BIOCHEMICAL AND BIOPHYSICAL STUDIES OF THE EGF RECEPTOR

表皮生长在信号转导中的一个关键早期事件 表皮生长因子(EGF)受体是酪氨酸内源蛋白的激活 受体的激酶活性。酪氨酸激酶活性的激活 是多种多肽有丝分裂途径中的共同成分 生长因子和癌基因。拟议研究的第一个目标是 为了更好地了解EGF的调节机制 在酶水平上的受体/酪氨酸激酶。生化，生物物理学， 并将结合分子生物学方法来研究 酪氨酸激酶激活和自磷酸化的结构基础 反应。这些研究的对象将是重组受体 酪氨酸激酶域(TKD)形式，它将在 杆状病毒/昆虫细胞系统。TKD是TKD的酶成分 受体，没有跨膜和生长因子的结合 全受体多肽的结构域。TKD受监管 由二价金属离子、某些碱性蛋白质和一种特定抗体 试剂。该计划的第二个目标是了解 参与信号形成的蛋白质-蛋白质相互作用 生长因子受体与蛋白质的转导复合体 加入src同源结构域2(SH2)。关于酸性物质的假设 EGF受体的结构域，相关的erbB3酪氨酸激酶和 转化中T抗原(MT)与SH2特异性相互作用 结构域将通过将单个结构域表达为细菌来进行测试 融合蛋白。将开发方便的检测方法来监测 融合蛋白之间的相互作用，使这些因子 调节这些蛋白质之间的相互作用是可以研究的。在……里面 特别是，蛋白质磷酸化的作用将被讨论。 最后，由于c-src酪氨酸激酶最近被认为与 EGF受体的信号转导，是典型的SH2结构域 蛋白质，将尝试重建功能相互作用 在体外EGF受体和c-src之间的关系。一个渗透性的细胞系统， 其中c-src激酶的调节磷酸化已经被 观察到的，似乎是这些调查的理想选择。希望是这样的 了解酪氨酸激酶的作用机制及方法 在这三个研究过程中发展起来的将具有广泛的意义 酪氨酸激酶在两者信号转导研究中的应用 正常的生长途径和转化的细胞。

A critical early event in signal transduction by the epidermal growth factor (EGF) receptor is the activation of the intrinsic protein tyrosine kinase activity of the receptor. Activation of tyrosine kinase activity is a common element in the mitogenic pathways of various polypeptide growth factors and oncogenes. The first goal of the proposed research is to better understand the mechanisms of regulation of the EGF receptor/tyrosine kinase at the enzyme level. Biochemical, biophysical, and molecular biological approaches will be combined to study the structural basis of tyrosine kinase activation and the autophosphorylation reactions. The subjects of these studies will be recombinant receptor tyrosine kinase domain (TKD) forms, which will be expressed in a baculovirus/insect cell system. The TKD is the enzyme component of the receptor and is free of the membrane-spanning and growth factor binding domains of the holoreceptor polypeptide. The TKD is subject to regulation by divalent metal ions, certain basic proteins, and a specific antibody reagent. A second goal of the plan is to understand the physical basis of the protein-protein interactions involved in the formation of signal transduction complexes between growth factor receptors and proteins incorporating src homology domain 2 (SH2). The hypothesis that the acidic domains of the EGF receptor, the related erbB3 tyrosine kinase, and the transforming middle T antigen (mT) interact specifically with the SH2 domain will be tested by expressing the individual domains as bacterial fusion proteins. Convenient assays will be developed for monitoring the interactions between the fusion proteins, so that the factors which modulate these protein-protein interactions can be studied. In particular, the role of protein phosphorylation will be addressed. Finally, as the c-src tyrosine kinase has been recently implicated in signal transduction by the EGF receptor and is the prototypical SH2 domain protein, attempts will be made to reconstitute functional interactions between the EGF receptor and c-src in vitro. A permeabilized cell system, in which the regulatory phosphorylations of the c-src kinase have been observed, would appear to be ideal for these investigations. It is hoped that the understanding of tyrosine kinase mechanism and the methods developed in the course of these three studies will have broad applications in the study of tyrosine kinase signal transduction in both normal growth pathways and transformed cells.