Structure Basis for Talin-mediated Integrin Activation

这项建议将继续在原子水平上研究整合素的激活--这是所有人的核心反应。 整合素介导的细胞粘附过程。二十多年前发现的整合素， 被广泛认为是介导多种细胞过程的主要细胞表面受体，包括细胞-细胞 基质（ECM）粘附、细胞迁移、细胞形状改变和细胞存活。整合素活化 通过独特的“由内而外”的信号传导过程发生，其中整合素胞质面首先感受到一种信号传导， 构象信号通过跨膜区传递到胞外结构域，因此 将所述受体从低亲和力状态转化为高亲和力状态。多年来，我们的实验室和其他许多实验室 试图了解这种由内而外的激活过程的分子细节。使用NMR 光谱作为一个主要的工具，结合协作功能的方法，我们一直专注于 研究在止血和血栓形成中起关键作用的血小板allb β 33 - a原型整合素。我们有 在一系列研究中显示，该受体的allb/p3胞质尾区（CTs）可以经历 扣合/解扣合过程，从而促进整联蛋白由内向外活化。我们进一步表明， 整联蛋白allbp的解扣过程由talin触发，talin是一种主要的细胞骨架衔接子， 作为整合素活化的基本成分。我们最新的数据表明， 塔林的活性也受到构象调节。我们的研究结果已经导致了一个全面的模型， 整合素活化，其中一系列能量依赖性构象变化需要在整合素上发生。 整合素在细胞内侧启动整合素跨膜信号传导及其高亲和力配体结合。 在这一延续建议中，我们将通过提出以下问题来积极测试这一模式： 整联蛋白胞内面的变化是否传播到其跨膜结构域，即中心区域 连接细胞内外的受体虽然两者的3D结构 已经报道了整合素的细胞外和细胞内结构域， 中心片仍然缺乏，（ii）什么是talin autoinhibition的原子基础，它是如何激活和调节，以触发整合素由内而外的信号？这些问题的答案对于深入了解整合素的功能至关重要，也是细胞生物学和信号转导的基础。我们将继续使用NMR光谱作为解决这些问题的核心技术。在与艾德普洛和其他项目负责人的持续合作中，我们将进行各种功能实验来证实我们基于NMR的发现。我们的结果，如果成功的话，将导致另一个重要的进步，了解整合素信号。它们也将促进对allb的认识和治疗|33介导的疾病，如血栓形成和动脉粥样硬化。

This proposal will continue the atomic level investigation of integrin activation - a central response for all integrin-mediated cell adhesive processes. Discovered more than two decades ago, integrins have been widely recognized as major cell surface receptors that mediate a variety of cellular processes including cellextracellular matrix (ECM) adhesion, cell migration, cell shape change, and cell survival. Integrin activation occurs via a distinct "inside-out" signaling process in which the integrin cytoplasmic face first senses a conformational signal that relays through the transmembrane region to the extracellular domain, thus converting the receptor from a low to a high affinity state. Over the years, our laboratory and many others have attempted to understand the molecular details of this inside-out activation process. Using NMR spectroscopy as a major tool, combined with collaborative functional approaches, we have been focusing on studying platelet allb(33 - a prototypic integrin that plays a key role in hemostasis and thrombosis. We have shown in a series of studies that the allb/p3 cytoplasmic tails (CTs) of this receptor can undergo clasping/unclasping process, thus promoting the integrin inside-out activation. We have further shown that the unclasping process of integrin allbps is triggered by talin - a major cytoskeletal adaptor that has been established as the essential component of the integrin activation. Our most recent data have indicated that the activity of talin is also conformationally regulated. Our findings have led to a comprehensive model for integrin activation where a series of energy-dependent conformational changes need to occur on the integrin intracellular side to initiate the integrin transmembrane signaling and its high affinity ligand binding. In this continuation proposal, we will vigorously test this model by asking the following questions: (i) How does the change of the integrin intracellular face propagate to its transmembrane domain, a central region that connects the intracellular and the extracellular sides of the receptor? While 3D structures of both extracellular and intracellular domains of integrins have been reported, an atomic view of this integrin central piece is still lacking, (ii) What is the atomic basis of the talin authoinhibition and how is it activated and regulated to trigger the integrin inside-out signaling? The answer to these questions is vital for a thorough understanding of the integrin function and is also fundamental for cell biology and signal transduction. We will continue to use NMR spectroscopy as a core technique to address these questions. In continued collaboration with Ed Plow and other project leaders, we will perform various functional experiments to corroborate our NMR-based findings. Our results, if successful, will lead to another significant advance for understanding the integrin signaling. They will also promote the understanding and treatment of allb|33-mediated diseases such as thrombosis and atherosclerosis.