Many studies have shown that Ras function is required for cell signalling by several growth factor receptors, but the role that Ras plays in these signalling pathways is still poorly defined. In this study, the dominant negative ras mutant, 116Y, was employed, which was previously found to inhibit ras proto-oncogene function, to investigate its effect on mitogenic factor stimulation, and to explore interactions of Ras targets in these signalling systems. A series of NIH3T3 cells expressing 116Y (6Y3, 6Y6) was established, and a revertant (F33) was isolated expressing a higher level of 116Y from a cell transformed by LTR-linked c-Ha-ras. It was found that 116Y cells failed to respond to EGF stimulation, even at a very high concentration, whereas little inhibition was observed for PDGF or fetal calf serum. This inhibition was tightly correlated with 116Y expression. As determined by a Scatchard analysis, it was surprising to find that binding of 125I-EGF to the EGF receptor was reduced to approximately 30% in 6Y3 and 6Y6, and to almost none in F33 cells. While positive receptor autokinase activity was obtained for NIH3T3 cells, none of the 116Y cells had significant phosphorylation of EGF-R. Loss of receptor function was not due to lack of EGF-R, since no appreciable decrease in EGF-R mRNA and membrane receptor proteins was observed. In addition, the intracellular activities of protein kinase C, which inhibits EGF-R function, were found to be lower in 116Y cells, as assessed by phosphorylation of the major p80 substrate upon phorbol ester treatment. These findings suggest that, under a physiological cellular condition, Ras function has a rather direct impact on the functioning of a key growth factor receptor.
