Structure Basis for Talin-mediated Integrin Activation

这项提议将继续对整合素激活的原子水平的研究-这是对所有人的中心反应 整合素介导的细胞黏附过程。整合素在二十多年前被发现，已经被 被广泛认为是主要的细胞表面受体，介导包括细胞外在内的各种细胞过程 基质(ECM)黏附、细胞迁移、细胞形态改变和细胞存活。整合素激活 通过一个独特的由内向外的信号过程发生，在这个过程中，整合素细胞质表面首先感觉到 通过跨膜区传递到胞外区的构象信号，因此 将受体从低亲和力状态转换为高亲和力状态。多年来，我们的实验室和许多其他实验室 试图了解这个由内而外的激活过程的分子细节。使用核磁共振 光谱作为一种主要工具，结合协作功能方法，我们一直专注于 研究血小板Allb(33)--一种在止血和血栓形成中起关键作用的典型整合素。我们有 一系列研究表明，这种受体的Allb/p3胞浆尾巴(CTs)可以经历 扣紧/解扣过程，从而促进整合素由内向外的激活。我们已经进一步表明， 整合素allbps的解锁过程是由talin触发的-talin是一种主要的细胞骨架适配器，已经被 被确定为整合素激活的基本成分。我们的最新数据表明， Talin的活性也受构象调控。我们的发现导致了一个全面的模型 整合素激活，需要发生一系列能量依赖的构象变化 整合素胞内侧启动整合素跨膜信号转导及其高亲和力配体结合。 在这份延续提案中，我们将通过提出以下问题来大力测试这一模式：(I)如何 整合素细胞内表面的变化是否传播到其跨膜区，即中心区 连接受体的胞内侧和胞外侧？而两者的3D结构 整合素的胞外和胞内结构域已被报道，对这种整合素的原子观点 中心片段仍然缺乏，(Ii)talin自我抑制的原子基础是什么，它是如何被激活和调节以触发整合素内向外信号的？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。我们将继续使用核磁共振波谱作为核心技术来解决这些问题。在与Ed Plow和其他项目负责人的持续合作下，我们将进行各种功能实验，以证实我们基于核磁共振的发现。我们的结果，如果成功，将在理解整合素信号方面取得又一重大进展。他们还将促进对Allb|33介导的疾病(如血栓形成和动脉粥样硬化)的理解和治疗。

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.