Activation of B3 Integrins

需要继续支持一项分析整合素allbB3激活的计划，这是正常止血和动脉血栓形成的核心过程。申请人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，并提供了对其中一些相互作用的结构洞察。他的研究证明了talin与整合素B细胞质结构域的结合是激活的最后一步，为talin如何激活整合素提供了见解，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。他现在将检验这样的假设，即talin和/或kindlin与整合素allbB3结合就足以 通过将整合素allbB3重组到脂质双层中并检测纯化的蛋白激活整合素的能力来激活。他将通过研究RIAM的片段，以及它们相互作用和与TALIN的相互作用对RAP的依赖，来检验RIAM包含RAP1调节的Talin结合位点的假设，以及Ram发现Talin的整合素结合的假设。此外，他将评估riam的talin结合片段与talin合作在细胞中诱导整合素激活的能力，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。第三个目的将通过分析哺乳动物细胞膜和脂双小体中这些跨膜结构域的相互作用来检验这一假设，即A和B跨膜结构域的相互作用调节整合素的激活。他将通过创造在血小板和白细胞中缺乏野生型Talin，并表达talin突变体的小鼠，保持与整合素和细胞质伙伴(如vinculin)的结合能力，但不能激活整合素，来测试Talin诱导的整合素激活在体内与talin介导的整合素与白细胞细胞骨架和血小板功能的不同作用。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的生物过程的范例。

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.