Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells.

Abstract

Receptor-mediated uptake and degradation of 1251-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37°C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface "'I-ASOR binding sites as well as a rapid decrease in 125 1-ASOR uptake and degradation . There is no effect of these amines on the binding of "'I-ASOR to the cell surface at 4°C or on the rate of internalization of prebound 125 1-ASOR . The loss of "'I-ASOR surface binding at 37°C is not attributable to altered affinity of ligandreceptor binding . In the presence of added ligand at 37°C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors . In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface . We examined the nature of this loss of 1251-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37°C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool .The selective uptake of macromolecular ligands such as proteins via receptor-mediated endocytosis is a common feature of eucaryotic cells (1, 2). Receptor-mediated endocytosis involves the specific binding of macromolecular ligands (e.g., virus, toxin, transferrin, asialoglycoprotein) to specialized cell surface receptors and their internalization via a coated pitcoated vesicle pathway. Many ligands dissociate from their receptor in a prelysosomal compartment and allow the receptor to recycle back to the cell surface (1, 2). Often, these ligands are then transported in a still unknown way to the lysosomes, wherein they are degraded. The asialoglycoprotein receptor (ASGP-R),' localized to the hepatic parenchymal cell, provides a well-characterized system for examination of the mechanisms involved in these processes (3) . A human hepatocyte-derived cell line, hepatoma HepG2, contains abundant ASGP-R (4) . Using these cultured cells as a model, we have recently defined the kinetics of receptor-mediated endocytosis of asialoglycoprotein ligands and ASGP-R recycling (5, 6). Our studies indicate that a single ASGP-R can recycle from the cell surface into "endosomal sorting compartments" (compartment of uncoupling receptor and ligand ' Abbreviations used in this paper. ASGP, asialoglycoprotein; ASGP-R, ASGP receptor; ASOR, asialoorosomucoid .[CURL]) within the cell and back to the cell surface within 8 min (5-7).The mechanisms responsible for ligand-receptor dissociation and the sorting and recycling of receptor molecules are not fully understood. Lysosomotropic agents (including the weak bases ammonium chloride and chloroquine) have been demonstrated to interfere with receptor-mediated uptake and degradation of numerous ligands, including asialoglycoproteins (8), mannose-6-phosphate-terminated ligands (9), alpha-2-macroglobulin-protease comple...

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