Beta2 Integrin Activation and Signaling

描述（由申请人提供）： 白细胞特异性β 2整联蛋白对这些细胞的生物学功能至关重要。β 2整联蛋白介导白细胞的二价金属离子依赖性粘附，包括归巢、牢固粘附、迁移、呼吸爆发和通过吞噬作用和细胞介导的杀伤清除病原体。β 2整联蛋白还导致许多非感染性疾病中的损伤，例如肾衰竭、同种异体移植排斥、心脏病发作、中风和自身免疫性糖尿病并发症，其中受体被病理激活。因此，β 2整联蛋白是炎症、自身免疫和移植中的重要治疗靶点。 整合素使用双向信号传导来调节细胞功能。由内而外的信号通过诱导整合素的胞外配体结合结构域的构象变化来激活整合素。整合素激活的结构基础知之甚少，是当前研究的热点。最近描述的高分辨率α V β 3整合素晶体结构突出了整合素二聚体的独特特征，并提供了对其激活性质的新见解。我们的目的是开发和测试特定的，基于结构的整合素激活的假设，重点是β 2整合素CD 11b/CD 18，通过使用α V β 3结构作为模型的CD 11b/CD 18整合素。我们将在整合素亚基中产生突变，并使用基于细胞的和生物化学测定来研究它们对整合素活化和生物功能的影响。在目标1中，我们将评估金属离子在α-Genu上配位的基础，并确定屈曲在变构整合素活化中的作用。α-β是位于大腿和小腿-1结构域之间的α-亚基中的膝结构。该结构作为一个高度灵活的铰链，是网站的弯曲的晶体结构，有一个二价金属离子与之关联。在目的2，我们将检查的死栓假说和研究β TD的作用，在由内而外激活整合素。β尾结构域（betaTD）是在整合素晶体结构中的β亚基中发现的新结构域。未配体和配体的整合素结构的比较表明，该结构域可以通过与β A结构域结合来调节整合素活化，从而作为固定栓（deadbolt）来保持整合素处于无活性形式。在目标3中，我们将确定细胞外近膜残基在整合素同源寡聚化和聚集中的功能。各种整合素之间的序列比对显示，在β TD和跨膜区之间存在许多保守残基。该残基可能在整合素活化或聚集中起作用。 鉴于β 2整联蛋白在白细胞功能的各个方面的重要性，这些研究将对细胞生物学产生深远的影响，并将有助于设计新的治疗方法来治疗由整联蛋白的病理调节引起的许多疾病。从这些研究中获得的见解也可能扩展到其他整合素。

DESCRIPTION (provided by applicant): The leukocyte specific beta2 integrins are central to the biological function of these cells. Beta2 integrins mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell-mediated killing, beta2 integrins also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications, where the receptors are pathologically activated. Therefore, beta2 integrins are important therapeutic targets in inflammation, autoimmunity, and transplantation. Integrins use bi-directional signaling to regulate cellular functions. Inside-out signals activate integrins by inducing conformational changes in their extracellular ligand-binding domains. Structural basis for integrin activation is poorly understood and is a focus of intense current research. Recently described high-resolution alphaVbeta3 integrin crystal structures highlight distinctive features of the integrin dimers and provide new insights into the nature of their activation. Our aim is to develop and test specific, structure-based hypotheses for integrin activation, with a focus on beta2 integrin CD11b/CD18, by using alphaVbeta3 structure as the model for the CD11b/CD18 integrin. We will generate mutations in integrin subunits and study their effect on integrin activation and biological function using cell-based and biochemical assays. In aim 1, we will evaluate the basis of metal ion coordination at alpha-Genu, and determine the role of flexion in allosteric integrin activation. Alpha-genu is the knee structure in the alpha-subunit that lies between the thigh and the calf-1 domains. The structure serves as a highly flexible hinge, and is the site of the bend in the crystal structure that has a divalent metal ion associated with it. In aim 2, we will examine the deadbolt hypothesis and study the role of betaTD in inside-out activation of integrins. Beta tail domain (betaTD) is a novel domain that was found in beta-subunit in the integrin crystal structure. Comparison of unliganded and liganded integrin structures shows that this domain may regulate integrin activation by binding with the betaA domain and thereby acting as a deadbolt to keep integrins in an inactive form. In aim 3, we will determine the function of a extracellular juxta-membrane residue in integrin homo-oligomerization and clustering. Sequence alignment between various integrins shows that there are a number of conserved residues between the betaTD and the transmembrane region. This residue may play a part in integrin activation or clustering. Given the importance of beta2 integrins in every aspect of leukocyte function, these studies will have a profound impact on cell biology and will facilitate design of novel therapeutics to treat the many diseases resulting from pathologic modulation of integrins. The insights gained from these studies are also likely to extend to other integrins.