Structure and Function of the Platelet Integrin

血小板整合素的结构和功能

• 批准号: 7876743
• 负责人: Joel S. Bennett
• 金额: $ 58.45万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7876743
• 关键词: Affinity; Binding; Binding Sites; Blood Platelets; Calcium; Cell physiology; Cell surface; Complex; Cytoplasmic Protein; Cytoplasmic Tail; Cytoskeletal Proteins; Endothelial Cells; Environment; Equilibrium; Fibrinogen; Figs - dietary; Glycoproteins; Hemostatic Agents; Homo; Integrins; Kinetics; Lasers; Ligand Binding; Ligands; Measures; Mediating; Membrane; Membrane Lipids; Molecular Conformation; Mutation; Nature; Pathologic; Peptides; Phospholipids; Platelet aggregation; Positioning Attribute; Process; Regulation; Relative (related person); Reporting; Specific qualifier value; Specificity; Structure; Surface Plasmon Resonance; System; Talin; Thermodynamics; Thrombus; Transmembrane Domain; base; 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.

该项目的目的是关联血小板膜的结构和功能 整合素Allb | 33。 AllB | 33是钙依赖性异二聚体，其结合位点（例如 纤维蛋白原和Von Willebrand因子因血小板刺激而暴露。配体与CDLB | 33是 负责血小板聚集，是止血血小板形成的关键步骤 塞子和病理动脉血栓。像AllB这样的整合素| 33居住在平衡中的细胞表面上 在低亲和力（非活性）和高亲和力（主动）构象之间。我们报告了 整合素跨膜结构域参与特定的异构体和同源性相互作用 这分别定义了他们的无效状态，并提出了一个“推拉”假设 解释整联蛋白活性如何调节。因此，稳定活性构象的过程 allb | 33将其推向激活状态，而过程更有利 当跨膜结构域分开时，将在相同方向上拉出平衡。这 该项目的目的将进一步描述“推扣”假设。在AIM 1中，我们将确定并 表征介导的界面介导的界面（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的相对位置的变化和（33 跨膜结构域改变了ALLB（33个激活状态。AIM 2将检查 跨膜结构域的分离和将Allb | 33与细胞质的相互作用的相互作用 蛋白质，重点是（33个细胞质结构域与细胞骨架蛋白塔林的相互作用。 我们将使用最近开发的系绳脂质膜表面等离子体共振系统 研究（33个细胞质结构域，塔林和磷脂）在天然膜类似的相互作用 环境。 ALLB的异源和同源复合物的NMR结构和（33 也将获得跨膜和细胞质结构域。在AIM 3中，我们将使用最近的 修改的激光镊子系统测量CDLB | 33位 - 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。