Structure Analysis of Integrin Activation

描述（由申请人提供）：心血管疾病是美国的主要死亡原因，主要是由于动脉粥样硬化斑块破裂导致的心肌梗死和随后的冠状动脉内血栓形成。刺激血栓形成的关键事件是血小板聚集，其由原型整合素aIIb <$3介导。整合素是调节细胞粘附（如血小板聚集）以及其它重要生物过程（如细胞迁移、分化、增殖和程序性细胞死亡）的异二聚体跨膜受体家族。整合素通过介导细胞外粘附分子和细胞内环境之间的动态联系来实现这些不同的功能。整联蛋白功能受跨膜信号传导调节，其可作为结合细胞外配体（“由外向内”信号传导）以及分子与细胞质结构域结合（“由内向外”信号传导）的结果而发生。我们建议使用电子冷冻显微镜，图像重建和分子建模来检查人血小板整合素aIIb <$3的结构。电子冷冻显微镜（cryo-EM）是一种强大的技术，通过它可以在天然的生理状态下检查大分子复合物，如膜蛋白。我们已经完成了一个3D结构的全长，人血小板整合素aIIb <$3在低亲和力，非活性构象。然后将α ν β 3胞外域的X射线结构对接到低分辨率cryo-EM图中。这种结合的方法使我们能够提出一个模型的结构重排与整合素激活。该项目的总体目标是测试该模型的整合素活化，我们的实验根据以下具体目标组织：目标1：继续进行图像分析，以提高低亲和力状态下aIIb <$3的3D重建分辨率;目标2：通过分析结合配体的aIIb <$3，获得高亲和力状态的3D图谱;目标3：抗体标记将用于定位高亲和力和低亲和力状态内的特定位点;目的4：将通过结合冷冻EM图谱、位点特异性抗体标记数据和单个整联蛋白结构域的同源性模型来推导高亲和力和低亲和力状态的分子模型。这些研究揭示的结构细节将提供深入了解整合素活化的分子基础，与新治疗剂的设计相关。

DESCRIPTION (provided by applicant): Cardiovascular disease is the major cause of mortality in the United States, primarily due to myocardial infarction resulting from rupture of an atherosclerotic plaque and subsequent thrombosis within a coronary artery. A key event that stimulates thrombus formation is platelet aggregation, which is mediated by the prototypical integrin aIIb¿3. Integrins are a family of heterodimeric transmembrane receptors which modulate cell adhesion, such as platelet aggregation, as well as other important biological processes such as cell migration, differentiation, proliferation and programmed cell death. Integrins accomplish these diverse functions by mediating dynamic linkages between extracellular adhesion molecules and the intracellular environment. Integrin functions are regulated by transmembrane signaling, which can occur as a consequence of binding extracellular ligands ("outside-in" signaling), as well as the binding of molecules to the cytoplasmic domains ("inside-out" signaling). We propose to use electron cryo-microscopy, image reconstruction and molecular modeling to examine the structure of the human platelet integrin aIIb¿3. Electron cryo-microscopy (cryo-EM) is a powerful technique by which macromolecular complexes such as membrane proteins can be examined in a native, physiological state. We have completed a 3D structure of the full-length, human platelet integrin aIIb¿3 in the low-affinity, inactive conformation. The X-ray structure of the aV¿3 ectodomain was then docked into the low resolution cryo-EM map. This combined approach allowed us to propose a model for the structural rearrangements associated with integrin activation. The overall goal of this project is to test this model for integrin activation, and our experiments are organized according to the following specific aims: Aim 1: Image analysis will be continued in order to improve the resolution of the 3D reconstruction of aIIb¿3 in the low-affinity state; Aim 2: A 3D map of the high-affinity state will be derived by analysis of aIIb¿3 with bound ligands; Aim 3: Antibody labeling will be used to localize specific sites within the high- and low-affinity states; Aim 4: Molecular models of the high- and low- affinity states will be derived by combining the cryo-EM maps, site-specific antibody labeling data, and homology models for individual integrin domains. The structural details revealed by these studies will provide insight into the molecular basis of integrin activation, relevant for the design of new therapeutic agents.