Structure and Function of the Platelet Integrin

血小板整合素的结构和功能

• 批准号: 7474406
• 负责人: Joel S. Bennett
• 金额: $ 57.82万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7474406
• 关键词: Affinity; Binding; Binding Sites; Blood Platelets; Calcium; Cell surface; Complex; Cytoplasmic Protein; Cytoplasmic Tail; Cytoskeletal Proteins; Environment; Equilibrium; Fibrinogen; Figs - dietary; Glycoproteins; Helix (Snails); Hemostatic Agents; Homo; Integrins; Kinetics; Lasers; Ligand Binding; Ligands; Measures; Mediating; Membrane; Membrane Lipids; Molecular Conformation; Mutation; Nature; Object Attachment; Pathologic; Peptides; Phospholipids; Platelet aggregation; Positioning Attribute; Process; Relative (related person); Reporting; Specific qualifier value; Specificity; Structure; Surface Plasmon Resonance; System; Talin; Thermodynamics; Thrombus; Transmembrane Domain; base; ear helix; von Willebrand Factor

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.

该项目的目标是将血小板膜的结构和功能关联起来 整合素allb|33。 allb|33 是一种钙依赖性异二聚体，其配体的结合位点包括 纤维蛋白原和血管性血友病因子通过血小板刺激而暴露。与 cdlb|33 结合的配体是 负责血小板聚集，是形成止血血小板的关键步骤 栓塞和病理性动脉血栓。像 allb|33 这样的整合素以平衡状态存在于细胞表面 低亲和力（非活性）和高亲和力（活性）构象之间的关系。我们曾报道过 整合素跨膜结构域参与特定的异聚和同聚相互作用 分别定义了它们的非活动和活动状态，并提出了“推拉”假设 解释整合素活性如何受到调节。因此，稳定活性构象的过程 allb|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，平衡会向同一方向拉动。这 该项目的目标将进一步表征“推拉”假设。在目标 1 中，我们将确定并 表征介导同聚和异聚关联的界面（33 跨膜螺旋，检查整合素跨膜特异性的结构基础 域相互作用，并确定 allb 和 (33 跨膜结构域改变 allb(33 激活状态。目标 2 将检查 跨膜结构域分离和寡聚化对 allb|33 与细胞质相互作用的影响 蛋白质，重点关注 (33 细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来 研究 (33 细胞质结构域、talin 和磷脂在天然膜样中的相互作用 环境。 allb 和 (33 跨膜和细胞质结构域也将获得。在目标 3 中，我们将使用最近的 改进的激光镊子系统来测量 cdlb|33-配体键的寿命，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。