Structure and Function of the Platelet Integrin

这个项目的目标是将血小板膜的结构和功能联系起来。 整合素Allb|33Allb|33是一种钙依赖的异二聚体，其与配体的结合位置如下 纤维蛋白原和血管性血友病因子通过血小板刺激暴露。与cdlb|33结合的配体是 负责血小板聚集，是止血血小板形成的关键步骤 血栓和病理性动脉血栓。像Allb|33这样的整合素以平衡的方式存在于细胞表面 在低亲和力(非活性)和高亲和力(活性)构象之间。我们已经报道了 整合素跨膜结构域参与特定的异构体和同聚体相互作用 分别定义了它们的非活动状态和活动状态，并提出了一种“推-拉”假说 解释整合素活性是如何被调节的。因此，稳定活性构象的过程 会将其推向激活状态，而更有利的进程 当跨膜结构域分离时，会将平衡拉向同一方向。这个 该项目的目标将进一步表征“推-拉”假说。在目标1中，我们将确定和 表征介导所述(33)的均相和异相缔合的界面 跨膜螺旋，研究整合素跨膜特异性的结构基础 结构域相互作用，并确定Allb和(33)的相对位置如何变化 跨膜结构域改变了Allb(33)的激活状态。目标2将考察以下方面的贡献 Allb|33与细胞质相互作用的跨膜区分离和齐聚 蛋白质，侧重于(33)胞质结构域与细胞骨架蛋白talin的相互作用。 我们将使用最近开发的系留类脂膜表面等离子体共振系统来 研究(33)胞质结构域、Talin和磷脂在天然膜状结构中的相互作用 环境。Al1b和(33)异构体和同构体配合物的核磁共振结构 还将获得跨膜区和细胞质结构域。在目标3中，我们将使用我们最近的 改进的激光镊子系统，测量cdlb|33-配位键的寿命，使我们能够 得出有关这种相互作用性质的定量热力学和动力学信息。

The objective of this project is to correlate the structure and function of the platelet membrane integrin allb|33. allb|33 is a calcium-dependent heterodimer whose binding site for ligands such as fibrinogen and von Willebrand factor is exposed by platelet stimulation. Ligand binding to cdlb|33 is responsible for platelet aggregation and is a critical step in the formation of hemostatic platelet plugs and pathologic arterial thrombi. Integrins like allb|33 reside on cell surfaces in an equilibrium between low affinity (inactive) and high affinity (active) conformations. We have reported that integrin transmembrane domains engage in both specific heteromeric and homomeric interactions that define their inactive and active states, respectively and have proposed a "push-pull" hypothesis to explain how integrin activity is regulated. Thus, processes that stabilize the active conformation of allb|33 would push it toward to its activated state, whereas processes that are more favorable when the transmembrane domains separate would pull the equilibrium in the same direction. The Aims of the project will further characterize the "push-pull" hypothesis. In Aim 1, we will identify and characterize the interface that mediates the homomeric and heteromeric association of the (33 transmembrane helix, examine the structural basis for the specificity of integrin transmembrane domain interactions, and determine how changes in the relative positions of the allb and (33 transmembrane domains alter the allb(33 activation state. Aim 2 will examine the contribution of transmembrane domain separation and oligomerization to the interaction of allb|33 with cytoplasmic proteins, focusing on the interaction of the (33 cytoplasmic domain with the cytoskeletal protein talin. We will use a recently developed tethered lipid membrane surface plasmon resonance system to study the interactions of the (33 cytoplasmic domain, talin, and phospholipids in a native membranelike environment. NMR structures for heteromeric and homomeric complexes of the allb and (33 transmembrane and cytoplasmic domains will be obtained as well. In Aim 3, we will use our recently modified laser tweezers system to measure the lifetime of cdlb|33-ligand bonds, enabling us to derive quantitative thermodynamic and kinetic information about the nature of this interaction.