Inhibitory effect of sodium arsenite and azide on asialoglycoprotein receptor mediated endocytosis in suspended rat hepatocytes

The inhibitory effect of sodium arsenite and azide on asialoorosomucoid endocytosis was tested using isolated rat hepatocytes. Under either continuous flux conditions or a single synchronous wave of ligand endocytosis we confirm that azide inhibits the recycling of the receptors and we provide evidence for the involvement of thiol groups in the internalization step. In addition pretreatment of hepatocytes with azide allows us to demonstrate that receptor endocytosis proceeds independently of the presence of any specific ligand.     

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.     

Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. Evidence for two potentially rate-limiting steps

The rate of endocytosis of cell surface-bound [3H]-asialo-orosomucoid was determined as a function of temperature. Freshly isolated rat hepatocytes were allowed to bind [3H]asialo-orosomucoid at 4 degrees C, washed to remove nonbound ligand, and internalization was then assessed by the resistance of cell-associated radioactivity to release by the Ca2+ chelator EDTA. At 10 degrees C or below, endocytosis is negligible. Above 10 degrees C, the rate of endocytosis is proportional to temperature but the increase of the rate of endocytosis with increasing temperature changes sharply at about 20 degrees C. From 10-20 degrees C, the apparent activation energy for endocytosis, calculated from an Arrhenius plot, is 45.9 kcal/mol and the temperature coefficient, Q10, is 15.6. However, between 20 and 41 degrees C, the calculated activation energy is 17.0 kcal/mol and the Q10 is 2.6. Although the rate of endocytosis of previously bound [3H]asialo-orosomucoid is very dependent on the temperature, the final extent of endocytosis is essentially temperature-independent between 14 and 37 degrees C. The results suggest that there are at least two steps in the overall process of endocytosis mediated by the asialoglycoprotein receptor on isolated hepatocytes which can be potentially rate-limiting, one at 10 degrees C and another at approximately 20 degrees C.     

Vasopressin-induced changes in receptor-mediated endocytosis of asialoglycoprotein in rat hepatocytes.

The ability of second messengers to modulate receptor-mediated endocytosis was studied on isolated rat hepatocytes. A 20-min preincubation with vasopressin was used as a modulation. We observed a 20% inactivation of both surface and intracellular receptors, with no change in the affinity of those remaining active. The internalization and dissociation of a synchronous wave of ligand was not affected, but its degradation was partially inhibited. Our observations suggest that second messengers such as intracellular calcium and diacylglycerol play a complex role in the intracellular trafficking associated with endocytosis.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.     

The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface. Following this clustering event, the ligand is internalized into a noncoated intracellular vesicle, the receptosome, which selectively avoids fusion with lysosomes and moves toward the Golgi region of the cell by saltatory motion. Using alpha 2-macroglobulin as the ligand and electron microscopic cytochemical methods, we have shown the unusual appearance of this previously undescribed organelle and have followed the labeled ligand in these vesicles in the cytoplasm. To accomplish this, cells were incubated with immunolabeled alpha 2-macroglobulin at 4C under conditions where ligand-receptor complexes cluster into coated pits on the cell surface. Formation of the receptosome occurs between 2 and 5 min after raising the temperature of cells to 37C. These labeled receptosomes were seen to associate with many small vesicular elements in the cytoplasm, and were often found near the Golgi or GERL region after 15 min. Between 15 and 30 min a significant transfer of labeled ligand occurred from the receptosomal population to a population of small uniform lysosomes. By 60 min, all of the label was contained in these small lysosomes. Immunocytochemical studies showed that the receptosomes were not associated with clathrin, actin, myosin or tubulin. This unique, short-lived, specialized organelle selectively delivers the products of receptor-mediated endocytosis to intracellular sites.     

Receptor mediated endocytosis of N-acetylglucosamine and mannose exposing molecules by cultured chick embryo hepatocytes.

We studied the receptor mediated endocytosis of a modified glycoprotein (N-acetylglucosamine-BSA) and mannan in cultured hepatocytes isolated from 19-days-old embryos. The binding sites for molecules exposing terminal N-acetylglucosamine (GlcNac) and mannose residues were localized and quantified at the ultrastructural level by means of protein-gold complexes. The binding sites were found to be randomly distributed as single gold particles on cultured hepatocyte cell surfaces not restricted to specialized areas of the plasma membrane. The gold ligands were internalized following a receptor mediated pathway, which was studied at different interval times (15, 30 and 60 min.) after incubating the cells with the electron dense markers.     

Copper and zinc ions differentially block asialoglycoprotein receptor-mediated endocytosis in isolated rat hepatocytes.

