Identification and analysis of a CA(2+)-dependent lactoferrin receptor in rat liver. Lactoferrin binds to the asialoglycoprotein receptor in a galactose-independent manner.

We identified a 45 kDa Ca(2+)-dependent Lf binding protein on rat hepatocytes. Dithiobis(sulfosuccimidylproprionate) (DTSSP)-crosslinked 125I-Lf to a 45 kDa adduct in a Ca(2+)-dependent manner on intact cells. The 125I-labeled crosslinked complexes were absent when either surface-bound 125I-Lf was stripped prior to crosslinking or an excess of unlabeled Lf was included in the DTSSP reaction. Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, and a 45 kDa polypeptide (p45) was eluted by EGTA. Anti-p45 sera blocked vigorously 125I-Lf endocytosis to intact rat hepatocytes, confirming that p45 functions as the Ca(2+)-dependent Lf receptor on hepatocytes. Two tryptic fragments of p45 showed 100% identity with internal sequences (Leu121-->Lys126 and Phe198-->Lys220) of the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Antisera against p45 and RHL-1 crossreacted equally well with each protein, and asialoorosomucoid blocked the binding of 125I-Lf to hepatocytes. We did not detect the minor subunits (RHL-2/3) of the rat asialoglycoprotein receptor in p45 preparations from Triton X-100-extracts of hepatocytes, and 125I-Lf bound to immobilized RHL-1 but not to RHL-2/3. Exoglycosidases were used to remove terminally-exposed NeuNAc and alpha- and beta-Gal from bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal Gal. Unexpectedly, deglycosylated Lf exhibited no loss in its ability to compete with unmodified Lf for binding to isolated hepatocytes. Moreover, beta-lactose but not sucrose competed vigorously for 125I-Lf endocytosis by hepatocytes, indicating that Lf binds at or near the carbohydrate-recognition domain of RHL-1. We conclude that RHL-1 is the Ca(2+)-dependent Lf receptor on hepatocytes and that it binds Lf in a Gal-independent manner.

Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 [correction of leptin-1] (RHL-1) oligomers.

The major subunit [rat hepatic lectin-1 (RHL-1)] of the asialoglycoprotein (ASGP) receptor mediates endocytosis of the iron-binding protein lactoferrin (Lf) by isolated rat hepatocytes, yet iron loading of cultured adult rat hepatocytes increases the binding and endocytosis of Lf while greatly inhibiting the uptake of desialylated ligand. In the present study, we determined whether the iron-induced Lf-binding site is RHL-1 and examined the nature of the iron-induced block in ASGP receptor endocytic function. Isolated rat hepatocytes increased their non-haem iron content from 70 to 470 p.p. b. following incubation with ferric ammonium citrate (<=100 microgram/ml). These conditions blocked internalization of 125I-asialo-orosomucoid (ASOR) by approximately 90% but increased 125I-Lf endocytosis by 40%. ASOR and anti-RHL-1 sera blocked the binding and endocytosis of 125I-Lf on control cells but not on iron-loaded cells, indicating that the iron-induced Lf-binding site on hepatocytes is not RHL-1. Iron-loading of hepatocytes in the presence or absence of excess ASOR did not significantly alter the number of active ASGP receptors on the cell surface. In contrast, iron-loading decreased the number of active intracellular receptors by 40% and blocked the uptake of 125I-ASOR prebound to the cells by approximately 80%. Under these conditions, we found an iron-dependent evolution of 88 and 140 kDa RHL-1-containing, beta-mercaptoethanol-sensitive multimers that constituted up to 34 and 23%, respectively, of total immunodetectable RHL-1. We propose that iron-induced formation of cystinyl-linked RHL-1-containing multimers inhibits ASGP receptor movement between cell surface and interior and disrupts acylation of intracellular receptors.

Identification and isolation of a 45-kDa calcium-dependent lactoferrin receptor from rat hepatocytes.

