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

gp 185 erbB-2与表皮生长因子的广泛同源性 受体（EGFR），促使他们的生物学比较研究， 行动和信号通路。分析erbB-2和EGFR激酶 特异性在可比的条件下，我们设计了一个嵌合 EGFR/erbB-2表达载体分子连接编码EGFR/erbB-2表达载体的序列 EGFR的胞外结合结构域与编码EGFR的胞外结合结构域的结合结构域结合。 erbB-2产物的细胞内部分。这样的一种嵌合体 在激酶活性方面比亲本gp 185受到严格调节 erbB-2并显示出类似的转化能力，然而， 依赖于向培养基中加入EGF（100倍以上）， 当在NIH/3 T3中过表达时，比EGFR更能诱导转化 成纤维细胞）。erbB-2激酶的转化能力越高， 就EGFR而言，可能是由于两者的不同内在能力， 激酶磷酸化细胞内底物。这种假设是 支持这一发现的是，活性erbB-2和EGFR激酶诱导酪氨酸 不同亚类的推定底物的磷酸化。 磷脂酶C-γ（PLC-γ）和GTP酶激活的分析 蛋白（GAP），两个主要的底物参与的转导， 促有丝分裂信号，揭示了这两种蛋白质的底物， erbB-2和EGFR激酶活性。没有定量或定性 两种受体磷酸化这些蛋白的能力的差异 基质被证明。为了识别和描述新的 细胞内底物，我们纯化EGF诱导的磷酸酪氨酸蛋白 来自NIH/3 T3成纤维细胞过度表达EGFR。纯化的蛋白质 然后用于免疫动物以产生多克隆血清。使用 通过获得的抗血清，我们能够鉴定出许多新的蛋白质 作为EGFR的底物，其中大多数似乎是特异性的， EGF处理后酪氨酸残基磷酸化。

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.