THE GP185 ERBB-2 AND EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALLING PATHWAYS

GP185 ERBB-2 和表皮生长因子受体信号传导途径

• 批准号: 3874772
• 负责人: P P DI FIORE
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874772
• 关键词: biological signal transduction; chimeric proteins; enzyme substrate; epidermal growth factor; fusion gene; growth factor receptors; guanosinetriphosphatases; human tissue; mitogens; oncogenes; phospholipase C; phosphoproteins; protein tyrosine kinase; proteins; receptor binding; transposon /insertion element

The extensive homology between gpl85 erbB-2 and epidermal growth factor receptor (EGFR) has prompted comparative studies of their biological actions and signalling pathways. To analyze erbB-2 and EGFR kinase specificities under comparable conditions, we engineered a chimeric EGFR/erbB-2 expression vector molecule joining sequences coding for the extracellular binding domain of EGFR to those encoding for the intracellular portion of the erbB-2 product. Such a chimera is more stringently regulated in terms of kinase activity than the parental gpl85 erbB-2 and displays similar transforming potency which is, however, dependent on the addition of EGF to the culture medium (100-fold more potent than EGFR in inducing transformation when overexpressed in NIH/3T3 fibroblasts). The higher transforming potency of the erbB-2 kinase, with respect to EGFR, might be due to a different intrinsic ability of the two kinases to phosphorylate intracellular substrates. This hypothesis is supported by the finding that active erbB-2 and EGFR kinase induce tyrosine phosphorylation of different subsets of putative substrates. Analysis of phospholipase C-gamma (PLC-gamma) and the GTPase activating protein (GAP), two major substrates implicated in the transduction of the mitogenic signal, revealed that both these proteins are substrates for the kinase activity of erbB-2 and EGFR. No quantitative or qualitative differences in the ability of the two receptors to phosphorylate these substrates were evidenced. In order to identify and characterize new intracellular substrates, we purified EGF-induced phosphotyrosine proteins from NIH/3T3 fibroblast overexpressing EGFR. The purified proteins were then used to immunize animals for the production of polyclonal sera. Using the antisera so obtained, we were able to identify a number of new proteins as substrates for EGFR, most of which appear to be specifically phosphorylated on tyrosine residues after EGF treatment.

Gpl85-erbB-2与表皮生长因子的广泛同源性 受体(EGFR)促进了对其生物学特性的比较研究 动作和信号通路。检测erbB-2和EGFR激酶的研究 在可比条件下，我们设计了一种嵌合体 EGFR/erbB-2表达载体分子连接序列编码 EGFR的胞外结合域与编码 ErbB-2产物的胞内部分。这样的嵌合体比 在激酶活性方面比亲本gpl85更严格地调控 ErbB-2，并表现出类似的转化效力，然而， 依赖于在培养基中添加EGF(100倍以上 在NIH/3T3中过表达时比EGFR更有效地诱导转化 成纤维细胞)。更高的erbB-2激酶的转化能力， 关于EGFR，可能是由于两者不同的内在能力 使细胞内底物磷酸化的激酶。这一假设是 由活性的erbB-2和EGFR激酶诱导酪氨酸的发现支持 假定底物的不同亚组的磷酸化。 磷脂酶C-γ(PLC-γ)与GTP酶激活的分析 蛋白质(GAP)，两种主要的底物，涉及转导 有丝分裂信号显示，这两种蛋白质都是 ErbB-2和EGFR的激酶活性。没有数量或质量 这两种受体磷酸化这些的能力的差异 底物也得到了证实。为了识别和描述新的 细胞内底物，我们纯化了EGF诱导的磷酸酪氨酸蛋白 来源于高表达EGFR的NIH/3T3成纤维细胞。纯化的蛋白质是 然后用于免疫动物以生产多克隆血清。vbl.使用 如此获得的抗血清，我们能够鉴定出一些新的蛋白质 作为EGFR的底物，其中大多数似乎是专门的 EGF处理后酪氨酸残基上的磷酸化。