INTERACTION OF GPIIB-IIIA WITH THE PLATELET CYTOSKELETON

整合素通过作为细胞粘附受体与细胞外基质和肌动蛋白细胞骨架相互作用而参与多种生物过程。 尽管talin被认为是整合素和肌动蛋白丝之间的连接蛋白，但对介导整合素-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传导事件中的作用尚未明确定义。 该建议的中心假设是，塔林与整合素的胞质结构域的相互作用在通过整合素受体的由内向外和由外向内的信号传导中起重要作用。以α Ⅱ b β 3为原型整合素，我们的目标是：1）进一步表征α Ⅱ b β 3胞质结构域中的talin结合位点。 通过α IIb β 3的靶向突变，我们将评估α IIb β 3胞质结构域的质膜区域中的碱性氨基酸残基在粘着斑和塔林结合中的作用。此外，我们将研究是否激活和alphaIIbbeta 3的配体占用将调节其结合亲和力塔林。 2)将整联蛋白结合位点定位在talin的N-末端头部和C-末端尾部结构域内。 通过化学交联研究，抑制性抗体的表位定位，和定点诱变，我们将确定塔林介导的相互作用与alphaIIbbeta 3的关键残基。 3)研究talin修饰对α IIb β 3-talin相互作用的调节作用。 我们将评估“由内而外”的信号传导过程的影响（即。例如，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-alphaIIb β 3相互作用的亲和力调节。 4)确定整合素-talin相互作用在整合素介导的“由外向内“信号传导中的功能意义。 在携带α IIb β 3的CHO细胞中talin头部结构域的过表达阻断了细胞铺展和粘着斑。 我们将利用异丙基硫代-β-半乳糖苷（IPTG）诱导的表达系统在成纤维细胞中检查的talin头部结构域的表达α 5 β 1介导的细胞扩散和粘着斑的显性负效应。 利用该系统，我们还将研究整合素-细胞骨架相互作用对通过pp 60 src、pp 125 FAK和MAP激酶的“outside-in”信号传导的作用。

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.