Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.     

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.     

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'-diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake.     

Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.     

Loss of surface galactosyl receptor activity on isolated rat hepatocytes induced by monensin or chloroquine requires receptor internalization via a clathrin coated pit pathway

We studied the effect of hyperosmotic inhibition of the clathrin coated pit cycle on the monensin- and chloroquine-dependent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes. Cells treated for 60 min without ligand at 37 degrees C with 25 microM monensin or 300 microM chloroquine in normal medium (osmolality congruent to 275 mmol/kg) bound 40-60% less 125I-asialo-orosomucoid (ASOR) at 4 degrees C than untreated cells. Cells exposed to monensin or chloroquine retained progressively more surface Gal receptor activity, however, when the osmolality of the medium was increased above 400 mmol/kg (using sucrose as osmolite) 10 min prior to and during drug treatment. Cells pretreated for 10 min with hyperosmolal media (600 mmol/kg) alone internalized less than or equal to 10% of surface-bound 125I-ASOR. Thus, the ligand-independent loss of surface Gal receptor activity on monensin- and chloroquine-treated hepatocytes requires internalization of constitutively recycling receptors via a coated pit pathway.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine

In this study, we characterized and compared the ligand-independent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes treated with monensin, chloroquine, microtubule depolymerizing agents, or NaN3 and NaF at 37 degrees C. Freshly isolated hepatocytes exhibit predominately one subset of surface Gal receptors, termed State 1 receptors (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). During equilibration at 37 degrees C, these cells also express a second subset of Gal receptors at the surface, termed State 2 receptors, and routinely double their total surface Gal receptor activity. Following equilibration at 37 degrees C and then inhibitor treatment, hepatocytes bound 40-60% less 125I-asialoorosomucoid (ASOR) at 4 degrees C than did untreated cells. Treated cells maintained a basal nonmodulated level of surface receptor activity regardless of temperature, perturbant concentration, or incubation time. Loss of surface Gal receptor activity on cells treated with multiple inhibitors simultaneously or sequentially was not additive. Thus, all treatments affected the same subpopulation of surface Gal receptors. None of these inhibitors decreased surface State 1 Gal receptor activity, but all prevented the normal appearance of State 2 Gal receptors on freshly isolated cells during incubation at 37 degrees C. The endocytic capability of residual surface State 1 Gal receptors on inhibitor-treated cells varied depending on the inhibitor. Hepatocytes treated first at 24 degrees C or with colchicine at 37 degrees C internalized greater than 85% of surface-bound 125I-ASOR. In contrast, monensin- or chloroquine-treated cells internalized approximately 50% of surface-bound 125I-ASOR. Azide-treated cells internalized less than 20% of surface-bound 125I-ASOR. We conclude that only surface State 2 Gal receptor activity is sensitive to these various perturbants. State 1 Gal receptor activity is not modulated. These data are consistent with the conclusion that only State 2 Gal receptors constitutively recycle.     

Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro.

We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation.

The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated.     

Dynamics of interleukin-6 internalization and degradation in rat hepatocytes.

We have investigated the dissociation, internalization, and degradation of 125I-interleukin-6 (125I-IL-6) by primary rat hepatocytes. Temperature shift experiments following saturation binding of 125I-IL-6 to cell surface receptors in hepatocytes showed a rapid loss of surface-bound 125I-IL-6 (t1/2 = 15 min), concomitant with a rapid rise in internalized radiolabeled ligand. After reaching a maximum by 30 min at 37 degrees C, the level of internalized 125I-IL-6 decreased with time and appeared in the culture media in a non-trichloroacetic acid-precipitable (degraded) state. The addition of the lysosomotropic agent chloroquine inhibited this receptor-mediated degradation of IL-6 without affecting ligand internalization. Polyacrylamide gel electrophoresis analysis of internalized 125I-IL-6 confirms these results. Additionally, we show that the IL-6.IL-6 receptor complex is stable, and dissociation of these two molecular species occurs at a pH below 5.0. In contrast to published results, data presented in this study clearly indicate that IL-6 is rapidly internalized and degraded within hepatocytes by a receptor-mediated mechanism.     

Binding and processing of (125)I-ACTH by isolated rat splenic lymphocytes

The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome.     

Recycling of the hepatic asialoglycoprotein receptor in isolated rat hepatocytes. Receptor-ligand complexes in an intracellular slowly dissociating pool return to the cell surface prior to dissociation

