 |
INTRODUCTION |
Cells require transition metals for a variety of crucial cellular
processes, but unregulated accumulation of these metals, such as iron
and copper ions, promotes damage to membrane lipids, proteins, and
nucleic acids (1, 2). As a result, eukaryotic cells acquire nutrient
metal ions by highly regulated mechanisms that also guard against
overaccumulation of these metals, sequester metal ions when they are
present in overabundance, and ameliorate the toxic effects of metals on
cellular macromolecules (for reviews, see Refs. 3-5).
We have been studying the hepatic metabolism of the iron-binding
protein lactoferrin and have found that rat hepatocytes take up
lactoferrin via the rat hepatic lectin (RHL)1 1 subunit of the ASGP receptor. Unlike other characterized ligands for
the ASGP receptor, lactoferrin binds at or near RHL1's carbohydrate recognition domain, yet in a carbohydrate-independent manner (6, 7). We
have also found that the iron status of hepatocytes alters the manner
in which hepatocytes bind and internalize lactoferrin. Iron loading of
hepatocytes by incubation of cells with ferric ammonium citrate
increases lactoferrin binding and endocytosis. This iron
ion-dependent increase in lactoferrin accumulation, however, is accomplished by non-RHL1 binding sites that are reversibly expressed on cells in an iron-dependent manner reminiscent
of iron-dependent regulation of ferritin (8, 9). In
contrast, the endocytic activity of the ASGP receptor is dramatically
inhibited by hepatocyte iron loading (8, 9). In addition, iron-replete hepatocytes accumulate up to half of their ASGP receptors in an inactive form, and these various effects correspond to the appearance of cystinyl-linked ASGP receptor RHL1 subunits (9). These findings represent a heretofore-unrecognized relationship between the ASGP receptor system and the iron status of hepatocytes.
Iron belongs to the borderline class of transition metals, which also
includes Mn2+, Co2+, Ni2+,
Cu2+, and Zn2+ (10). In general, borderline
transition metals interact readily with organic ligands and are
required by cells in trace amounts to carry out structural, catalytic,
or redox activities. Because iron ion treatment of hepatocytes promoted
formation of disulfide-bonded oligomers of RHL1 (9), we speculated that
the iron-dependent inhibition of ASGP receptor function was
related to the redox properties of iron ions. To address this matter,
we initiated a study to examine the effects of other borderline
transition metals (Mn2+, Cu2+, and
Zn2+) with various redox capacities on ASGP receptor
endocytic activity. Like iron ions, both manganese and copper ions can
mediate intracellular redox reactions whereas zinc ions are not redox
active. Here, we report that copper and zinc ions, but not manganese
ions, dramatically block ASGP receptor-mediated endocytosis and
receptor ligand binding activity. Moreover, zinc, but not copper,
partially blocked internalization of transferrin by hepatocytes, but
neither metal blocked intracellular processing of internalized ligand
nor pinocytosis. Our findings are the first demonstration of a specific
inhibition of receptor-mediated endocytosis by non-iron transition metals.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human orosomucoid, human holotransferrin,
neuraminidase (Clostridium perfringens), Lucifer Yellow
(dipotassium salt), and BSA (fraction V) were purchased from Sigma.
IODOGEN, IgG purification kit, and BCA protein assay reagent were
obtained from Pierce). Collagenase (type D) was from Roche Molecular
Biochemicals. Digitonin was obtained from Eastman Kodak.
Na125I (17 Ci/mg of iodine) was from NEN Life Science
Products. All other chemicals were reagent grade and obtained from
Fisher Biochemicals (Itasca, IL or Tustin, CA) or Sigma. BME was
obtained from Sigma and supplemented with 2.4 g/liter HEPES, pH 7.4, and 0.22 g/liter NaHCO3. BME-BSA is BME containing 0.1%
(w/v) BSA. HBS contains 150 mM NaCl, 3 mM KCl,
and 10 mM HEPES, pH 7.4. Buffer A contained HBS
supplemented with 5 mM CaCl2 and 5 mM MgCl2. Buffer B contains HBS supplemented
with 5 mM EGTA.
