Support is requested to continue an analysis of the activation of beta3 integrins. The applicant hypothesizes that the binding of a cytoskeletal protein, talin, to the beta3 integrin cytoplasmic domain (tail) is a final common step in the activation of integrin alphaIlb beta3 (platelet GPllb-llIa), a crucial event in platelet aggregation and in arterial thrombosis. To test this idea, he will assess the capacity of competitive inhibitors of talin binding to inhibit integrin activation. Furthermore, the applicant proposes that unmasking the integrin binding site in talin regulates integrin activation. To test this hypothesis, he will engineer talin for pre-designated conditional proteolytic cleavage and assess the effects of talin cleavage on integrin activation. Furthermore, he will examine the role of talin phosphorylation and polyphosphoinositide binding in the activation process. The applicant will collaborate in nuclear magnetic resonance spectroscopic analysis of the talin-integrin interaction to test the hypothesis that an extended surface of talin interacts with a membrane-proximal domain of the beta3 integrin subunit to induce activation. Furthermore, he proposes that this membrane-proximal interaction leads to a downward displacement of the trans-membrane domain in a piston like motion. He will test this idea by mapping the topography of this membrane-proximal domain by derivation of introduced cysteine residues. Finally, the studies proposed here will identify useful mutations of talin and integrin beta3 that perturb their interactions and/or integrin activation. Selected mutations will be introduced into megakaryocytes to test the role of integrin activation in platelet environment. These fundamental studies will provide novel insight into the regulation of platelet aggregation and will test and advance paradigms that apply to many integrin-dependent biological processes.
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