We recently reported that the dissociation of internalized receptor-125I-asialo-orosomucoid (ASOR) complexes by isolated hepatocytes is a biphasic process; most complexes dissociate rapidly but 25-50% dissociate slowly (Oka, J. A., and Weigel, P. H. J. Biol. Chem. 258, 10253-10262). Cells were allowed to endocytose a pulse of surface-bound 125I-ASOR, and were washed and then incubated at 37 degrees C in the presence or absence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Without EGTA, very little intact ASOR appeared in the medium. With EGTA present, a large amount of intracellular ligand appeared undegraded in the medium in a time-dependent manner. N-Acetylgalactosamine, but not ASOR, in the medium also caused release of intact 125I-ASOR. Within 15 min, more than 50% and by completion at least 80% of the internalized ligand in the slow dissociation compartment was released into the medium. If cells containing internalized ligand were incubated at 37 degrees C for increasing times before the addition of EGTA, then progressively less ligand accumulated in the medium. Experiments at 18 degrees C, a temperature at which neither degradation nor slow dissociation occurred, demonstrated that in the presence of EGTA the intracellular free 125I-ASOR pool did not change. The amount of receptor-bound ligand in the slowly dissociating pool decreased and the amount of intact ligand in the medium increased by essentially equal amounts. The temperature dependence for the return of internal 125I-ASOR to the cell surface was similar to that for endocytosis, with a cut-off temperature of about 12 degrees C. We conclude that a normal part of the endocytic process involves the return of receptor-ligand complexes to the cell surface from an internal slowly dissociating pool. This might reflect either an obligatory step or a reversible statistically random step in the endocytic/recycling pathway.     

Vanadate modulates the activity of a subpopulation of asialoglycoprotein receptors on isolated rat hepatocytes: active surface receptors are internalized and replaced by inactive receptors

In the absence of ligand, sodium vanadate causes a time- and dose-dependent loss of up to approximately 50% of the surface galactosyl receptor (GalR) activity in rat hepatocytes at 37 degrees C. The effect on total (surface plus intracellular) GalR activity is also dependent on exposure time and vanadate concentration. At less than 1 mM, vanadate induces a transient decrease and then partial recovery of cell surface GalR activity. At greater than 3 mM vanadate, surface GalR activity decreases rapidly (t1/2 approximately 2 min). Lost surface activity is initially recovered in digitonin-permeabilized cells, indicating that active surface GalRs redistribute to the cell interior. However, an antibody assay for GalR protein showed that although surface activity decreased, there was no decrease in surface receptor protein. The active intracellular GalRs then slowly inactivate over 30-60 min. With 8 mM vanadate, the loss of both surface and total cellular GalR activity is more rapid and coincident; no lag is observed. Maximal activity loss, however, was still only approximately 50%. Again, no net change was seen in the distribution of GalR protein between the cell surface and the interior. These results indicate that vanadate causes active GalRs to move from the surface to the inside and be replaced by inactive receptors moving from the inside to the cell surface. The Gal receptor system is comprised of two functionally different receptor subpopulations that operate via two distinct intracellular pathways. Only the State 2 GalRs, which recycle constitutively, are sensitive to modulation by vanadate. Consistent with this, vanadate inhibits the endocytosis of 125I-asialoorosomucoid (ASOR) only partially. The rate of uptake and the steady state level of ASOR intracellular accumulation were maximally inhibited by 50 and 70%, respectively, at 0.2 mM vanadate. The rate and extent of degradation of 125I-ASOR were also inhibited by 50-70%. Residual ASOR uptake and degradation is accounted for by the minor vanadate-resistant State 1 Gal receptor pathway.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

Metabolism of photoaffinity-labeled insulin receptors by adipocytes. Role of internalization, degradation, and recycling

Insulin receptors on isolated rat adipocytes were photoaffinity-labeled with a biologically active photo-derivative of insulin (iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin) in order to study the metabolism of surface receptors after binding insulin. Adipocytes were incubated with iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin (40 ng/ml) at 16 degrees C until specific binding reached equilibrium, subjected to photolysis, and then incubated at 37 degrees C to follow the metabolism of the covalent insulin-receptor complexes. Susceptibility of labeled insulin receptors to tryptic digestion was used to distinguish between receptors on the cell surface and those inside the cell. Following incubation of photoaffinity-labeled adipocytes at 37 degrees C, there was an initial rapid loss of insulin receptors from the cell surface. The internalization of insulin receptors occurred at a significantly faster rate than the loss of receptors from the cell, resulting in an accumulation of intracellular receptors. The proportion of surface-derived receptors inside the cell reached an apparent steady state after 30 min and represented about 20% of the labeled receptors originally on the cell surface. Chloroquine had no effect on the internalization of insulin receptors but inhibited their degradation. Cycloheximide inhibited both internalization and degradation of insulin receptors. After 60 min at 37 degrees C, the disappearance of insulin receptors from the cell surface slowed markedly and the overall loss of insulin receptors from the cell was minimal. If chloroquine was added at this time, there was a marked increase in the loss of receptors from the cell surface with a concomitant 2-fold increase in the intracellular pool of surface-derived receptors. From these observations, we conclude that 1) internalization is not rate-limiting in insulin receptor degradation, 2) chloroquine has no effect on the internalization of insulin receptors but inhibits the intracellular degradation of receptors, 3) cycloheximide interferes with both the internalization and degradation of insulin receptors, and 4) the plateau in the loss of labeled receptors from the cell surface after 60 min at 37 degrees C could be due to a new steady state balance between internalization and recycling of photoaffinity-labeled receptors.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.     

Recycling of 5''-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.