Activation of the beta-3 integrins: Role of the kindlins

描述（由申请人提供）：整合素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，aIIb¿3对于血小板聚集和血栓形成是不可或缺的。在内皮细胞上，av - 3是细胞粘附和迁移反应的主要调节剂，并影响血管生成。整合素在生物反应中的参与依赖于整合素之间的信号传导；由内向外的信号传导控制整合素的激活状态，由外向内的信号传导控制细胞骨架连接和细胞内的信号传导。3整合素和所有整合素的双向信号通路的两条臂都依赖于它们的短细胞质尾部（CT）与结合伙伴的相互作用。这一建议的重点是一个新认识的家族的结合伙伴的CT， kindlins，特别是在整合素激活的作用。最近的出版物（包括申请人实验室的出版物）表明，家族成员在亚投行和亚投行的功能中起着关键作用。要验证的主要假设是kindin -2和kindin -3调节整合素的激活。从机制上讲，假设这种活性依赖于点燃蛋白与CT远端膜区域的结合，在那里每个点燃蛋白都可以与第二个CT结合伙伴talin合作。提出了三个具体目标。目的1侧重于kindlin：整合素相互作用的要求和功能结果。kindlin结合整合素2亚基的选择性将被检验；这两种kindlins直接诱导整合素激活或与talin协同的能力将被确定；并求解了3型CT与引火剂复合时的结构。目的2阐述了kindlins共激活剂活性的潜在机制，并考虑了¿3 CT的磷酸化如何影响kindlin结合。目的3强调kindlins对完整细胞、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的kindlin水平和功能。体内kindlin-2水平降低的影响也将在小鼠中进行分析。综上所述，这些研究将为研究3整合素的生物反应是如何启动的提供关键的见解。这一信息可能反过来，建立更有效的方法来设计抗血栓和抗血管生成的药物靶向¿3整合素和其他整合素家族成员。公共卫生相关性：血小板聚集对血栓形成至关重要，因此是心脏病发作和中风等血栓性疾病的基础。同样，血管生成，即新血管的形成，也与癌症和受损心脏组织的修复有关。我们试图确定一组新的分子如何影响血小板和内皮细胞来控制聚集和血管生成，从而确定治疗和预防心血管疾病的新靶点。

DESCRIPTION (provided by applicant): The ¿3 integrins play critical roles in the biological responses of cells exposed to blood. On platelets, aIIb¿3 is indispensable for platelet aggregation and thrombus formation. On endothelial cells, av¿3 is a major regulator of the adhesive and migratory responses of the cells and influences angiogenesis. The involvement of the ¿3 integrins in biological responses 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 intracellular signaling. Both arms of the bidirectional signaling across ¿3 integrins as well as all integrins depend upon interaction of their short cytoplasmic tails (CT) with binding partners. This proposal focuses on a newly recognized family of binding partners of the ¿3 CT, the kindlins, and particularly on their role in integrin activation. Very recent publications, including ones from the applicant's laboratory, suggest a pivotal role of kindlin family members in the function of aIIb¿3 and av¿3. The primary hypothesis to be tested is that kindlin-2 and kindlin-3 regulate integrin activation. Mechanistically, it is postulated that this activity relies on binding of the kindlins to the membrane distal region of the ¿3 CT where each can cooperate with a second ¿3 CT binding partner, talin. Three specific aims are proposed. Aim 1 focuses on the requirements and functional outcomes of kindlin:integrin interaction. The selectivity of kindlin binding to integrin 2 subunits will be examined; the ability of the two kindlins to directly induce integrin activation or synergize with talin in inside-out signaling will be determined; and the structure that the ¿3 CT assumes when complexed with kindlins will be solved. Aim 2 addresses the mechanisms underlying the co-activator activities of kindlins and considers how phosphorylation of the ¿3 CT influence kindlin binding. Aim 3 emphasizes the effects of the kindlins on integrin-mediated responses in intact cells, platelets, megakaryocytes and endothelial cells. These analyses will involve modulation of 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. Taken together, these studies will provide key insights into the way biological responses of the ¿3 integrins are initiated. This information may, in turn, establish more effective ways to design anti-thrombotic and anti-angiogenic drugs that target the ¿3 integrins and other integrin family members. PUBLIC HEALTH RELEVANCE: Platelet aggregation is essential for thrombus formation and, hence, underlies the thrombotic diseases of heart attack and stroke. Similarly, angiogenesis, the formation of new blood vessels, is involved in cancer and the salvage of damaged heart tissue. We seek to define how a new set of molecules influence platelets and endothelial cells to control aggregation and angiogenesis, thereby identifying new targets for treatment and prevention of cardiovascular disease.