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

gpl85 erbB-2与表皮生长因子具有广泛的同源性

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.