Characterization of the asialoglycoprotein receptor in a continuous hepatoma line.

Schwartz, Alan L.; Fridovich, S E; Knowles, Barbara B.; Lodish, Harvey F.

doi:10.1016/s0021-9258(19)52477-2

Cited by 257 publications

(36 citation statements)

References 19 publications

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“…As measured by the sum of cell-associated and degraded 126I radioactivity, the overall rate of cellular uptake of ASOR is constant at 0.02-0.03 pmol m in-1 per 106 cells for at least 6 h (figure 1). In 28 independent experiments the rate of [126I]ASOR uptake at a concentration of 2 pg m l-1 at 37 °C, assessed over the first 60 min, averaged 0.029 ± 0.001 pmol min-1 per 106 cells, as we have reported earlier (Schwartz 1981a(Schwartz , 1982. Because the rate of ligand uptake (ligand flux) is dependent upon the total cell complement of functional receptors that participate in this process, we have determined the cell receptor distribution by destroying cell-surface receptors with protease.…”

Section: [ 123 ]supporting

confidence: 80%

Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence

Schwartz¹,

Geuze

1982

Phil. Trans. R. Soc. Lond. B

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One of the best documented systems of receptor-mediated endocytosis is the clearance of asialoglycoproteins (ASGP) from the blood plasma by liver parenchymal cells. There are 200 000-500 000 ligand binding sites per cell, which makes this system favourable for molecular studies of receptor function. By using both biochemical and immunocytochemical approaches, we have obtained evidence for receptor recycling. We have also localized the intracellular site at which the endocytosed receptor and ligand dissociate. The human hepatoma cell Hep G2 contains abundant ASGP receptors (approximately 225 000 per cell). In growing cells approximately 85% of the functional receptors are on the cell surface and the remaining 15% are internal. The maximal rate of ligand uptake in this cell system at 37 degrees C is approximately 30 000 molecules per cell per minute. Each functional receptor can therefore bind and internalize more than 50 ligand molecules during a 6 h period (in the absence of new receptor synthesis), or one ligand each 8 min. To follow both ligand and receptor during their common endocytosis and to visualize the compartment in which the dissociation of ligand from receptor occurs, we have used our recently developed double-labelling immunocytochemical electron microscopic techniques with purified antibodies against ASGP ligand and ASGP receptor. In normal rat hepatocytes, both ligand and receptor are taken up from the sinusoidal cell surface in clathrin-coated vesicles. Both receptor and ligand are associated with the membrane of small clathrin-coated vesicles close to the cell surface. Larger vesicles, farther removed from the surface, contain ligand accumulated within the lumen. The membranes of these larger vesicles contain little receptor, but receptor was concentrated in detached vesiculotubular extensions, which were largely free of ligand. These vesicles represent the compartment of uncoupling of receptor and ligand (CURL) during their common endocytosis. Ligand contained within the vesicle lumen is then transferred to multivesicular bodies and lysosomes; the tubular extensions may carry receptor back to the cell surface.

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Section: [ 123 ]supporting

confidence: 80%

Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence

Schwartz¹,

Geuze

1982

Phil. Trans. R. Soc. Lond. B

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“…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).…”

mentioning

confidence: 99%

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

Schwartz¹,

Bolognesi²,

Fridovich³

1984

The Journal of Cell Biology

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188

110

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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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“…(34) appeared that showed that 1-deoxynojirimycin inhibits both oligosaccharide processing and secretion of a 1-proteinase inhibitor (al-antitrypsin), one of the proteins we have studied, in primary hepatocytes. Growth and Labeling of HepG2 Cells : Culture dishes of 60mm diam were seeded with 1 .5 x 106 HepG2 cells, and incubated at 37°C in Eagle's minimal essential medium supplemented with 10% fetal calf serum (35). Cultures were generally fed on the second day after seeding and used on the fourth, at which time the cells had approximately doubled.…”

mentioning

confidence: 99%

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex.

Lodish¹,

Kong²

1984

The Journal of Cell Biology

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235

100

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ABSTRACT1-Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum . In a pulse-chase experiment, 1-deoxynojirimycin greatly reduced the rate of secretion of a1-antitrypsin and al -antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin . As judged by equilibrium gradient centrifugation, 1-deoxynojirimycin caused al-antitrypsin and al-antichymotrypsin to accumulate in the rough endoplasmic reticulum . The oligosaccharides on cell-associated al-antitrypsin and al-antichymotrypsin synthesized in the presence of 1-deoxynojirimycin, remained sensitive to Endoglycosidase H and most likely had the structure Glu 1 -3 Man 9 GIcNAC 2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins . Swainsonine, an inhibitor of the Golgi enzyme a-mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N-linked, partially complex, oligosaccharide . We conclude that the movement of al-antitrypsin and al-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9 GlcNAc 2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins .Secretory proteins and cell surface glycoproteins are synthesized on ribosomes attached to the rough endoplasmic reticulum membrane . Cotranslationally, the polypeptides are transported across or inserted into the rough endoplasmic reticulum membrane (1-3), and one or more oligosaccharides, of structure G1c 3 Man9GlcNAc2 , are transferred to specific asn residues (4) . By a series of membrane vesicles these proteins are transported first to the Golgi complex of membranes, then to the cell surface (1-3, 5, 6).How proteins move from the rough endoplasmic reticulum to the Golgi is not known . A common hypothesis is that small vesicles bud off from a specialized region of the rough endoplasmic reticulum, containing a sample of the luminal contents and membrane proteins. These vesicles subsequently fuse with vesicles of the cis-(or proximal) region of the Golgi cisternae (2, 3). In hepatic parenchymal cells albumin and lipoprotein particles are localized to different terminal dilations of the rough endoplasmic reticulum and cis-Golgi (7) . Thus it is possible that different vesicles transport different secretory products to the Golgi .It is generally believed that all secretory and plasma membrane glycoprotein proteins migrate through the same Golgi vesicles. A combination of cell-fractionation and immunoelectronmicroscopic approaches has shown that albumin, transferrin, and the vesicular stomatitis viral glycoprotein and other glycoproteins are found in the...

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Characterization of the asialoglycoprotein receptor in a continuous hepatoma line.

Cited by 257 publications

References 19 publications

Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence

Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence

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

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex.

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