Receptor Binding and Processing of Epidermal Growth Factor by Human Breast Cancer Cells*

Abstract

Epidermal growth factor (EGF) stimulates the growth of human breast cancer cells (MCF-7). To better define the mechanisms for these effects, we studied receptor binding and subsequent processing of [l25I]EGF in cells growing in monolayer culture under conditions in which an effect on growth is observed. Binding was temperature dependent and was higher at reduced temperature. At 37 C, binding rapidly reached a maximum (20–60 min) and then gradually decreased. Binding was slower at a reduced temperature. High affinity was demonstrated by the ability of unlabeled EGF to compete for [¹²⁵I]EGF binding; hah7 maximal displacement was observed at an EGF concentration that also resulted in half-maximal biological activity (1–2 ng/ml). Scatchard plots derived from these data or from those of studies using increasing concentrations of [¹²⁵I]EGF were curilinear and superimposable. Plots from binding data obtained at 0 C were also curvilinear, suggesting that the processing and degradation of tracer alone cannot explain this finding. Other peptides did not compete for EGF binding. During a 6-h incubation, progressive degradation of [¹²⁵I]EGF occurred on and/or in the cells, with prompt release of low molecular weight fragments into the medium. Furthermore, during this time, the bound intact EGF became more resistant to dissociation by excess unlabeled EGF. Reduced binding (50%), not due to simple occupancy, was also noted during a 2-h preincubation with unlabeled EGF, indicating down-regulation of receptor. These data suggest the following sequence of events. At 37 C, EGF rapidly binds to specific receptors on MCF-7 cells. The bound EGF becomes more and more difficult to dissociate from the cell, and the number of available receptors is reduced, implying sequestration in the cell membrane and/or internalization of the hormone-receptor complex. Gradually, EGF is degraded, and the products are released from the cell. Human breast cancer appears to be a target for EGF action.