Hepatocyte Preparation--
Male Sprague-Dawley rats (100-350
g, Harlan Sprague, San Diego, CA) were fed standard laboratory chow and
water ad libitum. The copper and zinc content of the chow
(Purina Rodent Laboratory Chow, formula 5001) were 13 and 70 ppm,
respectively, as assayed by the manufacturer. Hepatocytes were prepared
by a modification of a collagenase perfusion procedure (11) as
described previously (12). Cells were kept at approximately 30 °C
during the filtration and differential centrifugation steps. Final cell
pellets suspended in ice-cold BME-BSA were
85% viable and single
cells. Before experiments, cell suspensions (2-4 × 106 cells/ml in BME-BSA, 10% of the flask volume) were
incubated at 37 °C for 60 min to allow recovery from the isolation
procedure. Cell viability was determined microscopically by trypan blue
exclusion. For some experiments, isolated hepatocytes were plated on
unmodified tissue culture plates at a density of ~1000
cells/mm2 in William's E medium supplemented with 5%
(v/v) fetal bovine serum, 10 mM HEPES, 2 mM
L-glutamine, 0.48 µg/ml insulin, 0.5 µM
dexamethasone, 2.5 µg/ml amphotericin B, and 50 µg/ml gentamycin and incubated at 37 °C in 5% CO2 overnight prior to use.
Transferrin and ASOR Preparation--
Human holotransferrin and
human orosomucoid (
1-acid glycoprotein) were obtained
commercially. Human orosomucoid was desialylated with neuraminidase as
described previously (13), and removal of terminal
N-acetylneuraminyl groups was confirmed by lectin blotting
of neuraminidase-treated orosomucoid with Sambucus nigra agglutinin-digoxigenin conjugate (DIG glycan differentiation kit, Roche
Molecular Biochemicals) as reported elsewhere (7).
125I-Transferrin and 125I-ASOR, prepared by the
IODOGEN method (14), had specific activities of 90-130 dpm/fmol.
Homogeneity and structural integrity of 125I-transferrin
and 125I-ASOR were confirmed by denaturing electrophoresis
followed by autoradiography.
125I-ASOR Binding, Endocytosis and Degradation
Assays--
For 125I-ASOR binding assays, 2-4 × 106 cells/ml were routinely incubated with
125I-ASOR (1-2 µg/ml) at 4 °C for 60 min in either
BME-BSA or Buffer A. Cells incubated with 125I-ASOR were
washed free of unbound ligand by centrifugation in excess Buffer A (two
5-min washes, 4 °C). Washed cells were resuspended in 0.5-1.0 ml of
Buffer A, transferred to clean plastic tubes, and assayed for
radioactivity and protein. In some cases, hepatocytes were
permeabilized with 0.055% (w/v) digitonin prior to addition of
125I-ASOR in order to measure the surface and intracellular
pools of receptor activity on cells (15). To measure hepatocyte
endocytosis of 125I-ASOR, cells in BME-BSA were incubated
with 125I-ASOR at 37 °C for 45-60 min. Uptake was
stopped rapidly by diluting the cells into a 5-10-fold excess volume
of ice-cold Buffer A. To assay total cell-associated (surface and
intracellular) 125I-ASOR, cells were washed by
centrifugation twice in 2-4 ml of cold Buffer A. To assay internalized
125I-ASOR only, cells were washed by centrifugation twice,
5 min each wash, in 2-4 ml of Buffer B at 4 °C; EGTA present in
Buffer B chelates Ca2+ required for ASOR binding to ASGP
receptors, thereby dissociating cell surface receptor-ligand complexes.
After washing, cells were resuspended in 0.5-1.0 ml of Buffer A,
transferred to clean plastic tubes, and assayed for radioactivity and
protein. In some experiments, we determined the extent of
125I-ligand degradation by cells by measuring the amount of
acid-soluble radioactivity released into conditioned medium at
37 °C, as reported previously (16). Briefly, conditioned medium from
cells incubated with 125I-ASOR were collected and clarified
by centrifugation (400 × g, 2 min, 4 °C).
125I-Polypeptides in the conditioned media were
precipitated by the addition of two volumes of cold 10% (w/v)
phosphotungstic acid in 2 N HCl. After incubation for 15 min on ice, precipitated material was sedimented (4 °C, 4 min,
1000 × g); acid-precipitated pellets and acid-soluble
supernatants were assayed for radioactivity.