Asialoglycoprotein receptors on hepatocytes lose endocytic and ligand binding activity when hepatocytes are exposed to iron ions. Here, we report the effects of zinc and copper ions on the endocytic and ligand binding activity of asialoglycoprotein receptors on isolated rat hepatocytes. Treatment of cells at 37 degrees C for 2 h with ZnCl2 (0-220 microM) or CuCl2 (0-225 microM) reversibly blocked sustained endocytosis of 125I-asialoorosomucoid by up to 93% (t1/2 = 62 min) and 99% (t1/2 = 54 min), respectively. Cells remained viable during such treatments. Zinc- and copper-treated cells lost approximately 50% of their surface asialoglycoprotein receptor ligand binding activity; zinc-treated cells accumulated inactive asialoglycoprotein receptors intracellularly, whereas copper-treated cells accumulated inactive receptors on their surfaces. Cells treated at 4 degrees C with metal did not lose surface asialoglycoprotein receptor activity. Exposure of cells to copper ions, but not to zinc ions, blocked internalization of prebound 125I-asialoorosomucoid, but degradation of internalized ligand and pinocytosis of the fluid-phase marker Lucifer Yellow were not blocked by metal treatment. Zinc ions reduced diferric transferrin binding and endocytosis on hepatocytes by approximately 33%; copper ions had no inhibitory effects. These findings are the first demonstration of a specific inhibition of receptor-mediated endocytosis by non-iron transition metals.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Regulation of asialoglycoprotein receptor expression in the proliferative state of hepatocytes

It is necessary to proliferate hepatocytes and to increase the number of hepatocytes for development of bioartificial liver (BAL) and reconstitutive therapy. But usually the cell has a precarious balance between proliferation and differentiation: as the cell proliferation increases, functional differentiation decreases. Therefore, it is desirable for the hepatocytes to be functional by differentiation as a material for such clinical use not to be proliferative. In this study, we investigated the background of hepatocyte proliferation for the springboard of control between proliferation and differentiation of hepatocytes, and we focused attention to the asialoglycoprotein receptors (ASGP-R) of the hepatocytes. Partially hepatectomized (PH) rats were used as a model animal. When the isolated hepatocytes were plated onto the artificial extracellular matrix of poly-(N-p-vinylbenzyl-O-beta-d-galactopyranosyl-d-gluconamide) (PVLA) having galactose residues as cell-specific ligand, the rate of adhesion was decreased along with liver regeneration. Interestingly, the release of the ASGP-R from hepatocytes in serum after PH in vivo and reduction of ASGP-R of the hepatocytes in the proliferative state occurred due to cell growth in vitro. It is suggested that the ASGP-R on the hepatocyte surface during the differentiation was released in the proliferative state.     

Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.     

Effects of vasopressin on receptor-mediated endocytosis of asialoglycoprotein by hepatocytes from normal and diabetic rats

The hepatic asialoglycoprotein receptor is a membrane glycoprotein used as a model to study receptor-mediated endocytosis. In order to examine the ability of second messengers to modulate intracellular trafficking, we performed a comparative study on normal and diabetic rat hepatocytes exploring the effects of an in vivo modulation, streptozotocin-diabetes, and an in vitro modulator, vasopressin, which transduces signals via the phosphoinositide pathway. We studied three main experimental aspects: (1) constitutive endocytosis, (2) continuous ligand flux, and (3) a synchronous wave of ligand. In normal cells, vasopressin decreased ligand-binding capacity by 20%, without altering the mechanism of internalization, and decreased the level of degradation, without affecting the distribution of degradation products. Diabetic cells were characterized by a 50% decrease in cell-surface and intracellular receptor ligand-binding capacity, slowed internalization of a synchronous wave of ligand, and markedly reduced degradation with an altered distribution of degraded products. Vasopressin had no additive effect on the modification induced by diabetes. These results suggest that second messengers generated by hormones play a role in the regulation of receptor-mediated endocytosis. They also confirm that receptors are subdivided into those susceptible to modulation of any kind and those insensitive to modulation, although the boundary between the two subsets is variable.     

