NOVEL EFFECTORS OF GRB2 IN HUMAN GLIAL TUMORS

描述：(改编自《调查者摘要》)过度表达 受体酪氨酸激酶(RTK)是人脑胶质瘤的共同特征。 Grb2在RTK的信号通路中起着重要作用。它 作为一种适配蛋白，其中SH2结构域与许多 RTK的，而SH3域调节与SOS的交互，一种交换 上调RAS活性的因子。其他人的最新发现 实验室和首席调查员已经证明Grb2 与除SOS之外的其他几个潜在效应器交互。他们的 以前的工作已经发现这种效应器在 人类神经胶质瘤。几个与Grb2结合的克隆已经从 人脑胶质瘤基因表达文库。已经证实，有一个克隆人， 名为GAB1(用于Grb2关联的活页夹-1)，由Grb2和 转而可以沉淀Grb2。它在不同物种之间高度保守， 还发现了一个相关的cDNAs。出于这些原因，它是 推测GAB1在信号转导中具有重要作用。在……里面 这一目标，重点将是克隆完全开放的阅读框架 GAB1，并对其全长进行了测序。兔多克隆 将制备抗体，并进行免疫沉淀实验 执行以验证GAB1和Grb2在体内相互作用。抗体 还将生成GAB1的两种可选剪接形式的特定 这三种抗体将用于免疫印迹法以确定 天然蛋白质的大小，这种蛋白质的分布，以及 三种形式的相对表达水平。北方斑点 将用于确定剪接变体是否与 一定的文字记录大小。GAB1基因定位到的染色体 也将被确定。确定GAB1的功能将是关键 了解它是如何参与信号转导途径的 来自RTK的。线索可能来自与已知蛋白质的同源性。 该蛋白在NIH-3T3细胞中的过表达将揭示GAB1是否可以 影响细胞生长或导致细胞转化，如果EGF增加 任何能看到的效果。酪氨酸或丝氨酸/苏氨酸的磷酸化 将确定GAB1的地位。信息也将通过以下方式获得 使用细胞分离和免疫荧光研究来确定 GAB1是否与颗粒物分数或移位有关 随着生长因子的加入而转移到细胞核。如果这些方法是 不确定，确定GAB1与什么关联可能会产生洞察力 转化为势能函数。最后，GAB1的表达水平在 将对一系列高级别和低级别肿瘤进行检查，以确定是否有 与肿瘤分级和Southern印迹分析有相关性吗 ，以查看是否有任何删除、重新安排或 放大。将确定Grb2对GAB1的亲和力， 但分离出几个与Grb2结合的克隆提供了 有机会解决有关该组织性质的几个问题 Grb2的SH3结构域与其靶标的相互作用。 逐步的氨基和羧基截断突变将在所有 GAB克隆及其与Grb2和SH3单株的亲和力 将向这些底物测量结构域。最小的区域 将定义仍保留高亲和力绑定的 对多个克隆的评估精炼的共识结合位点可以 获得。这一分析还将揭示是否所有的Gab克隆人都拥有 多个目标站点，两个SH3域是否具有相同的 共识结合站点，如果两个域都需要给予高 亲和力结合，以及靶标是否延伸了二级结构 是赋予高亲和力结合所必需的。其他实验将会 以确认这两个域是否会导致协同绑定 效应器。该信息可用于预测Grb2相互作用 并设计新型Grb2抑制剂。

DESCRIPTION: (Adapted from Investigator's Abstract) Overexpression of receptor tyrosine kinases (RTK's) is a common feature of human gliomas. Grb2 has a fundamental role in the signaling pathways of RTK's. It functions as an adapter protein where the SH2 domain binds to numerous RTK's while the SH3 domains mediate interaction with SOS, an exchange factor that upregulates ras activity. Recent discoveries by other laboratories and the principal investigator have shown that Grb2 interacts with several other potential effectors aside from SOS. Their previous work has found specific overexpression of such effectors in human gliomas. Several clones that bind to Grb2 have been isolated from a glioma cDNA expression library. It has been verified that one clone, named Gab1 (for Grb2 associated binder-1), is recognized by Grb2 and in turn can precipitate Grb2. It is highly conserved across species and a related cDNA has also been found. For these reasons, it is hypothesized that Gab1 has an important role in signal transduction. In this aim, the focus will be on cloning the full open reading frame of Gab1 and sequencing the cDNA in its entirety. A rabbit polyclonal antibody will be made and immunoprecipitation experiments will be performed to verify that Gab1 and Grb2 interact in vivo. Antibodies specific for two alternative splice forms of Gab1 also will be generated and this trio of antibodies will be used on Western blots to determine the size of the native protein, the distribution of this protein, and the relative levels of expression of the three forms. Northern blots will be used to determine if the splice variants are associated with a certain transcript size. The chromosome to which the Gab1 gene maps also will be determined. Determining the function of Gab1 will be key to understanding how it participates in the signal transduction pathway from RTK's. Clues could arise from homologies with known proteins. Overexpression of the protein in NIH-3T3 cells will reveal if Gab1 can affect cell growth or cause cellular transformation and if EGF augments any effect seen. The tyrosine or serine/threonine phosphorylation status of Gab1 will be ascertained. Information will also be gained by using cell fractionation and immunofluoresence studies to determine whether Gab1 associates with the particulate fraction or translocates to the nucleus upon growth factor addition. If these approaches are inconclusive, identifying what Gab1 associates with may yield insights into potential functions. Finally, the levels of expression of Gab1 in a series of high and low grade tumors will be examined to see if there is any correlation with ascending tumor grade and Southern blot analysis will be done to see if there are any deletions, rearrangements or amplifications. The affinity of Grb2 towards Gab1 will be determined, but the isolation of several clones that bind to Grb2 affords the opportunity to resolve several questions about the nature of the interaction beween the SH3 domains of Grb2 and their targets. Progressive amino and carboxy truncation mutants will be made in all the Gab clones and the affinity to Grb2 and those of the individual SH3 domains will be measured towards these substrates. The smallest region that still retains high affinity binding will be defined and from the evaluation of multiple clones a refined consensus binding site can be obtained. This analysis also will reveal if all the Gab clones possess more than one target site, whether the two SH3 domains have the same consensus binding site, if the two domains are necessary to give high affinity binding, and whether extended secondary structure of the target is necessary to confer high affinity binding. Other experiments will be done to confirm if the two domains result in co-operative binding to the effector. This information can be used to predict Grb2 interactions and to design novel inhibitors of Grb2.