Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation

Abstract

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell linme,o st of the p2lras warsa pidly 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 (CH0)-derivecde ll lines expressing varying numbers of insulin receptors. In the parental CH09 cells, expressing only 5.103 insulin receptors, insulin stimulation for 3 min increased Ras*GTP levels with 10%.U pon increasing the numbero f insulin receptors in these cells, Ras-GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. Thesed ata show that receptor overexpression is not a prerequisite for insulin-stimulated Ras-GTP formation. Thye ield of Ras-GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CH09- and NIH3T3-derived cell linesre, - spectively. These values argue against signal-amplifying processes between the insulin receptor and p2 lras. To determine whether receptor autophosphorylation is required for Rase GTP formation, NIH3cTe3ll s overexpressing insulin receptors were stimulated witah monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser’200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects CinH O cells, were used. In both cases, no Ras. GTP formation was observed. Furthermore, Rat- 1-derived cell lines expressing mutant palras, 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.