LDL受体介导的血浆低密度脂蛋白胆固醇的内吞

胆固醇是动物细胞细胞膜的重要组成成分,其做为细胞和环境之间的屏障调节细胞膜的流动性。胆固醇是体内所有的类固醇激素和胆酸合成的前体物质,参与体内代谢。同时胆固醇在神经系统的发育中也起着重要的作用。在血浆中胆固醇以低密度脂蛋白和高密度脂蛋白这两种胆固醇运载血脂蛋白的形式运输。动物细胞通过细胞表面的低密度脂蛋白受体(LDL receptor,LDLR)介导的内吞可以从血液中摄取富含胆固醇的低密度脂蛋白,当细胞表面的LDLR的功能缺陷时,可以导致高胆固醇血症,继而引起动脉粥样硬化、冠心病和中风等严重疾病。本文综述了LDL受体的概述及其通过内吞调节血液中低密度脂蛋白胆固醇水平的作用,并对LDL受体的调节进行了阐述。     

The pathways for fluid phase and receptor mediated endocytosis in rat hepatocytes are different but thermodynamically equivalent

In isolated rat hepatocytes fluid phase endocytosis, determined by the uptake of the fluorescent dye lucifer yellow (LY), and receptor mediated endocytosis, determined using a ligand for the asialoglycoprotein receptor (asialo-orosomucoid; ASOR), are different pathways based on their different sensitivities to hyperosmolarity induced by sucrose (Oka and Weigel, J. Cell. Biol. 105, 311a, 1987). LY uptake was unaffected by 0.2 M sucrose at all temperatures tested between 12 degrees and 37 degrees C whereas the uptake of 125I-ASOR was completely inhibited at any temperature. Since the two probes are taken up by different pathways it was possible to determine independently the activation energies (Ea) for the fluid phase versus the receptor mediated coated pit endocytic process. The Ea was 26.4 +/- 3.5 and 25.8 +/- 1.9 kcal/mole for, respectively, receptor mediated and fluid phase endocytosis. These values are not significantly different, and we conclude that the fluid phase and receptor mediated pathways are thermodynamically equivalent even though they are independent.     

Modification of triantennary glycopeptide into probes for the asialoglycoprotein receptor of hepatocytes

Triantennary glycopeptide was oxidized with galactose oxidase to convert the -CH2OH group on terminal galactose residues to the aldehyde group (oxo-form). Kinetic profiling by reverse phase high performance liquid chromatography allowed termination of the reaction when intermediate mono-oxo- and di-oxo-triantennary glycopeptides had been produced. The mixture of the oxo-glycopeptides was derivatized with 2,4-dinitrophenylhydrazine for efficient separation, and each isomeric triantennary hydrazone was separated by reverse phase high performance liquid chromatography. The purified hydrazones were reverted to three original isomeric mono-oxo- and di-oxo-glycopeptides, and a single tri-oxo-glycopeptide. Each of these isomers was characterized by proton NMR by a downfield shift in the anomeric signals of 6-oxo-Gal residue(s). The functionalized glycopeptides were successively modified with dansyl and naphthyl groups through the 6-oxo-Gal residue and the amino terminus of the peptide to prepare three isomeric glycopeptide probes suitable for conformation studies by fluorescence energy transfer measurements. Alternatively, glycopeptides were derivatized by attaching t-butyloxycarbonyl-L-tyrosine to the amino terminus of the peptide, and reductive amination of the 6-oxo-Gal residue, provided three isomeric triantennary photoaffinity probes which allow photolyzable groups to be attached to the newly introduced 6-amino-Gal residue.     

Immature human dendritic cells express asialoglycoprotein receptor isoforms for efficient receptor-mediated endocytosis.

In a search for genes expressed by dendritic cells (DC), we have cloned cDNAs encoding different forms of an asialoglycoprotein receptor (ASGPR). The DC-ASGPR represents long and short isoforms of human macrophage lectin, a Ca(2+)-dependent type II transmembrane lectin displaying considerable homology with the H1 and H2 subunits of the hepatic ASGPR. Immunoprecipitation from DC using an anti-DC-ASGPR mAb yielded a major 40-kDa protein with an isoelectric point of 8.2. DC-ASGPR mRNA was observed predominantly in immune tissues. Both isoforms were detected in DC and granulocytes, but not in T, B, or NK cells, or monocytes. DC-ASGPR species were restricted to the CD14-derived DC obtained from CD34(+) progenitors, while absent from the CD1a-derived subset. Accordingly, both monocyte-derived DC and tonsillar interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type cells did not. Furthermore, DC-ASGPR is a feature of immaturity, as expression was lost upon CD40 activation. In agreement with the presence of tyrosine-based and dileucine motifs in the intracytoplasmic domain, mAb against DC-ASGPR was rapidly internalized by DC at 37 degrees C. Finally, intracellular DC-ASGPR was localized to early endosomes, suggesting that the receptor recycles to the cell surface following internalization of ligand. Our findings identify DC-ASGPR/human macrophage lectin as a feature of immature DC, and as another lectin important for the specialized Ag-capture function of DC.     

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.