ACTIVATION OF THE PLATELET FIBRINOGEN RECEPTOR

血小板整合素GPIIb-IIIa的胞质结构域(或 在接收激动剂的信号方面起着关键作用。 刺激血小板，导致GPIIb-IIIa的激活构象。 因此，我们试图确定与细胞结合的蛋白质。 胞质结构域和受调控的GPIIb-IIIa激活。我们已经获得了 一种新的约25 kDa的结合蛋白的全序列 利用酵母双杂交系统，将其克隆到GPIIb胞质结构域。这 蛋白质与调节亚基钙调蛋白和 钙调神经磷酸酶B，有两个EF手区，结合Ca/45，表达于 血小板，并与抗体捕获的天然GPIIb-IIIa结合。我们有 因此将该蛋白命名为“CIB”，意为钙离子和整合素结合蛋白。 在具体目标#1中，我们建议进一步探索结构和 CIB的功能，尤指与整合素功能有关的。具体而言 目标2，我们建议绘制通向(1)的信号转导通路 激活和(2)维持GPIIb-IIIa的激活。绘制地图 GPIIb-IIIa激活途径，我们将使用CHO细胞表达 成分活性R-RAS，因为GPIIb-IIIa在这些 Ruosiahti和同事最近报道的细胞，并由 我们。具体地说，我们将(A)进一步确定其他分子是否与 与R-RAS相关的基因(如TC21等)也激活了这些基因中的GPIIb-IIIa 细胞；(B)微量注射R-RAS蛋白和其他分子鉴定 激活GPIIb-IIIa，以确定它们是否立即并因此 更直接地影响GPIIb-IIIa功能，而不是潜在地 诱导合成其他实际诱导激活的蛋白质； 以及(C)通过以下方式剖析R-RAS和整合素之间的信号通路 表达可能的信号分子的显性负向版本 在R-RAS下游。(D)最后，绘制必要的信号通路图 维持GPIIb-IIIa的激活我们将用CHO细胞表达 突变的GPIIb-IIIa，存在于一个能量依赖的，结构性的 活动状态，其方法与上述方法类似。因此，这些 研究将使我们能够剖析“由内而外”的整合素信号通路 在分子水平上，使用不能用于无核的方法 血小板。

The cytoplasmic domains of the platelet integrin GPIIb-IIIa (or alphaIIbBeta3) play a critical role in receiving signals from agonist- stimulated platelets, resulting in a activated conformation of GPIIb-IIIa. We have therefore attempted to identify proteins that bind to the cytoplasmic domains and regulated GPIIb-IIIa activation. We have obtained the complete sequence of a novel approximately 25 kDa protein that binds to the GPIIb cytoplasmic domain, using the yeast two-hybrid system. This protein is homologous to the regulatory subunits calmodulin and calcineurin B, has two EF hand domains and binds Ca/45, is expressed in platelets, and binds to antibody-captured, native GPIIb-IIIa. We have therefore named this protein "CIB) for Ca2+ and integrin binding protein. In specific aim #1, we propose to further explore the structure and function of CIB, especially as related to integrin function. In specific aim #2, we propose to map signal transduction pathways leading to (1) the activation and (2) the maintenance of activation of GPIIb-IIIa. To map pathways for GPIIb-IIIa activation, we will use CHO cells expressing constitutively active R-Ras, since GPIIb-IIIa becomes active in these cells as reported recently by Ruosiahti and coworkers and reproduced by us. Specifically we will (a) further determine if other molecules closely related to R-Ras (e.g. TC21 and others) also activate GPIIb-IIIa in these cells; (b) microinject R-Ras protein and other molecules identified that activate GPIIb-IIIa, to determine whether they immediately and therefore more directly affect GPIIb-IIIa function, as opposed to potentially inducing synthesis of other proteins that actually induce the activation; and (c) dissect the signaling pathway between R-Ras and the integrin by expressing dominant negative versions of likely signaling molecules downstream of R-Ras. (d) Finally, to map signaling pathways necessary for the maintenance of GPIIb-IIIa activation we will use CHO cells expressing mutant GPIIb-IIIa that exists in an energy dependent, constitutively active state with approaches similar to those described above. Thus these studies will allow us to dissect "inside-out" integrin signaling pathways on a molecular level, with approaches that cannot be used in the anucleate platelet.