Regulation of integrin function by divalent cations

说明(申请人提供)血小板聚集是血栓形成的中心细胞事件，对于防止损伤部位失血过多是必不可少的，但同时也是血栓性疾病的基础，包括心肌梗死、不稳定心绞痛和中风，这些都是工业化国家的主要死亡原因。在分子水平上，血小板聚集依赖于黏附配体(如血浆蛋白纤维蛋白原)与血小板表面主要膜蛋白整合素α-β3(GPllb-111a)的结合。AlphalibBeta3与AlphavBeta3共享相同的Beta亚基。这种整合素分布更广泛，参与包括骨吸收、骨质疏松、血管生成和肿瘤转移在内的生理和病理生理反应。纤维蛋白原与这两种β3整合素的结合以及大多数配体与大多数整合素(有20多个整合素)的结合是二价离子依赖的，整合素中的二价阳离子与配体结合部位之间存在密切但尚未解决的关系。HL54924的目的2研究了配体与alphallbBeta3结合的分子基础。FIRCA试图扩展这些研究，以检查二价阳离子与AlphalibBeta3结合的分子基础，并确定为什么二价阳离子不同地影响两种Beta3整合素的功能。申请FIRCA的动力来自三个来源。首先，Plow博士和Cierniewski博士在研究二价阳离子与AlphalibBeta3的相互作用方面进行了长期而富有成效的合作。其次，最近，他们的研究导致了一些发现，这些发现有可能为二价阳离子如何调节整合素功能提供新的见解。第三，整合素AlphavBeta3胞外区的晶体结构刚刚公布，这提供了一个重大突破和机会来设计精辟的方法来精确地定义二价阳离子如何影响配体结合。应用程序中开发的初步数据支持这样的假设，即Beta3亚基的一小部分，Beta3(109-352)，包含一个非选择性的二价阳离子结合位点，配体必须占据该结合位点，一个配体胜任(LC)位点，以及一个钙专一性的抑制配体结合的(I)位点。这一假设将通过表达重组片段在FIRCA中得到验证，在重组片段中，这两个阳离子结合的配位位置通过第一个特定目的的系统突变来定位。第二个具体目标将通过对杆状病毒系统中表达的部分α和β亚基组成的“迷你受体”进行突变研究，来解决为什么钙对AlphallbBeta3和AlphavBeta3的功能产生不同的影响。综上所述，这些研究将为二价阳离子在调节Beta整合素的配体结合功能中的作用提供深入的见解。这些研究将补充父母助学金的目标。同时，这两个应用将提供与具有生物医学重要性的整合素功能相关的基本分子机制的基本见解。

DESCRIPTION (provided by applicant) The aggregation of blood platelets, the central cellular event in thrombus formation, is essential to prevent excessive blood loss at sites of injury; but, at the same time, underlies the thrombotic diseases including myocardial infarction, unstable angina, and stroke, the leading causes of death in industrialized countries. At a molecular level, platelet aggregation depends upon the binding of adhesive ligands, such as the plasma protein fibrinogen, to the major membrane protein on the platelet surface, integrin alphalibBeta3 (GPllb-llla). alphalibBeta3 shares the same beta subunit with alphavBeta3. This integrin is more broadly distributed and is involved in both physiologic and pathophysiologic responses including bone resorption, in osteoporosis, angiogenesis and tumor metastasis. Binding of fibrinogen to these two beta3 integrins, as well as most ligands to most integrins (there are more than 20 integrins), is divalent ion dependent, and there is an intimate, but unresolved relationship between the divalent cation and ligand binding sites in the integrins. Aim 2 of HL54924 examines the molecular basis for ligand binding to alphallbBeta3. This FIRCA seeks to expand these studies to examine the molecular basis for divalent cation binding to alphalibBeta3 and to determine why divalent cations differentially influence the functions of the two Beta3 integrins. The impetus for this FIRCA application comes from three sources. First, Drs. Plow and Cierniewski have had a longstanding and productive collaboration in examining the interaction of divalent cations with alphalibBeta3. Second, recently, their investigations have led to findings which have the potential to provide novel insights into how divalent cations regulate integrin function. Third, the crystal structure of the extracellular domain of integrin alphavBeta3 has just been published, providing a major breakthrough and an opportunity to design incisive approaches to precisely define how divalent cations influence ligand binding. The preliminary data developed in the application support the hypothesis that a small segment of the Beta3 subunit, Beta3 (109-352), contains a non-selective divalent cation binding site, which must be occupied for ligands to bind, a ligand competent (LC) site, and a calcium-specific, inhibitory (I) site which inhibits ligand binding. This hypothesis will be tested in the FIRCA by expressing recombinant fragments in which the coordination sites for these two cation bindings are localized by systematic mutagenesis in the first Specific Aim. The second Specific Aim will address why calcium exerts differential effects on the function of alphallbBeta3 and alphavBeta3 by performing mutagenesis studies on "mini receptors" composed of portions of the alpha and beta subunits expressed in baculovirus systems. Taken together, these studies will provide insights into the role of divalent cations in regulating the ligand binding functions of the Beta integrins. These studies will complement the goals of the parent grant. In concert, the two applications will provide fundamental insights into the basic molecular mechanisms associated with the functions of integrins that are of biomedical importance.