Isolated rat hepatocytes bind, internalize, and degrade bovine lactoferrin (Lf) via high-affinity Ca2+-dependent sites [<10(6) sites/cell; McAbee et al., (1993) Biochemistry 32, 13749-13760]. In this study, we identified a 45-kDa Ca2+-dependent Lf binding protein on rat hepatocytes by three independent approaches. First, dithiobis(sulfosuccimidylproprionate) (DTSSP) cross-linked 125I-Lf to a 45-kDa adduct in a Ca2+-dependent manner on intact cells. The 125I-labeled cross-linked complexes were absent when either surface-bound 125I-Lf was stripped prior to cross-linking or an excess of unlabeled Lf was included in the DTSSP reaction. Second, 125I-Lf bound to a 45-kDa hepatocyte polypeptide in a Ca2+-dependent fashion following incubation with SDS-PAGE fractioned hepatocyte membrane proteins absorbed on nitrocellulose. Third, when Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, a 45-kDa polypeptide (p45) was eluted by EGTA. Column fractions enriched in p45--but not those depleted of p45--possessed soluble Lf receptor activity as determined by competition binding assay. Monospecific polyclonal anti-p45 IgG detected p45 in crude hepatocyte ghost homogenates and blocked vigorously 125I-Lf binding and endocytosis to intact rat hepatocytes. We conclude, therefore, that p45 constitutes the Ca2+-dependent Lf receptor on isolated rat hepatocytes.

Iron-loading of cultured adult rat hepatocytes reversibly enhances lactoferrin binding and endocytosis.

Isolated rat hepatocytes bind and internalize bovine lactoferrin (Lf) protein and Lf-bound Fe3+ via Ca2+-dependent recycling Lf binding sites (McAbee, 1995, Biochem. J., 311:603-609). In this study, we determined if iron loading of primary cultures of adult rat hepatocytes altered their ability to bind and internalize Lf. Rat hepatocytes were cultured 16-24 h with or without ferric ammonium citrate (FAC) and then assayed for Ca2+-dependent 125I-Lf binding at 4 degrees C or 125I-Lf endocytosis at 37 degrees C. Cells pretreated with FAC (5 microg/mL) internalized two- to sixfold more 125I-Lf than did control cells. The FAC-induced increase in 125I-Lf endocytosis required 4-8 h of culture at 37 degrees C and was fully reversible if cells were incubated an additional 24 h without FAC either in the presence or absence of the Fe3+ chelator desferrioxamine. Maximal endocytic rates for untreated and FAC-treated cells were 370 and 2,300 molecules 125I-Lf cell(-1) sec(-1), respectively. Both 125I-Lf binding at 4 degrees C and endocytosis at 37 degrees C increased up to sixfold between 0.3 10 microg/mL FAC, indicating that iron-induced enhancement of 125I-Lf uptake was due to an increase in the number of Lf receptors present on the cells. 125I-Lf bound to untreated and FAC-treated cells at 4 degrees C with similar affinities (K(d) approximately 1.5 microM). Cycloheximide but not actinomycin D blocked the FAC-induced increase in 125I-Lf binding, indicating that the increase in the number of Lf binding sites required translation but not transcription. Notably, iron loading blocked endocytosis of asialoorosomucoid by hepatocytes by up to 80%, reducing the number of active intracellular asialoglycoprotein receptors >65% without altering the number of active cell surface receptors. We conclude from these studies that Lf receptor activity on hepatocytes is regulated posttranscriptionally by the iron status of the cells.

Isolated rat hepatocytes acquire iron from lactoferrin by endocytosis.

