Modulation of epidermal growth factor‐dependent protein phosphorylation in cell membrane preparations by receptor down regulation

Abstract We have reported previously [6] that epidermal growth factor (EGF)‐induced down regulation of EGF receptors in normal rat kidney (NRK) cells results in a selective decrease in the in vitro EGF‐dependent 32P‐phosphorylation of two membrane phosphoproteins of Mr I70K and Mr I50K. In this report, we further characterized the modulation of 32P‐phosphorylation of the 170K‐ and 150K‐dalton proteins by down regulation with EGF in NRK cells. While EGF binding to its receptors was a necessary condition to induce loss of EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton proteins, it was not sufficient. Thus, reduction in the temperature of the incubation of cells with EGF from 37°C to 4°C abolished the loss of EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton membrane proteins. When EGF was removed from the medium the EGF‐dependent phosphorylation of the 170K‐ and l50K‐dalton proteins was quickly replenished; by 3 hr one‐half of the “down regulated” phosphorylation was restored. All EGF‐dependent phosphorylating capacity of the 170K‐ and l50K‐dalton protein bands returned by 6 hr after removal of the growth factor. The loss of EGF‐dependent phosphorylation of the 170K‐ and I50K‐dalton proteins occurred at physiological EGF concentrations (0.25–25 ng/ml) that span the concentration range which is mitogenic for NRK cells. Exposure of confluent nondividing NRK cells to 1 ng/ml EGF, followed by incubation for 5 hr at 37°C. led to a 50% reduction in the EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton proteins. Maximal reduction (∼95%) in the EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton proteins was noted with 10 ng/ml EGF for 5 hr. The EGF‐induced loss of EGF‐dependent phosphorylation was specific: several other growth factors did not produce phosphorylation loss of the 170K‐