Insulin and epidermal growth factor stimulate a conformational change in Rap1 and dissociation of the CrkII-C3G complex

Insulin and epidermal growth factor (EGF) stimulation of Chinese hamster ovary cells expressing the human insulin and EGF receptors resulted in a time-dependent decrease in the ability of a Rap1 antibody (amino acid epitope 121-136) to immunoprecipitate Rap1 from whole cell detergent extracts. This was due to an apparent masking of Rap1 as heat denaturation of the whole cell detergent extracts (5 min at 100 °C) resulted in equal immunoprecipitation of Rap1 with this epitope-specific antibody. The time-dependent change in Rap1 immunoreactivity was paralleled with an insulin-stimulated dissociation of the CrkII-C3G complex. Similarly, EGF treatment also resulted in a time- dependent dissociation of the CrkII-C3G complex that occurred concomitant with the masking of the 121-136 Rap1 epitope. Furthermore, pretreatment of the cells with the tyrosine kinase inhibitor, genistein, decreased both the basal and insulin-stimulated tyrosine phosphorylation of CrkII that directly correlated with the amount of CrkII that was immunoprecipitated with C3G. Together, these data suggest that insulin and EGF stimulation result in the dissociation of the CrkII-C3G complex, thereby inducing an apparent conformation change in Rap1.