些目标的实现将检验塔林本身的范式转移假设 止血和血栓形成中血小板功能的主要RAP1效应子，并评估 Rap1-Talin相互作用在白细胞和内皮细胞中的重要性。