EGF RECEPTOR FUNCTION AND CONTROL BY PHOSPHORYLATION

关于参与细胞生长控制的分子机制的信息是 对于了解围绕正常发育和 那些潜在的致癌因素。表皮生长因子(EGF)是一种 快速EGF诱导的配体刺激蛋白酪氨酸激酶 在其极端的羧基-(C-)末端进行自我磷酸化。EGF 受体与逆转录病毒转化蛋白v-erbB同源，并且 它在某些人类肿瘤中过度表达。令人感兴趣的是两个一般 增强其致癌能力的erbB基因突变：氨基末端 删除移除EGF结合位点和各种C-末端 截断。在这方面，我们的初步研究表明 C末端自磷酸化结构域的去除量降低很高 亲和力结合，增加体内酪氨酸激酶活性并破坏 受体与细胞骨架的联系。此外，这些和其他 研究强烈表明，受体C末端结构域和 受体与细胞骨架的相互作用都可以控制 表皮生长因子受体结合和激酶活性。因为EGF受体激酶 活动对于正常的生物功能是必不可少的，以下是 具体目标被提出：1)检查EGF通过哪些机制 受体C末端调节蛋白酪氨酸激酶的活性。这些 研究将涉及底物特异性、动力学的分析 正常和C-蛋白激活剂的作用机制及敏感性 EGF受体末端突变。2)描述EGF的作用 受体C末端促进细胞骨架附着，并确定 细胞骨架结合影响受体高亲和力的过程 结合和蛋白酪氨酸激酶活性。这项工作将集中在 C末端结构域和蛋白激酶C激活对受体的影响 细胞骨架的分布，并将评估细胞骨架的影响 受体高亲和力结合、酪氨酸激酶活性与 底物专一性。3)鉴定和分析涉及的特定蛋白质 在使用直接结合研究的EGF受体-细胞骨架结合中， 以及免疫共沉淀和交联分析。这些 预计经验将在以下方面更严格地建立这些机制 哪些特定区域，以及它们与细胞的相互作用 细胞骨架在EGF受体的调节中起关键作用 在正常和异常细胞生长中都起作用。

Information on the molecular mechanisms involved in cell growth control is important for understanding the events surrounding normal development and those underlying carcinogenesis. The epidermal growth factor (EGF) is a ligand-stimulated protein-tyrosine kinase that undergoes rapid EGF-induced self-phosphorylation in its extreme carboxy- (C-) terminus. The EGF receptor is homologous to the retroviral transforming protein v-erbB, and it is overexpressed in certain human tumors. Of interest are two general mutations in erbB that increase its oncogenic capacity: an amino-terminal deletion that removes the EGF binding site, and various C-terminal truncations. In this regard, our initial studies have suggested that removal of the C-terminal self-phosphorylation domain decreases high affinity binding, increases tyrosine kinase activity in vivo and disrupts receptor association with the cell cytoskeleton. Moreover, these and other investigations strongly suggest that the receptor C-terminal domain and receptor interaction with the cell cytoskeleton can both exert control over EGF receptor binding and kinase activities. Because EGF receptor kinase activity is essential for proper biological function, the following specific aims are proposed: 1) Examine the mechanisms by which the EGF receptor C-terminus regulates protein-tyrosine kinase activity. These studies will involve an analysis of the substrate specificity, kinetic mechanism and sensitivity to protein activators of both normal and C- terminally mutated EGF receptors. 2) Characterize the role of the EGF receptor C-terminus in promoting cytoskeletal attachment, and ascertain the processes by which cytoskeletal association affects receptor high affinity binding and protein-tyrosine kinase activity. This work will focus on the influence of C-terminal domains and protein kinase C activation on receptor cytoskeletal distribution, and will also asses the effects of cytoskeletal association on receptor high affinity binding, tyrosine kinase activity and substrate specificity. 3) Identify and analyze specific proteins involved in the EGF receptor-cytoskeletal association using direct binding studies, as well as co-immunoprecipitation and crosslinking analyses. These experiences are expected to more rigorously establish the mechanisms by which specific domains, and their interaction with the cellular cytoskeleton, can play a key role in the regulation of EGF receptor function in both normal and abnormal cell growth.