Structure Analysis of Integrin Activation

描述（由申请人提供）：心血管疾病是美国死亡的主要原因，主要是由于冠状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血栓形成。刺激血栓形成的一个关键事件是血小板聚集，这是由原型整合素介导的。整合素是一个异二聚体跨膜受体家族，它调节细胞粘附，如血小板聚集，以及其他重要的生物过程，如细胞迁移、分化、增殖和细胞程序性死亡。整合素通过介导细胞外粘附分子与细胞内环境之间的动态联系来实现这些不同的功能。整合素的功能受跨膜信号的调控，这可能是结合细胞外配体（“外向内”信号）以及分子与细胞质结构域结合（“内向外”信号）的结果。我们建议使用电子冷冻显微镜、图像重建和分子模型来研究人血小板整合素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。我们已经完成了全长、低亲和力、无活性构象的人血小板整合素aIIb¿3的三维结构。然后将aV¿3外畴的x射线结构与低分辨率低温电镜图对接。这种结合的方法使我们能够提出与整合素激活相关的结构重排模型。本项目的总体目标是对该模型进行整合素激活测试，我们的实验是按照以下具体目标组织的：目标1：继续进行图像分析，以提高aIIb¿3在低亲和状态下的三维重建分辨率；目标2：通过对结合配体的aIIb¿3进行分析，得出高亲和状态的3D图；目标3：抗体标记将用于定位高亲和力和低亲和力状态下的特定位点；目标4：高亲和力和低亲和力状态的分子模型将通过结合冷冻电镜图谱、位点特异性抗体标记数据和单个整合素结构域的同源性模型来推导。这些研究揭示的结构细节将为整合素激活的分子基础提供见解，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.