Structure and Function of the Platelet Integrin

本课题的目的是研究血小板膜的结构和功能 整联蛋白allb| 33.阿尔布|33是钙依赖性异二聚体，其与配体如 纤维蛋白原和血管性血友病因子通过血小板刺激暴露。配体与cdlb的结合|33是 负责血小板聚集，是止血血小板形成的关键步骤 栓塞和病理性动脉血栓。整合素如allb| 33以平衡的方式驻留在细胞表面上 低亲和力（非活性）和高亲和力（活性）构象之间的差异。我们已经报道了 整联蛋白跨膜结构域参与特异性异聚和同聚相互作用 分别定义了它们的非活动状态和活动状态，并提出了“推拉”假说 来解释整合素活性是如何调节的。因此，稳定活性构象的过程 所有b| 33会将其推向激活状态，而更有利的过程 当跨膜结构域分离时，会将平衡拉向相同的方向。的 该项目的目标将进一步说明“推拉”假设的特点。在目标1中，我们将确定和 表征介导（33）的同聚和异聚缔合的界面 跨膜螺旋，检查整合素跨膜特异性的结构基础 结构域的相互作用，并确定如何改变的相对位置的allb和（33 跨膜结构域改变A11 b β 3活化状态。目标2将审查 跨膜结构域分离和寡聚化对allb相互作用的影响|33例细胞质 蛋白质，重点是β 3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脂质膜表面等离子体共振系统， 研究β 3胞质结构域、talin和磷脂在天然膜样蛋白中的相互作用。 环境A1 b和（33）的异聚和同聚络合物的NMR结构 也将获得跨膜和胞质结构域。在目标3中，我们将使用最近 改进的激光光镊系统测量cdlb寿命|33-配体键，使我们能够 导出关于这种相互作用的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.