Activation Mechanisms of betaIII Integrins

β 3整联蛋白在暴露于血液的细胞的生物反应中起关键作用。在血小板上，allb 33对于血小板聚集和血栓形成是不可缺少的，并且是抗血栓药物的靶点。在内皮细胞上，α V β 3是血管生成的主要调节因子，也是抑制血管生成药物的靶点。两种β 3整联蛋白在生理和病理生理过程中的参与取决于整联蛋白之间的信号传导;由内向外信号传导以控制整联蛋白的活化状态，由外向内信号传导以控制细胞骨架连接和无数细胞内反应。 跨β 3整联蛋白的双向信号传导的两个臂以及所有整联蛋白取决于它们的胞质尾（CT）与结合配偶体的相互作用。该提议集中于β 3整联蛋白的两种特定结合配偶体：三个成员Kindlin家族中的两个（Kindlin-2和Kindlin-3）和scr家族激酶之一Fyn。待检验的假设是，两种Kindlin是导致整联蛋白活化的由内而外信号的关键调节剂，并且Fyn与scr不同地结合至β 3 CT，从而在控制由外而内的信号传导事件中发挥不同的作用。提出了三个具体目标来检验这些假设。具体目标1侧重于控制Kindlin-2和Kindlin-3与B3 BT和其他整合素之间相互作用的结构-功能关系。将检查Kindlin直接诱导整联蛋白活化或与talin由内而外信号传导协同作用的能力;将确定Kindlin的共活化剂活性的潜在机制和Kindlin-B3 CT相互作用的结构细节。具体目标2将强调Kindlin对血小板、巨核细胞和内皮细胞中整合素介导的反应的影响。这些分析将涉及使用siRNA和膜渗透肽调节这些细胞中Kindlin的水平和功能。还将在小鼠中分析体内Kindlin-2水平降低的影响。特异性目的3是基于最近观察到Fyn与B3 CT膜近端区域中的位点结合。Fyn和Scr与B3 CT的差异性相互作用的分子细节以及Fyn与该位点结合的功能后果将在血小板和内皮细胞中以及Fyn和Scr缺陷小鼠中的血栓形成和血管生成的体内测定中确定。总之，这些研究将为B3整合素的反应启动和传播方式提供关键见解。这些信息反过来可能建立更有效的方法来设计靶向B3整联蛋白和其他整联蛋白家族的抗血栓形成和抗血管生成药物 成员

The Beta3 integrins play critical roles in the biological responses of cells exposed to blood. On platelets, allb33 is indispensable for platelet aggregation and thrombus formation and is a target for anti-thrombotic drugs. On endothelial cells, aV(33 is a major regulator of in angiogenesis and is also the target for drugs that suppress angiogenesis. The involvement of the two (B3 integrins in physiological and pathophysiological processes depends upon signaling across the integrins; inside-out signaling to control the activation state of the integrins and outside-in signaling to control cytoskeletal connections and a myriad of intracellular responses. Both arms of the bidirectional signaling across (B3 integrins as well as all integrins depend upon interaction of their cytoplasmic tails (CT) with binding partners. This proposal focuses on two particular binding partners of the (B3 integrins: two (Kindlin-2 and Kindlin-3) of the three member Kindlin family and one of the scr family kinases, Fyn. The hypotheses to be tested are that the two Kindlins are key regulators of the inside-out signals that lead to integrin activation and that Fyn binds differently than scr to the (B3 CT and, thereby, exerts distinct effects in controlling outside-in signaling events. Three specific aims are proposed to test these hypotheses. Specific Aim 1 focuses on the structure-function relationships that govern the interactions between Kindlin-2 and Kindlin-3 and B3 BT and other integrins. The ability of the Kindlins to directly induce integrin activation or to synergize with talin inside-out signaling will be examined; the mechanisms underlying the co-activator activities of the Kindlins and the structural details of the Kindlin-B3 CT interaction will be determined. Specific Aim 2 will emphasize the effects of the Kindlins on integrin-mediated responses in platelets, megakaryocytes and endothelial cells. These analyses will involve in modulating Kindlin levels and functions in these cells using siRNA and membrane permeable peptides. The effects of reduced levels of Kindlin-2 in vivo will also be analyzed in mice. Specific Aim 3 is predicated on the recent observation that Fyn binds to a site in the membrane proximal region of the B3 CT. The molecular details of the differential interactions of the Fyn and Scr with the B3 CT and the functional consequences of Fyn binding to this site will be determined in platelets and endothelial cells and in vivo assays of thrombosis and angiogenesis in Fyn and Scr deficient mice. Taken together, these studies will provide key insights into the way responses of the B3 integrins are initiated and propagated. This information may, in turn, establish more effective ways to design anti-thrombotic and anti-angiogenic drugs that target the B3 integrins and other integrin family members.