Activated receptors with a tyrosine kinase activity induce a variety of responses like changes in the differentiation and mitogenic status of cells. These responses are mediated in part by p21ras. Some of these activated receptors induce in certain cell types a pronounced, but transient, increase in Ras-GTP. We have stimulated cells with insulin, epidermal growth factor (EGF), and fetal calf serum (FCS), and the mitogenic response, as reflected by stimulation of [3H]thymidine incorporation, was compared with the magnitude of the transient increase in Ras-GTP levels. Cell lines were used that expressed both physiological and elevated numbers of p21ras and receptors for insulin and EGF, respectively. In all the examined cell lines 9% FCS did not induce a marked increase in Ras-GTP despite its high mitogenic potency. Pronounced increases in Ras-GTP levels were observed in insulin-stimulated CHO cells which overexpress insulin receptors whereas in the parental CHO cells only a small increase is seen. Insulin (1 microM) and FCS (9%) stimulate [3H]thymidine incorporation in parental CHO cells to a similar high level whereas in insulin receptor overexpressing CHO cells the maximum of insulin-stimulated [3H]thymidine incorporation is only 55% of the level reached by 9% FCS. In those cells the maximum is already reached at low (1 nM) insulin concentrations. Remarkably, at higher insulin concentrations stimulation of [3H]thymidine incorporation starts to decrease strongly despite the fact that the magnitude of the transient increase in Ras-GTP and subsequent MAPkinase activation increases. Similarly, when EGF receptors are overexpressed in Rat-1 cells, the mitogenic response is also decreased at higher EGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell line, most of the p21ras was rapidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109). To determine whether this process also occurs in cells expressing physiologically relevant numbers of insulin receptors, insulin stimulated Ras.GTP formation was quantitated in Chinese hamster ovary (CHO)-derived cell lines expressing varying numbers of insulin receptors. In the parental CHO9 cells, expressing only 5.10(3) insulin receptors, insulin stimulation for 3 min increased Ras.GTP levels with 10%. Upon increasing the number of insulin receptors in these cells, Ras.GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. These data show that receptor overexpression is not a prerequisite for insulin-stimulated Ras.GTP formation. The yield of Ras.GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CHO9- and NIH3T3-derived cell lines, respectively. These values argue against signal-amplifying processes between the insulin receptor and p21ras. To determine whether receptor autophosphorylation is required for Ras.GTP formation, NIH3T3 cells overexpressing insulin receptors were stimulated with a monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser1200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects in CHO cells, were used. In both cases, no Ras.GTP formation was observed. Furthermore, Rat-1-derived cell lines expressing mutant p21ras, which is permanently in the active GTP-bound form, still responded to insulin by increasing the glucose uptake. These results support our hypothesis that Ras.GTP formation is activated by the tyrosyl-phosphorylated insulin receptor and suggest that an active Ras.GTP complex does not mediate metabolic signaling.

Ras-proteins are guanine nucleotide binding proteins, which, in the GTP bound state emit a strong mitogenic signal. In the GDP bound state, the protein appears inactive. We have found that stimulation by insulin of cells expressing elevated levels of insulin receptors results in a rapid conversion of Ras-GDP into Ras-GTP. This process is part of the signalling pathway leading to immediate-early gene expression and a mitogenic response. There seems to be no involvement of Ras-GTP formation in the process of insulin stimulated glucose transport. Though the precise mechanism by which Ras is converted to the GTP bound state remains to be established, a tight correlation exists between receptor autophosphorylation and Ras-GTP formation.