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

摘要 Rap 1是连接激动剂刺激与塔林募集至整联蛋白的中央信号节点，这是一种 整合素激活的最后一个共同步骤。在白细胞中，RIAM是一种Rap 1效应子， talin依赖性激活;然而，在血小板中，这种Rap 1效应物的身份尚不清楚 这是我们认识上的一个关键空白。初步数据表明，一种新的进化保守的 范例，塔林包含两个Rap 1结合位点，使其能够作为Rap 1效应器。的 申请人假设talin本身是主要的，并且可能是唯一的，涉及Rap 1效应子 血小板整合素活化。其次，他认为talin-Rap 1相互作用可能 甚至在含有大量Rap 1效应子RIAM的白细胞中也有助于整合素功能 以及在含有大量片状脂质蛋白（一种RIAM蛋白）的内皮细胞中。审查 这些想法：具体目标1将测试Rap 1-talin直接相互作用的假设， 在血小板整合素活化中的核心作用。小鼠体内的巨核细胞和血小板 将分析Rap 1结合位点之一或两个都失活的表达talin的血小板结构， 功能和形成。将在各种试验中检测止血和血栓形成。一 生物膜力探针将表征Rap 1-talin相互作用在个体中的作用， β IIb β 3整联蛋白-配体键。具体目标2将检验直接Rap 1-Talin 相互作用在RIAM充满的细胞中是重要的。与项目2集成合作 白细胞的活化、构象和拓扑结构将在携带每种 Aim 1中描述的三个talin等位基因。每种突变与RIAM组合的效果 缺失或RIAM过表达将测试这两个Rap 1效应子的相对作用。 具体目标3将检验talin-Rap 1相互作用在以下假设： 内皮细胞选择性表达三种突变talin等位基因之一的小鼠， 将研究内皮的出血和发育表型。对整合素的影响 将在体外评估原代肺和脑中的活化、扩散和细胞-细胞连接。 微血管内皮细胞板脂蛋白缺失和过表达将使分析成为可能。 的关系lamellipodin和Rap 1结合talin在内皮细胞功能。在一起， 这些目标的实现将检验塔林本身作为 血小板在止血和血栓形成中的主要Rap 1效应子，并评估 Rap 1-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.