BIOCHEMICAL AND BIOPHYSICAL STUDIES OF THE EGF RECEPTOR

表皮生长信号转导的一个关键早期事件 表皮生长因子（EGF）受体是激活的内在蛋白酪氨酸 受体的激酶活性。 酪氨酸激酶活性的激活 是多种多肽的促有丝分裂途径中的共同元件 生长因子和致癌基因。 拟议研究的第一个目标是 更好地了解EGF的调节机制 受体/酪氨酸激酶在酶水平。 生物化学，生物物理， 和分子生物学方法相结合， 酪氨酸激酶激活和自磷酸化的结构基础 反应. 这些研究的主题将是重组受体 酪氨酸激酶结构域（TKD）形式，其将以 杆状病毒/昆虫细胞系统。 TKD是蛋白质的酶组分， 受体，并且不含跨膜和生长因子结合 全受体多肽的结构域。 将军澳区受规管 二价金属离子、某些碱性蛋白质和特异性抗体 试剂. 该计划的第二个目标是了解 参与信号形成的蛋白质间相互作用 生长因子受体和蛋白质之间的转导复合物 引入src同源结构域2（SH 2）。 假设酸性物质 EGF受体的结构域，相关的erbB 3酪氨酸激酶，以及 转化中T抗原（mT）与SH 2特异性相互作用 将通过将各个结构域表达为细菌的形式来测试结构域。 融合蛋白 将开发方便的检测方法， 融合蛋白之间的相互作用，使因子， 调节这些蛋白质-蛋白质相互作用。 在 特别是，蛋白质磷酸化的作用将得到解决。 最后，由于c-src酪氨酸激酶最近被牵连到 EGF受体的信号转导，是原型SH 2结构域 蛋白质，将尝试重建功能相互作用 EGF受体与c-src的关系 一种透化细胞系统， 其中c-src激酶的调节性磷酸化已经被 观察到，似乎是这些调查的理想选择。 希望 对酪氨酸激酶机制的理解和研究方法 在这三项研究的过程中， 在酪氨酸激酶信号转导研究中的应用， 正常生长途径和转化细胞。

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.