Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester.

Abstract

Treatment of human K562 cells with 4f3 phorbol 12-myristate 13-acetate (PMA) resulted in an approximately 50% reduction in cell surface transferrin receptors within 30-45 min as judged by binding of both ligand and antireceptor antibody. The affinity of the remaining surface receptors for diferric transferrin appeared to be unaltered. The time-dependent loss in transferrin receptors was also dependent upon PMA concentration, with a half-maximal effect observed at approximately 1 nM. The kinetic parameters for the binding, internalization, intracellular residency, and recycling of 2'I-labeled transferrin were unchanged by PMA treatment, as were the rate and extent of internalization of anti-receptor antibody. Moreover, despite the decrease in surface receptors, uptake of 59Fe from transferrin proceeded at a rate comparable to that seen in untreated cells. Accounting for this observation was the fact that ligand induced a reduction in surface receptors in untreated but not PMA-treated cells. Quantitative immunoprecipitation of transferrin receptors from surface-iodinated K562 cells revealed that little receptor internalization occurred in untreated cells in the absence of ligand, but internalization of ligand-occupied receptors in these cells was readily detected. In contrast, PMA treatment resulted in the rapid internalization of surface receptors irrespective of occupancy. Thus, binding of ligand appeared to trigger the internalization of receptors that were relatively static in their unoccupied state, and a signal for receptor internalization was also provided by PMA treatment. The possibility that this signal involves phosphorylation of the transferrin receptor is discussed.Phorbol esters have been studied for several decades, largely because of their action as tumor promoters (reviewed in ref. 1). Cells undergo a wide variety of changes after treatment with phorbol esters, including alterations in the synthesis and turnover of phosphatidylcholine, prostaglandins, polyamines, proteins, DNA, and RNA. Recently, interest has been heightened by the identification of specific phorbol ester receptors (2), the discovery that phorbol esters activate protein kinase C (3), and the likely role of this kinase as the receptor (4-6). How activation of this kinase leads to the many cellular events triggered by phorbol esters is unknown. Whereas multiple changes in the cell surface upon treatment with phorbol esters have been described (1), it is not clear that all such changes are attributable to phosphorylation events. Phorbol esters may alter, directly or indirectly, a wide variety of cell surface receptors. Exposure to phorbol esters leads to a loss of high-affinity binding sites for epidermal growth factor (EGF) (7) and somatomedin (8) and has been shown to induce phosphorylation of the receptor for EGF (9), insulin, and somatomedin C (10). In this report, we present evidence for a specific alteration in the dynamics of the human transferrin (Tf) receptor that accompanies treatment of K562 cells with 43-phorbol 1...

Phorbol ester-induced regulation of transferrin receptors in human leukemia K562 cells.

“…Subsequent decrease of the receptors on cell surface has been observed which could be responsible for the inhibition of cell growth by TPA treatment. Klausner et al reported that the decrease seemed to involve sequestering of the receptors to the inside of the cells during TPA treatment (18). However, no detailed study on the regulation of the receptors in TPA-treated cells has been done Here we report that the treatment of human erythroleukemia (K562) cells with TPA causes the decrease of transferrin receptors on the cell surface as well as in the cell interior.…”

“…It has also been observed that the phosphorylation of the transferrin receptor is markedly stimulated at that time (18,20,26). Subsequent decrease of the receptors on cell surface has been observed which could be responsible for the inhibition of cell growth by TPA treatment.…”

“…Cochet et al (4) and Iwashita and Fox (14) have described the phosphorylation of epidermal growth factor receptors by phorbol. Klausner et al (18) and May et al (26) have demonstrated that the phosphorylation of transferrin receptors in human leukemic cell lines is stimulated by phorbol esters. Furthermore, we have shown that the enhanced phosphorylation by TPA took place only on the cell-surface transferrin receptors (20).…”

Characterization of Rapid Membrane Internalization and Recycling

“…In HepG2 hepatoma cells (26) or in phorbol 12-myristate 13-acetate-treated K562 cells (27), the rate of iron uptake is almost twice as fast as that measured for transferrin. We propose that rapid recycling of transferrin through acidic sorting endosomes could account for the additional iron delivered to the cells, since a rapid recycling would have caused an underestimation of the rate of transferrin uptake.…”

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

“…PKC-mediated phosphorylation has been implicated in the internalization of numerous receptors including the EGFR, CD4, TfR, and the asialoglycoprotein receptor (ASGPR) (Klausner et al, 1984;McCaffrey et al, 1984;Beguinot et al, 1985;Fallon and Schwartz, 1988;Shin et al, 1991). It has been demonstrated that PKC phosphorylates the EGFR at T654 (Hunter et al, 1984).…”