Insulin and epidermal growth factor receptors regulate distinct pools of Grb2-SOS in the control of ras activation

Insulin and epidermal growth factor (EGF) stimulate a rapid but transient increase in the amount of GTP bound to Ras that returns to the basal GDP bound state within 10-30 min, Although insulin stimulation resulted in a dissociation of the Grb2 . SOS complex, EGF did not affect the Grb2 . SOS complex but instead induced dissociation of Grb2-SOS from tyrosine-phosphorylated Shc. The dissociation of Grb2-SOS from Shc was not due to dephosphorylation as She remained persistently tyrosine-phosphorylated during this time, Furthermore, there was no decrease in the extent of insulin receptor substrate 1, insulin receptor, or EGF receptor tyrosine phosphorylation. Surprisingly, however, despite the EGF-induced decrease in the amount of Grb2-SOS bound to Shc, the extent of Grb2 associated with Shc remained constant, and there was a concomitant increase in the amount of SOS associated with Grb2, In addition, after the insulin-stimulated dissociation of Grb2 from SOS, EGF treatment induced the reassociation of the Grb2 SOS complex. Quantitative immunoprecipitation demonstrated that only a small fraction of the total cellular pool of Grb2 was associated with SOS, Similarly, only a small fraction of SOS and Grb2 were co-immunoprecipitated with Shc. Together, these data suggest the presence of distinct Grb2-SOS pools that are independently utilized by insulin and EGF in their recruitment to tyrosine-phosphorylated Shc.