Regulation of integrin function by divalent cations

描述（由申请人提供）血小板的聚集是血栓形成中的中心细胞事件，对于防止损伤部位的过度失血是必不可少的;但同时，血小板聚集是血栓性疾病的基础，包括心肌梗塞、不稳定型心绞痛和中风，这些疾病是工业化国家的主要死亡原因。在分子水平上，血小板聚集取决于粘附配体（如血浆蛋白纤维蛋白原）与血小板表面上的主要膜蛋白整合素β 3（GPIIb-IIIa）的结合。alibBeta 3与alphavBeta 3共享相同的β亚基。这种整合素分布更广泛，并参与生理和病理生理反应，包括骨吸收、骨质疏松、血管生成和肿瘤转移。纤维蛋白原与这两种β 3整联蛋白以及大多数配体与大多数整联蛋白（有超过20种整联蛋白）的结合是二价离子依赖性的，并且在二价阳离子和整联蛋白中的配体结合位点之间存在密切但未解决的关系。HL 54924的目的2检查了配体与β 1b β 3结合的分子基础。该FIRCA旨在扩展这些研究，以检查二价阳离子与β 3整合素结合的分子基础，并确定为什么二价阳离子差异地影响两种β 3整合素的功能。FIRCA应用程序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β 3的相互作用方面进行了长期而富有成效的合作。其次，最近，他们的研究已经导致了有可能提供新的见解二价阳离子如何调节整合素功能的发现。第三，整合素α v β 3胞外结构域的晶体结构刚刚发表，这为设计精确定义二价阳离子如何影响配体结合的精辟方法提供了重大突破和机会。本申请中开发的初步数据支持以下假设：β 3亚基的一小部分β 3（109-352）含有一个非选择性二价阳离子结合位点（必须被配体占据才能结合）、一个配体感受态（LC）位点和一个抑制配体结合的钙特异性抑制（I）位点。将在FIRCA中通过表达重组片段来检验这一假设，其中这两种阳离子结合的配位位点通过第一个特定目的中的系统性诱变来定位。第二个具体目标将通过对由杆状病毒系统中表达的α和β亚基部分组成的“微型受体”进行诱变研究，阐明钙对α 1b β 3和α v β 3的功能产生差异效应的原因。总之，这些研究将提供洞察二价阳离子在调节β整合素的配体结合功能中的作用。这些研究将补充父母补助金的目标。在演唱会上，这两个应用程序将提供与整合素的功能，具有生物医学重要性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.