Human Hepatoma Cell Mutant Defective in Cell Surface Protein Trafficking

Abstract

To isolate a mutant liver cell defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was devised. Ovalbumin-gelonin and asialoorosomucoid (ASOR)-gelonin were incubated with mutagenized HuH-7 cells, and a rare survivor termed trafficking mutant 1 (Trf1) was isolated. Trf1 cells were stably 3-fold more resistant than the parental HuH-7 to both toxic conjugates. The anterograde steps of intracellular endocytic processing of ASOR, including internalization, endosomal acidification, and ligand degradation, were unaltered in Trf1 cells. In contrast, retrograde diacytosis of asialoglycoprotein receptor (ASGR).ASOR complex back to the cell surface was enhanced by about 250%. Selective labeling revealed an approximately 46% reduction in cell surface-associated ASGR in Trf1 cells, although their total cellular ASGR content was essentially equivalent to that in HuH-7. Similar results were obtained with the transferrin receptor. Binding of 125I-ASOR and 125I-transferrin was reduced in Trf1 cells to 49 +/- 2.5% and 30 +/- 2%, respectively, of HuH-7 cells. The methionine transporter was also reduced in Trf1 cells, as revealed by a 2-fold reduction in Vmax with no change in apparent Km. Pretreatment with monensin, sodium azide, or colchicine reduced surface binding of 125I-ASOR in HuH-7 cells by 50% but had no effect on binding to Trf1 cells. This result is predicted for a cell that expresses only State 1 ASGRs, which are resistant to modulation by metabolic and cytoskeletal inhibitors in contrast to State 2, which are responsive to these agents (Weigel, P. H., and Oka, J. A. (1984) J. Biol. Chem. 259, 1150-1154). The Trf1 mutant, having lost the ability to express State 2 receptors, provides genetic evidence for the existence of these two receptor subpopulations and an approach to identifying the biochemical mechanism by which they are generated.

The Minor Subunit Splice Variants, H2b and H2c, of the Human Asialoglycoprotein Receptor Are Present with the Major Subunit H1 in Different Hetero-oligomeric Receptor Complexes

“…Stockert et al (22) independently validated the existence of this two-pathway endocytosis system for a variety of receptor types targeted to coated pits when they fortuitously isolated a trafficking mutant (designated Trf1) from HuH-7 cells. The Trf1 mutant is defective in endocytosis mediated by the State 2 pathway and its pleiotropic phenotype is exactly what was predicted for cells lacking a functional State 2 pathway (5, 40).…”

“…HepG2, HuH-7, COS-7, and SK-Hep-1 cell lines were obtained from ATCC. Trf1, a trafficking mutant cell line, which was derived from HuH-7 cells (22), was a generous gift from Dr. Richard Stockert (Albert Einstein College of Medicine, New York). All established cell lines were maintained in complete medium containing Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Summit Biotechnology), 2 mM L-glutamine, and 100 units/ml each of penicillin and streptomycin (Invitrogen).…”

Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7

“…18 Alteration of the transferrin and mannose receptors, as well as the ASGPR, by the Trf1 cell line strongly suggests a common regulatory element. 15 To determine whether the Trf1 mutation affected trafficking of other types of cell-surface proteins, the distribution, function, and posttranslational processing of the gap junction protein connexin43 (Cx43) was investigated in Trf1 cells and the parental HuH-7 cell line. Our findings indicate alterations in the trafficking pathway of Cx43 in these 2 cell lines.…”

“…15 Endocytic activity of 2 different carbohydratespecific receptors of the human hepatoma cell line HuH-7 were simultaneously selected against using gelonin, an inhibitor of protein synthesis, conjugated to galactose-and mannoseterminating glycoproteins. Because there was no significant reciprocal inhibition between the mannose-and galactoseterminating glycoproteins and their respective gelonin conjugates, the mutation harbored by Trf1 cells appeared to affect a common step in their endocytic pathway.…”

Rab1a regulates sorting of early endocytic vesicles

“…ASOR was prepared as described previously (70) by hydrolysis of human ␣ 1-orosomucoid (Sigma) in 0.1 N sulfuric acid at 75°C for 1 h. The solution was neutralized with 1 N NaOH and dialyzed against water, and protein concentration was determined. Fluorescent ASOR was prepared by conjugation with Alexa 488-carboxylic acid succinimidyl ester (Molecular Probes, Eugene, OR) following the manufacturer's instructions.…”