Resolution of high and low affinity epidermal growth factor receptors. Inhibition of high affinity component by low temperature, cycloheximide, and phorbol esters.

Abstract

Receptors for the mitogen epidermal growth factor (EGF) have been implicated as mediators of cellular transformation and tumor promotion.12-0-Tetradecanoylphorbol-13-myristate (PMA) causes immediate but transient reductions in the ability of human KB cells to internalize EGF by decreasing the affinity of cell surface EGF receptors 7-to 12-fold.After 90 min of PMA exposure, complete recovery of the high affinity apparatus occurs.Evidence is presented that the decreased binding capacity to "%EGF is caused by delaying the appearance at the plasma membrane of cryptic receptor sites of high affinity.Exposure of these high affinity binding sites is temperature dependent and is blocked by pretreatment of cells with inhibitors of de nouo protein synthesis.After short incubation periods (0 to 60 min) with '''I-EGF, up to 90% of the internalized '261-EGF can be rapidly dissociated from cells by the addition of PMA.This treatment kinetically reveals a trypsin-insensitive, nondissociating component of cell-associated Iz6I-EGF.With PMA, both the rate (maximal at 4 h with PMA as compared to 24 h) and extent (&fold greater with PMA) of transfer of '"I-EGF into this trypsinstable compartment is enhanced and accounts for the severalfold stimulation in ''%EGF accumulation.This evidence suggests that the internalization of '"I-EGF can be resolved into two components, one into which EGF immediately partitions and is freely exchangeable with the extracellular medium and that is disrupted by PMA.A second and previously unknown compartment retains '"I-EGF in the presence of PMA after a short delay to a considerably greater extent than that of untreated cells.Thus, without PMA, EGF is internalized into this nondissociating intracellular compartment, but the transfer is only 5-10% as efficient.These results suggest that the accumulation of stable intracellular mitogen-receptor complexes may be directed by a population of very high affinity receptors which may be an element involved in the control of cellular growth and tumor promotion.Epidermal growth factor promotes the growth and differentiation of epithelial and fibroblastic cells both in vivo and in vitro (1-3).Although it is generally accepted that EGF'