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

摘要 Rap1是一个连接激动剂刺激和Talin募集到整合素的中枢信号节点，a 整合素激活的最后一个共同步骤。在白细胞中，RIAM是一种RAP1效应器，它介导 Talin依赖的激活；然而，在血小板中，这种Rap1效应器的身份尚不清楚 这是我们理解上的一个关键差距。初步数据表明，一种新的进化保守的 该范式认为talin包含两个Rap1结合位点，使其能够作为Rap1效应器。这个 申请人假设Talin本身是牵连的主要且可能是唯一的Rap1效应器 在血小板整合素的激活中。其次，他认为Talin-Rap1的相互作用可能 有助于整合素功能，即使在含有丰富的RAP1效应器的白细胞中也是如此 而在含有大量片层脂蛋白的内皮细胞中，这是一种RIAM类似物。检视 这些想法：特定目标1将检验直接的Rap1-talin相互作用发挥作用的假设 在血小板整合素活化中起核心作用。小鼠的巨核细胞和血小板 具有任一个或两个RAP1结合位点被禁用的表达TALIN将被分析血小板结构， 功能和编队。止血和血栓形成将通过各种化验进行测试。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整合素-配位键。《特定目标2》将检验一种假说，即引导Rap1-Talin 相互作用在充满RIAM的细胞中很重要。与项目2整合协作 白细胞的激活、构象和拓扑结构将在携带每一种 Aim1中描述的三个Talin等位基因。每种突变与RIAM联合应用的效果 缺失或RIAM过度表达将测试这两个RAP1效应器的相对作用。 特定目标3将检验Talin-Rap1相互作用在 内皮细胞。选择性表达三种Talin突变等位基因之一的小鼠 将研究血管内皮细胞的出血和发育表型。对整合素的影响 将在体外评估原发肺和脑的激活、扩散和细胞-细胞连接。 微血管内皮细胞。LamelLipodin的缺失和过度表达将使分析成为可能 内皮细胞功能中层脂蛋白和Rap1结合与talin的关系。一起， 这些目标的实现将检验塔林本身作为范式转换假说 血小板功能中RAP1的主要效应分子在止血和血栓形成中的作用 Rap1-talin相互作用在白细胞和内皮细胞中的重要性。

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.