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

描述(由申请人提供)：整合素在暴露在血液中的细胞的生物学反应中发挥关键作用。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是细胞黏附和迁移反应的主要调节因子，并影响血管生成。整合素在生物反应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细胞内信号。跨3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短细胞质尾巴(CT)与结合伙伴的相互作用。这项建议集中在一个新发现的结合伙伴家族的3 CT，Kindlins，特别是它们在整合素激活中的作用。最近发表的文章，包括申请人实验室的文章，表明kindlin家族成员在AIIB？3和av？3的功能中起着关键作用。需要检验的主要假设是kindlin-2和kindlin-3调节整合素的激活。从机制上说，这种活动依赖于Kindlins与CT膜远端区域的结合，在那里每个Kindlins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重点研究Kindlin：整合素相互作用的要求和功能结果。将检测Kindlin与整合素2亚基结合的选择性；将确定这两种Kindlins直接诱导整合素激活或在内向外信号转导中与talin协同作用的能力；并解决与kindlins结合时所呈现的结构。目的2探讨Kindlins共激活子活性的潜在机制，并考虑3CT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和膜通透性多肽来调节这些细胞中Kindlin的水平和功能。体内Kindlin-2水平降低的影响也将在小鼠身上进行分析。综上所述，这些研究将为整合素的生物反应的启动方式提供关键的见解。这些信息反过来可能建立更有效的方法来设计针对整合素和其他整合素家族成员的抗血栓和抗血管生成药物。公共卫生相关性：血小板聚集是血栓形成的关键，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挽救有关。我们试图定义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.