Anti-RHL-1 Immunoglobulin Purification and Cell Surface RHL1
Protein Assay--
Anti-RHL1 sera were generated and analyzed as
described elsewhere (6, 7). The whole Ig fraction from sera (immune and nonimmune) was isolated by chromatography on Protein A-agarose (Pierce)
and iodinated with Na125I (14) to a final specific activity
of 70-110 dpm/fmol. Hepatocytes in BME-BSA were incubated with or
without CuCl2 (75 µM) or ZnCl2 (225 µM) for 2 h at 37 °C, then chilled on ice.
The cells were washed free of metals by centrifugation in cold excess
BME, then incubated with 125I-anti-RHL1 Ig or
125I-Ig (nonimmune) for 90 min at 4 °C with or without a
100-fold molar excess of unlabeled anti-RHL1 Ig or nonimmune Ig. The
cells were collected by centrifugation, washed twice with excess cold Buffer A, then assayed for cell-associated radioactivity.
Pinocytosis Assay--
Control, CuCl2-treated, and
ZnCl2-treated hepatocytes were incubated in the presence or
absence of metals with BME-BSA supplemented with 0.2 mg/ml Lucifer
Yellow for up to 1 h at 37 °C. Cells were chilled on ice,
washed twice with excess cold BME-BSA, 10 min/wash, then solubilized at
4 °C for 20 min in 0.05% Triton X-100 containing 0.1% (w/v) BSA.
Insoluble debris was sedimented by centrifugation (13,000 × g, 15 min, 4 °C), and supernatants were assayed for Lucifer Yellow fluorescence (excitation at 430 nm, emission at 540 nm)
using a Fluoromax-2 spectrofluorimeter (Jobin Yvon Spex, Instruments
S.A., Inc., Edison, NJ).
125I-Transferrin Binding and Endocytosis
Assay--
Adult rat hepatocytes were plated on unmodified tissue
culture plates and incubated overnight as described above. Prior to experiments, cells were incubated in BME-BSA (no serum present) with or
without CuCl2 or ZnCl2 for 2 h at
37 °C. To determine surface binding of 125I-transferrin
by hepatocytes, cells were chilled on ice and incubated 2 h with
125I-transferrin (2 µg/ml) in BME at 4 °C. The
radioactive mixture was removed, and the cells were rinsed four times
with cold Buffer A. The cell-bound radioactivity was solubilized by
incubating the cells in 2 ml of Buffer A containing 2% (v/v) Triton
X-100 at 37 °C for 2 h. Detergent-released
125I-radioactivity was transferred to clean tubes and
assayed by
spectroscopy. To determine 125I-transferrin
endocytosis by hepatocytes, cells in BME-BSA were supplemented with
125I-transferrin (2 µg/ml) and incubated at 37 °C for
60 min, after which the cells were chilled on ice. Radioactive media
were removed, and the cells were assayed for total bound
125I-transferrin as described above. To determine the
amount of internal 125I-transferrin, rinsed cells at
4 °C were stripped of surface-bound 125I-transferrin by
a 30-s exposure to 0.25 M acetic acid, 0.5 M NaCl, after which an equal volume of ice-cold 0.5 M
Na2HPO4 was added to bring the pH to 7.5. Acid-released radioactivity was aspirated off the cells, and the cells
were rinsed an additional three times with cold Buffer A. Acid-resistant radioactivity, deemed intracellular, was assayed
following Triton X-100 solubilization as described above.
General Procedures--
Protein was determined by the
bicinchoninic acid protein assay procedure using BSA as standard
(Pierce). Centrifugation of cell suspensions was at 400 × g for 2 min at 4 °C using a Beckman GS-6R centrifuge
equipped with a GH-3.8 rotor (Beckman Instruments, Inc., Fullerton,
CA). 125I radioactivity was determined using a Packard
Cobra Auto-Gamma counting system (model 5002; Packard Instrument Co.,
Downers Grove, IL). Spectroscopic measurements were done using a
Shimadzu UV-160 spectrophotometer (Kyoto, Japan).
 |
RESULTS |
Zinc and Copper Block Sustained ASGP Receptor-dependent
Endocytosis--
In the first series of experiments, we examined the
effects of MnCl2, ZnCl2, or CuCl2
on sustained endocytosis (binding and internalization) of
125I-ASOR by hepatocytes. Cells exposed to
MnCl2 (0-220 µM) exhibited no alteration in
sustained endocytosis of 125I-ASOR (data not shown). In
contrast, continuous internalization of 125I-ASOR by cells
incubated with 0-220 µM ZnCl2 (Fig.
