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.     

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.     

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.     

Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes

We have investigated the effects of hyperosmolarity induced by sucrose on the fluid phase endocytosis of the fluorescent dye lucifer yellow CH (LY) and the endocytosis of 125I-asialo-orosomucoid (ASOR) by the galactosyl receptor system in isolated rat hepatocytes. Continuous uptake of LY by cells at 37 degrees C is biphasic, occurs for 3-4 h, and then plateaus. Permeabilized cells or crude membranes do not bind LY at 4 or 37 degrees C. Intact cells also do not accumulate LY at 4 degrees C. The rate and extent of LY accumulation are concentration- and energy-dependent, and internalized LY is released from permeabilized cells. Efflux of internalized LY from washed cells is also biphasic and occurs with halftimes of approximately 38 and 82 min. LY is taken up into vesicles throughout the cytoplasm and the perinuclear region with a distribution pattern typical of the endocytic pathway. LY, therefore, behaves as a fluid phase marker in hepatocytes. LY has no effect on the uptake of 125I-ASOR at 37 degrees C. The rate of LY uptake by cells in suspension is not affected for at least 30 min by up to 0.2 M sucrose. The rate of endocytosis of 125I-ASOR, however, is progressively inhibited by increasing the osmolality of the medium with sucrose (greater than 98% with 0.2 M sucrose; Oka and Weigel (1988) J. Cell. Biochem. 36, 169-183). Hyperosmolarity completely inhibits endocytosis of 125I-ASOR by the galactosyl receptor, whereas fluid phase endocytosis of LY is unaffected. Cultured hepatocytes contained about 100 coated pits/mm of apical membrane length as assessed by transmission electron microscopy. In the presence of 0.4 M sucrose, only 17 coated pits/mm of membrane were observed, an 83% decrease. Only a few percent of the total cellular fluid phase uptake in hepatocytes is due to the coated pit endocytic pathway. We conclude that the fluid phase and receptor-mediated endocytic processes must operate via two separate pathways.     

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.     

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.     

The hepatic galactosyl receptor system: two different ligand dissociation pathways are mediated by distinct receptor populations

After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.     

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

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees 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 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

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.     

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.     

ATP-dependent inactivation and reactivation of constitutively recycling galactosyl receptors in isolated rat hepatocytes

Isolated rat hepatocytes depleted of ATP with NaN3 without ligand lose galactosyl (Gal) receptors from the cell surface and accumulate inactive receptors within the cell [McAbee, D. D., & Weigel, P. H. (1987) J. Biol. Chem. 262, 1942-1945]. Here, we describe the kinetics of receptor redistribution and inactivation after ATP depletion with NaN3 and of receptor redistribution and reactivation after ATP recovery. Only intact cells (greater than 98% viable) isolated from Percoll gradients were assayed. Gal receptor activity and protein were measured by the binding of 125I-asialoorosomucoid (125I-ASOR) and 125I-anti-Gal receptor IgG (125I-IgGR), respectively, at 4 degrees C. Surface and total (surface and intracellular) cellular Gal receptors were measured in the absence or presence, respectively, of digitonin. Following ATP depletion, 60-70% of Gal receptor activity and protein were lost from cell surfaces with first-order kinetics (t1/2 = 6.5 min, k = 0.107 min-1) at an initial rate of 11,000 125I-ASOR binding sites cell-1 min-1. Lost cell-surface Gal receptors were transiently recovered still active inside the cell. After a short lag, total cellular receptor inactivation then proceeded with first-order kinetics (t1/2 = 13 min, k = 0.053 min-1) at an initial rate of 14,000 125I-ASOR binding sites cell-1 min-1. Up to half of all cellular Gal receptors were inactivated by 40 min. 125I-IgGR binding to NaN3-treated, permeable cells, however, was virtually constant. The distribution of total cellular receptors changed from 35% on the cell surface initially to 10% after 40 min of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Surface and internal galactosyl receptors are heterooligomers and retain this structure after ligand internalization or receptor modulation

We have developed a specific chemical affinity reagent for the hepatic galactosyl receptor (GalR) by derivatizing asialoorosomucoid (ASOR) with the homobifunctional N-hydroxysuccinimide (NHS) ester cross-linker disuccinimidyl suberate [Herzig, M. C. S., & Weigel, P. H. (1989) Biochemistry 28, 600]. NHS-ASOR cross-links with 30-50% efficiency to the three GalR subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Here, we examined the subunit structure of both surface and internal receptors of two functionally distinct GalR subpopulations, designated state 1 or state 2 GalR. Freshly isolated cells, referred to as state 1 cells, kept at 4 degrees C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 degrees C, they then express both state 1 GalR and state 2 GalR on their surface. These cells are referred to as state 1,2 cells. After incubation at 4 degrees C with NHS-125I-ASOR, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-ASOR into all three RHL subunits. As analyzed by autoradiography of SDS-PAGE, radiolabeling was identical for all conditions and was in a ratio of 1:1:1 for RHL 1:2:3. Native GalR structure was also examined by first cross-linking nonradiolabeled NHS-ASOR at 4 degrees C to surface or internal receptors of state 1 or state 1,2 hepatocytes. These cells were then washed with EGTA, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid antibody, and subjected to Western blot analysis. Antisera specific for RHL 1 or RHL 2/3 detected cross-linked complexes of Mr congruent to 85K or congruent to 90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was greater than or equal to 5:1 for RHL 1 and less than or equal to 0.5:1 for RHL 2/3. Internalized GalR had the same ratio of free to cross-linked subunits as noninternalized GalR. Depletion of ATP either before or after cross-linking GalR to NHS/ASOR also did not alter the ratio of free cross-linked RHL subunits. We conclude that the surface and internal GalR of the two functionally distinct GalR populations have the same heterooligomeric subunit composition and that this GalR structure persists following endocytosis or ATP depletion.     

The effects of chronic ethanol administration on the rates of internalization of various ligands during hepatic endocytosis.

The purpose of the present study was to further characterize the ethanol-induced impairments in hepatic endocytosis. Specifically, we examined the effects of ethanol treatment on receptor-ligand internalization via the coated and noncoated pit pathways. Insulin, epidermal growth factor (EGF) and asialoorosomucoid (ASOR) were used as model ligands to study internalization by isolated hepatocytes. ASOR and EGF are thought to be internalized strictly in coated pit regions of the cell membrane, while insulin may be internalized in both coated and uncoated membrane regions. Ethanol administration for 5-7 weeks decreased internalization of ASOR and EGF while internalization of insulin was unchanged during a single round of endocytosis of surface-bound ligand. Similarly, a more quantitative measure of endocytosis, the endocytic rate constant, was decreased for EGF and ASOR but not for insulin in livers of experimental rats. When endocytosis of Lucifer yellow, a fluorescent dye known to be internalized in the cell by fluid-phase endocytosis was examined, the initial rates of dye uptake were not significantly altered by alcohol administration. These results indicate that ethanol may selectively impair internalization occurring by coated pits while it has a minimal effect on initial uptake of molecules which are internalized by noncoated membrane regions.     

Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.     

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.     

Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes.

We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules.