Activation Mechanisms of betaIII Integrins

β3整合素在暴露于血液中的细胞的生物学反应中起着关键作用。在血小板上，allb33是血小板聚集和血栓形成所必需的，也是抗血栓药物的靶点。在内皮细胞上，AV(33)是血管生成的主要调节因子，也是抑制血管生成的药物的靶点。这两种整合素(B3整合素)在生理和病理生理过程中的参与依赖于跨整合素的信号；由内向外的信号控制整合素的激活状态，由外向内的信号控制细胞骨架连接和无数的细胞内反应。 B3整合素和所有整合素的双向信号传递的两个臂都依赖于它们的细胞质尾巴(CT)与结合伙伴的相互作用。这项建议侧重于(B3整合素的两个特定结合伙伴：三个成员Kindlin家族的两个(Kindlin-2和Kindlin-3)和SCR家族的一个激酶Fyn。需要检验的假设是，这两个Kindlin是导致整合素激活的内向外信号的关键调节因子，Fyn与(B3CT)结合的方式不同于SCR，因此在控制外向内信号事件方面发挥了不同的作用。为了检验这些假说，本文提出了三个具体目标。具体目标1侧重于支配Kindlin-2和Kindlin-3和B3、Bt和其他整合素之间相互作用的结构-功能关系。将研究Kindlins直接诱导整合素激活或与talin内向外信号协同的能力；Kindlins共激活活性的潜在机制以及Kindlin-B3CT相互作用的结构细节将被确定。具体目标2将强调Kindlins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和膜通透性多肽来调节这些细胞中的Kindlin水平和功能。体内Kindlin-2水平降低的影响也将在小鼠身上进行分析。特异性目标3是基于最近的观察结果，即Fyn与B3CT的膜近端区域的一个位置结合。Fyn和Scr与B3CT的不同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确定，并将在体内对Fyn和Scr缺陷小鼠的血栓形成和血管生成进行分析。综上所述，这些研究将为B3整合素的反应的启动和传播提供关键的见解。这些信息反过来可能建立更有效的方法来设计针对B3整合素和其他整合素家族的抗血栓和抗血管生成药物 会员。

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.