1A) or 0-225 µM
CuCl2 (Fig. 1B) diminished up to at least 94%.
Half-maximal inhibition of endocytosis (Fig. 1B,
inset) was observed at 39 µM CuCl2
and 53 µM ZnCl2 although significant
inhibition exerted by ZnCl2 occurred only at concentrations
>20 µM. Inhibition of continuous 125I-ASOR
endocytosis by ZnCl2 (220 µM) or
CuCl2 (75 µM) was time-dependent (Fig. 2). The first-order half-time rates
for loss of 125I-ASOR internalization were 62 min for zinc
ions and 54 min for copper ions (Fig. 2, A and B,
insets). Overall, the loss of 125I-ASOR binding
and uptake followed similar dose-response curves (Fig. 1) and kinetics
(Fig. 2). Regardless of the treatment, cell viability was not altered,
as determined by trypan blue exclusion by control and metal-treated
cells (data not shown). We also found that the effects of copper and
zinc ions on continuous uptake of 125I-ASOR were additive
(Fig. 3). Binding and internalization of 125I-ASOR were more greatly inhibited when cells were
treated with a combination of ZnCl2 (220 µM)
and CuCl2 (75 µM) (91% and 95% for zinc and
copper, respectively) than when treated with either metal alone. We
also found that the effects of zinc and copper ions on ASOR endocytosis
were not ameliorated by co-incubation with the general reductant
ascorbate (Table I). Notably, ascorbate (0.1 mM) enhanced the copper-dependent
inhibition of ASOR binding and internalization compared with cells
treated with copper ions alone.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Continuous endocytosis as a function of metal
concentration. Hepatocytes (2 × 106 cells/ml)
were incubated in BME-BSA with and without the designated
concentrations of ZnCl2 (A) or CuCl2
(B) at 37 °C for 2 h. The cells were supplemented
with 125I-ASOR (2 µg/ml), and the incubations were
continued at 37 °C for an additional 60 min. The cells were chilled
at 4 °C and assayed for total ( ) and internalized ( )
125I-ASOR. Symbols reflect the mean of duplicate
samples; error bars reflect the standard
deviations of the means. Results shown in panels
A and B were from experiments done on different
days using different hepatocyte and 125I-ASOR preparations.
The extent of inhibition (inset) was calculated according to
the equation (Xmax XC)/(Xmax Xmin), where Xmax = maximal inhibition, XC = inhibition at metal
concentration C, and Xmin = levels of
125I-ASOR total bound or internalized in the absence of
added metal ions. Symbols represent extent of inhibition of
125I-ASOR binding (surface and intracellular; , ) and
internalization ( , ) in the presence of copper ( , ) or zinc
( , ) ions.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
Kinetics of endocytosis inhibition.
Hepatocytes (2 × 106 cells/ml) were incubated in
BME-BSA with and without 225 µM ZnCl2
(A) or 75 µM CuCl2 (B)
for the indicated times at 37 °C. The cells were supplemented with
125I-ASOR (2 µg/ml), and the incubation was continued for
an additional 60 min at 37 °C. The cells were chilled at 4 °C and
assayed for total bound ( ) and internalized ( )
125I-ASOR. Symbols reflect the mean of duplicate
samples; error bars reflect the standard
deviations of the means. The metal-dependent inhibition of
total binding and internalization of 125I-ASOR compared
with nontreated control cells (% Control) was also plotted
(insets). The inhibition of 125I-ASOR
internalization followed first-order kinetics for both
ZnCl2 (t1/2 = 62 min;
r = 0.98, n = 6) and
CuCl2 (t1/2 = 54 min;
r = 0.97, n = 6).
|
|

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of zinc and copper co-treatment on
ASOR endocytosis. Hepatocytes (2 × 106 cells/ml)
were incubated with BME-BSA with and without the indicated
concentrations of ZnCl2 or CuCl2 at 37 °C
for 2 h. The incubations were supplemented with
125I-ASOR (2 µg/ml) and the incubations continued at
37 °C for an additional 60 min. The cells were chilled at 4 °C,
washed, and assayed for total and internalized 125I-ASOR.
