We investigated the effect of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the binding of epidermal growth factor (EGF) to Rat-1 fibroblasts. Utilizing Scatchard analysis of specific 125I-EGF binding to surface receptors at 4°C, we observed a decrease in EGF receptor number and affinity in the presence of TPA as compared to controls. The curvilinear Scatchard plot in the control cells was explained by the presence of two classes of receptors with differing affinities, in which case the effect of TPA was to eliminate binding to the high affinity receptors. Incubation in TPA for up to 8 h, followed by Scatchard analysis, revealed that the initial TPA-induced decrease in affinity was maximal at 1 h and was overcome with time, such that the high affinity EGF receptors regained their ability to bind EGF. Degradation of TPA by the cells was not responsible for the recovery of the high affinity EGF receptors. The effect of TPA on EGF metabolism was determined by the conversion of 125I-EGF to its major metabolite, monoiodotyrosine, as measured by gel permeation chromatography. TPA did not alter the rate at which bound EGF was degraded. However, at low EGF concentrations (0.2 ng/ml), TPA reduced the amount of EGF that was metabolized over a 12-h period, resulting in 50% more intact EGF in the medium of TPA-treated cells compared to controls. At higher EGF concentrations (5 and 200 ng/ml), TPA had a negligible effect on EGF degradation. The ability of TPA to spare EGF from degradation at low EGF concentrations resulted from the initial TPA-induced reduction in EGF binding. The increased amounts of EGF remaining in the medium of cells exposed to 0.2 ng/ml EGF plus TPA for 12 h resulted in significantly more EGF binding in the TPA-treated cells than in control cultures. We suggest that the ability of TPA to induce DNA synthesis with EGF synergistically may rely on the unique ability of TPA to decrease EGF affinity only temporarily, thus reducing EGF binding and subsequent degradation. When receptor affinity has been restored, EGF concentrations would be greater in culture medium of TPA-treated cells than in control cells. These higher EGF concentrations would be sufficient to induce a greater population of cells to enter DNA synthesis.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1980|
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