Mechanism of Transferrin Receptor Down-regulation in K562 Cells in Response to Protein Kinase C Activation (*)

Abstract

Treatment with phorbol esters increases endocytosis of the transferrin receptor in K562 cells (Klausner, R. D., Harford, J., and van Renswoude, J.(1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3005-3009). In this report, we demonstrate that this effect is reversible within early times of protein kinase C activation (<2 h) but that prolonged exposure to phorbol esters results in a net loss of receptors. These effects are not due to the differentiation response of K562 cells to phorbol esters since bryostatin-1 also down-regulates the endocytosis of the transferrin receptor and shut downs receptor synthesis, but does not induce differentiation (Hocevar, B. A., Morrow, D. M., Tykocinski, M. L., and Fields, A. P.(1992) J. Cell Sci. 101, 671-679). We have characterized the early stages of receptor down-regulation which occur due to stimulation of receptor internalization from the cell surface. The fact that fluid-phase pinocytosis is also enhanced upon protein kinase C activation indicates that this effect is not specific for the transferrin receptor itself, but is a rather general cellular response to tumor-promoting phorbol esters. The fate of down-regulated transferrin receptors was followed in morphological and subcellular fractionation studies that demonstrate localization of this pool of receptors in early endocytic and recycling compartments. Our results exclude the possibility that transferrin receptor down-regulation results in trafficking of the receptor to lysosomal compartments for degradation. This idea is consistent with the observations that the time course of transferrin receptor degradation is not enhanced in stimulated K562 cells, while transferrin receptor synthesis is shut down. Our results rigorously demonstrate that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two stages: acute regulation of early steps in endocytosis that results in an immediate reduction of 40% in cell surface number of receptors and a more chronic reduction in transferrin receptor synthesis upon prolonged exposure to phorbol esters (>15 h).