Epidermal-growth-factor receptors generate Ras.GTP more efficiently than insulin receptors

Activation of the Ras proto-oncogene contributes in general to mitogenic activation of cells. We show here that epidermal growth factor (EGF) stimulates Ras.GTP formation very efficiently in a variety of cell lines expressing endogenous EGF receptors only. Maximal activation of the receptor converts up to 65% of cellular p21ras from the GDP form into the active GTP-bound state. This efficient activation occurs also in cultured primary human fibroblasts. Maximal insulin-induced Ras.GTP formation is less but in cells overexpressing the insulin receptor a similar high response of Ras.GTP formation is observed after insulin stimulation. Not only the efficiency but also the kinetics by which the EGF and insulin receptors stimulate Ras.GTP formation are quite distinct. In the Rat-1-derived cell line, H13IR2000, overexpressing both p21Ha-ras and the insulin receptor, the activated insulin receptor generates approximately 1 mol Ras.GTP/mol activated insulin receptor. The activated EGF receptor amplifies the signal, resulting in the activation of approximately 40 mol p21ras/mol receptor. Moreover, EGF-stimulated generation of Ras.GTP is transient with a maximum after 2 min of hormone stimulation and diminishes to near basal levels within 1 h whereas the insulin-induced Ras.GTP levels are maximal at 5-10 min and decline only slowly to half-maximal in 1 h. Desensitization of the EGF pathway by prolonged EGF stimulation, prevents subsequent stimulation of Ras.GTP formation by newly added EGF but not by insulin. Vice versa, in cells preincubated with insulin for 1 h, EGF stimulates Ras.GTP formation to near maximal values. These observations indicate that desensitization by prolonged hormone incubation does not involve inactivation of common signaling intermediates but rather components, specific for each pathway, like the particular receptors. The rapid down regulation of EGF receptors compared to insulin receptors corroborate this possibility. The observed high potency of EGF receptors to generate Ras.GTP may explain the, in general, stronger mitogenic activity of EGF compared to insulin.