Activation of B3 Integrins

整合素allbB3的活化分析是正常止血和动脉血栓形成的核心过程，需要继续得到支持。申请人已经开发了分析细胞内整合素信号的方法，确定了整合素细胞质表面的蛋白质-蛋白质相互作用，并提供了一些相互作用的结构见解。他的研究证明了talin结合到整合素B细胞质结构域是激活的最后一步，提供了talin如何激活整合素的见解，展示了Rapl如何与talin合作激活整合素，并验证了干扰talin- b3整合素相互作用的抗血栓潜力。他现在将测试talin和/或kindlin与整合蛋白allbB3结合的假设

Continued support is requested for a program to analyze activation of integrin allbB3, a process central to normal hemostasis and to arterial thrombosis. The applicant has developed approaches for analyzing intracellular integrin signaling, identified protein-protein interactions atthe integrin cytoplasmic face, and provided structural insight into some of those interactions. His studies proved that talin binding to the integrin B cytoplasmic domain is a final step in activation, provided insights into how talin activates integrins, showed how Rapl cooperates with talin to activate integrins, and validated the anti-thrombotic potential of interfering with the talin-B3 integrin interaction. He will now test the hypothesis that talin and/or kindlin binding to integrin allbB3 is sufficient for activation by reconstituting integrin allbB3 into lipid bilayers and examining the capacity of purified proteins to activate the integrin. He will test the hypothesis that RIAM contains a Rapl-regulated talin binding site and that RIAM uncovers talin's integrin binding by studying fragment of RIAM, and the Rap dependence of their interactions with each other and with talin. In addition he will assess the ability of talin-binding fragments of RIAM to cooperate with talin to induce integrin activation in cells and will use bimolecular fluorescence complementation to assess the capacity of these RIAM fragments to promote the integrin-talin interaction. A third aim will test the hypothesis that interactions of the a and p transmembrane domains regulate integrin activation by analyzing the interactions of these transmembrane domains in mammalian cell membranes and in lipid bicelles. He will test the hypothesis that talin-induced integrin activation plays a distinct role from talin-mediated linkage of integrins to the cytoskeleton in leukocyte and platelet function in vivo by creating mice that either lack wild type talin in platelets and leukocytes and that express talin mutants that maintain the ability to bind to integrins and cytoplasmic partners such as vinculin, but cannot activate integrins. He will assess platelet and leukocyte function in these animals in collaborations with Drs. Ruggeri, Groisman, and Ley. These fundamental studies will provide novel insight into the regulation of platelet aggregation and will test and advance paradigms that apply to many integrindependent biological processes.