The iron-binding protein lactoferrin (Lf) present in blood is metabolized by the liver. Isolated rat hepatocytes vigorously endocytose bovine Lf via recycling Ca2(+)-dependent binding sites, but the uptake of iron from Lf by hepatocytes has not been examined. In this study, isolated rat hepatocytes were incubated with radiolabelled bovine Lf (125I-Lf, 59Fe-Lf or 125I-59Fe-Lf) at 37 degrees C, then washed at 4 degrees C in the presence of dextran sulphate with either Ca2+ or EGTA to distinguish between total bound and internal radioactivity respectively. Cells internalized 125I-Lf protein and Lf-bound 59Fe at maximal endocytic rates of 1700 and 480 mol.cell-1.s-1 respectively. When Lf was normalized for 59Fe content, these endocytic rates were equivalent and reflected an uptake potential of at least 3400 mol of iron.cell-1.s-1. Cells prebound with 125I-59Fe-Lf to Ca2+(-)dependent sites at 4 degrees C internalized more than 80% of both 125I-Lf protein and Lf-bound 59Fe approx. 6 min after warming to 37 degrees C at similar rates (125I-Lf: k(in) = 0.276 min-1, 59Fe: k(in) = 0.303 min-1). Within 4 h at 37 degrees C, cells had released 25% or less internalized Lf protein in the form of acid-soluble 125I-by-products but retained all the Lf-delivered 59Fe. Hyperosmotic disruption of clathrin-dependent endocytosis blocked the uptake of 125I-Lf and Lf-bound 59Fe. Incubation of cells with 125I-59Fe-Lf and a 100 molar excess of diferric transferrin reduced slightly the endocytosis of 125I-Lf protein and 59Fe accumulation. Treatment of cells with the ferric chelator desferrioxamine did not alter uptake of 125I-Lf protein or Lf-bound 59Fe, but the ferrous chelator bathophenanthroline disulphonate slightly elevated endocytosis of 125I-Lf protein and Lf-bound 59Fe. These findings indicate that Lf does not release its bound iron before endocytosis. It was concluded from this study that hepatocytes take up iron from Lf at high rates by a process that requires endocytosis of Lf-iron complexes.

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.

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)

ATP depletion causes a reversible redistribution and inactivation of a subpopulation of galactosyl receptors in isolated rat hepatocytes.

Isolated rat hepatocytes, treated with metabolic energy poisons such as NaN3 in the absence of exogenous ligand, lose surface galactosyl (Gal) receptor activity (Clarke, B. L., and Weigel, P. H. (1985) J. Biol. Chem. 260, 128-133). We have used 125I-labeled asialo-orosomucoid and affinity-purified anti-receptor IgG to quantitate, respectively, the activity and the amount of Gal receptor protein. Cells were treated with NaN3 at 37 degrees C and the surface or total (surface and intracellular) binding of these two probes was measured at 4 degrees C, respectively, in intact cells or in cells permeabilized with digitonin. As a function of NaN3 concentration, both surface receptor activity and protein decreased in parallel by 50-80%. Virtually all of the lost surface receptor protein was found inside the cell, but only about 50% of all cellular Gal receptors were active. As determined by equilibrium binding studies, this decreased receptor activity reflected an overall loss of ligand binding sites with little change in binding affinity of the remaining Gal receptors for asialo-orosomucoid. When ATP was restored, normal surface receptor activity and number completely recovered even in the absence of protein synthesis. We conclude that a subpopulation of Gal receptors constitutively recycles and undergoes an inactivation/reactivation cycle. In the absence of ligand, these receptors are normally internalized and then inactivated. Loss of cellular ATP blocks receptor reactivation, prevents the reappearance of receptors at the cell surface and redistributes Gal receptors as inactive receptors accumulate intracellularly.

Thiol-sensitive sites in cell adhesion. Decreased entry of SH-binding reagents into attached BHK cells.

Studies were carried out to learn more about the critical SH groups involved in cell spreading. Pretreatment of suspended baby hamster kidney (BHK) cells with 3 mM-iodoacetate or iodoacetamide for 10 min at 4 degrees C completely inhibited the ability of the cells to spread on fibronectin-coated substrata. If, however, BHK cells were permitted to attach and spread before being treated with the SH-binding reagents, and then harvested by trypsinization and assayed for spreading on fibronectin-coated substrata, there was no inhibition of cell spreading. The extent of prior attachment required before the cells became insensitive to the SH-binding reagents was tested and was found to occur early during the cell adhesion process, before any cell spreading was observed. In analytical experiments, there did not appear to be any difference in the total number of SH groups between suspended or spread cells as determined with 5,5'-dithiobis-(2-nitrobenzoic acid). The uptake of radiolabelled iodoacetate into intact spread cells, however, was found to be 3.5 times less than that found with suspended cells. On the other hand, the distribution of incorporated radioactivity into suspended and spread cells was similar. Most of the radioactivity (approximately 70%) was incorporated into small molecules (e.g. glutathione and cysteine), less (approximately 20%) was incorporated into cytoplasmic proteins, and the least incorporation (approximately 10%) was into the cell cytoskeleton. The data are interpreted to indicate there is a decreased permeability of spread cells to the SH-binding reagents.