Mechanism of the phorbol ester-mediated redistribution of asialoglycoprotein receptor: selective effects on receptor recycling pathways in Hep G2 cells.

We have investigated the effect of phorbol dibutyrate on intracellular routing of the asialoglycoprotein receptor (ASGP-R) in a human hepatoma cell line, Hep G2. We have previously shown that this agent causes a net redistribution of 50% of cell surface receptors to the cell interior (Fallon, R.J., and A.L. Schwartz, J. Biol. Chem. 261: 15081-15089 (1986)). To explore the mechanism of this effect, we measured the rate constants of receptor and ligand movement during internalization, ligand-receptor uncoupling, sorting of ligand to degradative sites or return to the extracellular medium, and return of receptor to the plasma membrane. The rate of internalization of bound asialoorosomucoid (ASOR) is identical in phorbol ester-treated and control cells, over a range of ASOR concentrations from 5 to 125 nM. The pathway of ligand recycling returns approximately 30% of internalized ASOR undegraded to the extracellular medium; phorbol esters do not modify the extent of this pathway in Hep G2 cells nor the kinetics of recovery of undegraded ASOR in the medium (t1/2 = 20 min). The rate of ligand-receptor uncoupling is similarly unaltered by phorbol esters, as measured by the amount of free ASOR that accumulates intracellularly and exits the cell after saponin permeabilization. In contrast, phorbol esters cause a rapid (less than 5 min) 50% decrease in receptor return to the cell surface from internal sites. This suggests that 1) phorbol esters interfere with selected specific sites in receptor and ligand pathways of receptor-mediated endocytosis and 2) the apparent net "internalization" of ASGP-R by phorbol esters results from an inhibition of receptor recycling to the cell surface and not from a direct stimulation of the internalization process.