INTERACTION OF GPIIB-IIIA WITH THE PLATELET CYTOSKELETON

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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.