Activation of B3 Integrins

需要继续支持一个程序，以分析激活的整合素allbB 3，一个过程的核心正常止血和动脉血栓形成。申请人已经开发了用于分析细胞内整联蛋白信号传导的方法，鉴定了整联蛋白胞质面处的蛋白质-蛋白质相互作用，并提供了对其中一些相互作用的结构见解。他的研究证明了talin与整联蛋白B胞质结构域的结合是激活的最后一步，提供了talin如何激活整联蛋白的见解，显示了Rapl如何与talin合作激活整联蛋白，并验证了干扰talin-B3整联蛋白相互作用的抗血栓形成潜力。他现在将检验talin和/或kindlin与整联蛋白allbB 3结合足以 通过将整联蛋白allbB 3重组到脂质双层中并检测纯化蛋白激活整联蛋白的能力来检测活化。他将通过研究RIAM的片段，以及它们相互作用和与talin相互作用的Rap依赖性，来验证RIAM包含Rap 1调节的talin结合位点和RIAM揭示talin的整合素结合的假设。此外，他还将评估RIAM的talin结合片段与talin合作诱导细胞中整合素活化的能力，并将使用双分子荧光互补来评估这些RIAM片段促进整合素-talin相互作用的能力。第三个目的是通过分析哺乳动物细胞膜和脂质双胞中这些跨膜结构域的相互作用来检验α和B跨膜结构域的相互作用调节整联蛋白活化的假设。他将测试的假设，塔林诱导的整合素激活发挥了独特的作用，从塔林介导的连接的整合素的细胞骨架在白细胞和血小板的功能在体内，通过创建小鼠，要么缺乏野生型塔林在血小板和白细胞和表达塔林突变体，保持能力结合到整合素和细胞质的合作伙伴，如黏着斑蛋白，但不能激活整合素。他将与Ruggeri，Groisman和Ley博士合作评估这些动物的血小板和白细胞功能。这些基础研究将为血小板聚集的调节提供新的见解，并将测试和推进适用于许多整合素依赖性生物过程的范例。

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 at the 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 Rap1-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 B 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 integrin-dependent biological processes.