INTERACTION OF GPIIB-IIIA WITH THE PLATELET CYTOSKELETON

整合素作为细胞粘附受体参与多种生物过程，与细胞外基质和肌动蛋白细胞骨架相互作用。虽然talin被认为是连接整联蛋白和肌动蛋白之间的连接蛋白，但对于调节整联蛋白与talin相互作用的分子机制知之甚少。此外，talin在整合素介导的信号事件中的作用尚未明确定义。该建议的中心假设是talin与整合素细胞质结构域的相互作用在通过整合素受体的内向外和外向内信号传导中起重要作用。以alphaIIbbeta3为原型整合素，我们的目的是：1)进一步表征alphaIIbbeta3细胞质结构域的talin结合位点。通过靶向诱变alphaIIbbeta3，我们将评估alphaIIbbeta3细胞质域近膜区碱性氨基酸残基在局灶黏附和talin结合中的作用。此外，我们将研究alphaIIbbeta3的激活和配体占用是否会调节其与talin的结合亲和力。2)将整合素结合位点定位在talin的N端头部和c端尾部区域。通过化学交联研究、抑制抗体表位定位和位点定向诱变，我们将确定talin介导与alphaIIbbeta3相互作用的关键残基。3)研究talin修饰对alphaIIbbeta3-talin相互作用调控的影响。我们将评估“由内向外”信号传导过程（即PKC介导的talin磷酸化和PtdIns(4,5) p2诱导的talin-vinculin复合物形成）对talin- alphaiibbeta3相互作用的亲和调节的影响。4)确定整合素-talin相互作用在整合素介导的“outside-in”信号传导中的功能意义。在携带alphaIIbbeta3的CHO细胞中，talin头部结构域的过表达会阻碍细胞的扩散和局灶性粘附。我们将利用异丙基硫- β -半乳糖苷（IPTG）诱导的成纤维细胞表达系统来研究talin头部结构域的表达对α 5beta1介导的细胞扩散和局灶性粘附的主要负面影响。利用该系统，我们还将研究整合素-细胞骨架相互作用通过pp60src、pp125FAK和MAP激酶在“外向内”信号传导中的作用。

It is well established that integrins are involved in diverse biological processes by serving as cell adhesion receptors that interact with both the extracellular matrix and the actin cytoskeleton. Although talin has been implicated to serve as a linkage protein between integrins and actin filaments, little is known about the molecular mechanism(s) mediating integrin-talin interaction. Furthermore, the role of talin in integrin-mediated signaling events has not been clearly defined. The central hypothesis of this proposal is that the interaction of talin with the cytoplasmic domains of integrins plays an important role in inside-out and outside-in signaling through integrin receptors. Using alphaIIbbeta3 as the prototype integrin, we aim: 1) To further characterize the talin binding site in the cytoplasmic domain of alphaIIbbeta3. By targeted mutagenesis of a alphaIIbbeta3, we will evaluate the role of the basic amino acid residues in the juxtamembrane region of the alphaIIbbeta3 cytoplasmic domain in focal adhesion and talin binding. Furthermore, we will examine whether activation and ligand occupancy of alphaIIbbeta3 would modulate its binding affinity to talin. 2) To localize the integrin binding site(s) within the N- terminal head and C-terminal tail domains of talin. By chemical cross-linking studies, epitope mapping of inhibitory antibodies, and site directed mutagenesis, we will determine the critical residues in talin mediating interaction with alphaIIbbeta3. 3) To investigate the effect of talin modifications on the regulation of alphaIIbbeta3-talin interaction. We will evaluate the effect of "inside-out" signaling processes (i. e., PKC- mediated phosphorylation of talin and PtdIns(4,5)P2-induced talin-vinculin complex formation) on affinity modulation of talin-alphaIIbbeta3 interaction. 4) To determine the functional significance of integrin-talin interaction in integrin-mediated "outside-in"signaling. Overexpression of the talin head domain in CHO cells bearing alphaIIbbeta3 blocks cell spreading and focal adhesion. We will utilize an isopropylthio-beta- galactoside (IPTG)-inducible expression system in fibroblasts to examine the dominant negative effect of the expression of the talin head domain on alpha5beta1-mediated cell spreading and focal adhesion. Using this system, we will also investigate the role of integrin-cytoskeleton interaction on "outside-in" signaling through pp60src, pp125FAK and MAP kinase.