Values reflect the means of triplicate samples; error
bars reflect the standard deviation of the means.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Effect of ascorbate, Zn2+, and Cu2+ treatment on ASGP
receptor endocytic activity
Hepatocytes (2 × 106 cells/ml) in BME-BSA were incubated
with or without the designated amounts of ZnCl2, CuCl2,
or ascorbate for 2 h at 37 °C. Cells were chilled at 4 °C,
washed twice in BME-BSA to remove excess metal, then prebound with
125I-ASOR at 4 °C for 1 h. After cells were washed free
of unbound 125I-ASOR, cells were resuspended in fresh BME-BSA
with or without ZnCl2, CuCl2, or ascorbate (similar to
that during the original 2-h incubation) and incubated at 37 °C for
15 min. The cells were chilled at 4 °C and assayed for total bound
and internalized 125I-ASOR. Values represent the mean ± standard deviation of duplicate samples. Values as a function of a
percentage of untreated control cells are included in parentheses.
|
|
The inhibition of ASGP receptor-dependent endocytosis by
metal treatment was reversible. Cells treated with ZnCl2 or
CuCl2 exhibited >60% loss in total (surface and
intracellular) bound (Fig. 4A)
and internalized (Fig. 4B) 125I-ASOR. When cells
were washed free of metals and incubated in the absence of exogenous
zinc and copper ions, they recovered their ability to bind and take up
125I-ASOR. These results indicate that the metal-sensitive
sites required for endocytosis were either not irreversibly blocked by
metal treatment or were readily replaced following de novo synthesis. In either case, these data support the conclusion that the
block in endocytosis was not due to a loss of cell viability.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Reversibility of metal inhibition of ASOR
endocytosis. Hepatocytes (4 × 106 cells/ml) in
BME-BSA were treated with or without ZnCl2 (220 µM) or CuCl2 (75 µM) for 2 h at 37 °C. A portion of these cells were supplemented with
125I-ASOR (2 µg/ml) and incubated at 37 °C for 30 min,
after which they were assayed for total bound (A) and
internalized (B) 125I-ASOR (2 h ± metal).
The remainder of the cells was chilled at 4 °C, washed twice in BME
to remove excess metal, then resuspended into fresh BME-BSA and
incubated 1 or 2 h at 37 °C. At each of these recovery times,
some cells were supplemented with 125I-ASOR (2 µg/ml) and
incubated an additional 60 min at 37 °C, after which they were
assayed for total bound (A) and internalized (B)
125I-ASOR. Values represent the mean of duplicate samples;
error bars reflect the standard deviations of the
means. , no additions; , 220 µM ZnCl2;
, 75 µM CuCl2.
|
|
In each of the preceding experiments, zinc- or copper-treated
hepatocytes were examined for their ability to take up
125I-ASOR continuously during a 1-h assay period following
a 2-h metal treatment period. As a result, inhibition of continuous endocytosis could reflect a block in one or more events including internalization of receptor-ligand complexes at the cell surface, intracellular processing of receptor-ligand complexes, or recycling of
ASGP receptors. To determine if zinc or copper ions blocked internalization of receptor-ligand complexes at the cell surface, we
examined the ability of metal-treated hepatocytes to internalize 125I-ASOR prebound on the cell surface (Fig.
5). We found that the amount of
125I-ASOR bound to the cells following zinc and copper
treatment was reduced by 56% and 35%, respectively, reflecting a loss
in the number of active ASGP receptors on the surfaces of metal-treated cells. Zinc-treated cells internalized progressively less
125I-ASOR as a function of ZnCl2 concentration but
the percentage of bound 125I-ASOR that was internalized
remained the same (~30%) regardless of the ZnCl2
concentration (Fig. 5A). In contrast, the percentage of
prebound 125I-ASOR internalized by cells was reduced from
33% on control cells to <8% on cells treated with 75 µM CuCl2 (Fig. 5B). These results indicate that, although both metals reduced the number of active surface ASGP receptors, copper ions, but not zinc ions, blocked the
ability of cells to internalize receptor-ligand complexes at the cell
surface. These data also suggest that at least some of the sites
sensitive to these two metals are different.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of zinc and copper on internalization
of receptor-ligand complexes. Hepatocytes (2 × 106 cells/ml) in BME-BSA were incubated with and without
0-225 µM ZnCl2 (A) or 0-75
µM CuCl2 (B) for 2 h at
37 °C. Cells were chilled at 4 °C, washed once in BME to remove
excess metal, then incubated with 125I-ASOR at 4 °C for
1 h to bind surface ASGP receptors. After they were washed free of
unbound 125I-ASOR, cells were resuspended in fresh BME-BSA
containing ZnCl2 or CuCl2 (at concentrations
identical to those during the original 2-h incubation) and incubated at
37 °C for 10 min. The cells were chilled at 4 °C and assayed for
total bound ( ) and internalized ( ) 125I-ASOR.
Symbols represent the mean of duplicate samples;
error bars reflect the standard deviations of the
means. The percentage of total bound 125I-ASOR internalized
by the cells ( ) equaled (125I-ASOR internal) (125I-ASOR total)×100.
|
|
Zinc and Copper Reduce Hepatocyte Surface ASGP Receptor
Activity--
The previous experiments (Fig. 5, Table I) showed that
copper- and zinc-treated hepatocytes reduced the number of active ASGP
receptors on their surfaces. To address this issue more directly, we
examined the ligand binding activity of surface and intracellular populations of ASGP receptors on metal-treated hepatocytes. Surface ASGP receptor activity was assayed on intact cells, whereas both surface and intracellular ASGP receptor activity was assayed on digitonin-permeabilized hepatocytes (15, 17). Treatment of cells with
ZnCl2 or CuCl2 reduced surface binding of
125I-ASOR by up to 44% (Fig.
6A) and 48% (Fig.
6B), respectively. Permeable metal-treated cells also bound
progressively less 125I-ASOR than did nontreated control
cells. Notably, the absolute molar reduction of 125I-ASOR
binding to permeable cells was similar to that observed for intact
cells, such that the calculated number of active intracellular ASGP
receptors was not altered by metal treatment (Fig. 6, A and B, closed circles). Thus, both copper
and zinc ions induced a loss of surface ASGP receptor activity with no
net change in the amount of intracellular receptor activity.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of zinc and copper on ASGP receptor
ligand binding activity. A and B,
hepatocytes (2 × 106 cells/ml) in BME-BSA were
incubated with or without the designated concentrations of
ZnCl2 (A) or CuCl2 (B) at
37 °C for 2 h. Cells were chilled on ice. Half of the cells
were permeabilized with 0.06% (w/v) digitonin for 10 min at 4 °C,
after which they were pelleted by centrifugation; supernatants were
discarded. Intact ( ) and permeable ( ) cells were assayed for
binding of 125I-ASOR at 4 °C. Symbols
represent the means of duplicate samples; error
bars reflect the standard deviations of the means.
125I-ASOR bound to intracellular ASGP receptors ( ;
Internal) was calculated as the difference of
125I-ASOR bound to permeable v. intact cells. C
and D, hepatocytes were incubated with or without 75 µM CuCl2 or 225 µM
ZnCl2 for 2 h at 37 °C, after which the cells were
chilled on ice. The cells were then assayed for 125I-ASOR
binding at 4 °C (C) or for 125I-anti-RHL1 IgG
binding at 4 °C (Total Binding; D).
To determine nonspecific 125I-anti-RHL1 IgG binding to
hepatocytes, incubations containing 125I-anti-RHL1 Ig were
done in the presence of a 100-fold excess of unlabeled anti-RHL1 Ig;
specific binding was calculated as the difference between total binding
and nonspecific binding. Values represent the means of duplicate
samples; error bars reflect the standard
deviations of the means.
|
|
The loss of receptor-ligand binding activity observed in the previous
experiment does not necessarily reflect the loss of receptor protein
from the cell surface. Thus, we examined the surfaces of metal-treated
cells for the presence of immunodetectable ASGP receptor protein by
assaying their ability to bind 125I-anti-RHL1 Ig. As
expected, CuCl2 (75 µM) and ZnCl2
(225 µM) reduced 125I-ASOR bound to cells by
40% and 70%, respectively (Fig. 6C). Accordingly,
zinc-treated cells bound ~50% less 125I-anti-RHL1 Ig
than did control cells indicating that RHL1 protein was lost from the
cell surface. In contrast, copper-treated cells exhibited no loss of
125I-anti-RHL1 Ig binding (Fig. 6D). Treatment
of cells with zinc or copper ions at 4 °C either before or during
the 125I-anti-RHL1 Ig binding assay did not alter
125I-anti-RHL1 Ig binding to the
cells,2 indicating that
copper and zinc ions per se did not interfere with antibody
binding to the receptor. Taken together, these data indicate that
copper-treated cells accumulated inactive ASGP receptors at the cell
surface with no significant change in the distribution of RHL1 subunits
between the cell surface and cell interior. Because zinc ion-treated
cells lost surface ASGP receptor activity and protein without an
increase in intracellular ASGP receptor activity (Fig. 6A),
we conclude that zinc-treated cells accumulated inactive ASGP receptors intracellularly.
In other experiments, we found that the metal-induced reduction of
surface ASGP receptor activity was temperature-dependent. Cells treated with various concentrations of zinc or copper ions at
4 °C showed no loss of surface binding of 125I-ASOR
(Fig. 7A) compared with cells
pretreated at 37 °C with either CuCl2 or
ZnCl2 alone or in combination (Fig. 7B). These data indicated that metal-induced losses of surface receptor activity results from perturbations in one or more cellular processes rather than direct interference of metals on the ability of ASGP receptors to
bind ligand per se. Notably, significant
metal-dependent reductions of surface 125I-ASOR
binding were evident only when cells were exposed to metal ions at or
above 22 °C (Fig. 7C), and 125I-ASOR binding
was actually higher on samples treated with different levels of
CuCl2 than on control cells. In other experiments, we found
that the ASGP receptor binding of ASOR was partially reduced for
zinc-treated 125I-ASOR (Table
II). When cells were incubated with
125I-ASOR and zinc or copper ions simultaneously at
4 °C, binding of 125I-ASOR was the same as control
cells. If, however, 125I-ASOR was mixed with
ZnCl2 at 22 °C for 1 h prior to addition to
untreated hepatocytes at 4 °C, cells bound 28% less
125I-ASOR compared with non-pretreated
125I-ASOR (Table II). In contrast, 125I-ASOR
pretreated with 75 µM CuCl2 maintained >90%
of its cell binding activity. The inhibitory effects of zinc ions on
125I-ASOR were reversed if zinc-treated
125I-ASOR was dialyzed free of zinc ions prior to addition
to hepatocytes. These data suggest that zinc ions bind reversibly to
ASOR, possibly in a temperature-dependent manner, and
reduce at least partially its ability to bind ASGP
receptors.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of temperature on metal-induced loss
of surface ASGP receptor activity. A, hepatocytes
(2 × 106 cells/ml) in BME-BSA were incubated with and
without ZnCl2 or CuCl2 at the indicated
concentrations for 2 h at 4 °C. Cells were washed once in BME,
then assayed for 125I-ASOR binding at 4 °C.
Symbols represent means of duplicate samples;
error bars reflect standard deviations of the
means. B, hepatocytes (2 × 106 cells/ml)
in BME-BSA were incubated for 2 h with or without 225 µM ZnCl2 or 75 µM
CuCl2 at either 4 °C or 37 °C. Cells were chilled at
4 °C, washed twice in HBS to remove excess metal, then assayed for
bound 125I-ASOR. Symbols represent means of
duplicate samples; error bars reflect standard
deviations of the means. C, hepatocytes (2 × 106 cells/ml) in BME-BSA were incubated for 2 h with
or without 225 µM of ZnCl2 or 75 µM of CuCl2 at the indicated temperatures.
The cells were chilled, washed twice to remove excess metal, then
assayed for 125I-ASOR binding at 4 °C.
Symbols represent means of duplicate samples;
error bars reflect standard deviations of the
means.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Effect of Zn2+ and Cu2+ on ASGP receptor ligand-binding
activity
In experiment 1, hepatocytes (2 × 106) were incubated
with 125I-ASOR in the presence or absence of 220 µM ZnCl2 or 75 µM CuCl2 for
1 h at 4 °C, after which the cells were assayed for bound
radioactivity. In experiment 2, 125I-ASOR was preincubated with
or without 220 µM ZnCl2 or 75 µM
CuCl2 for 1 h at 22 °C, dialyzed overnight against HBS
to remove metals, then assayed for binding on hepatocytes (4 × 106 cells/ml) at 4 °C for 1 h. Values represent the
means of duplicate samples ± standard deviation.
|
|
Effect of Zinc and Copper on Intracellular Processing of ASGP
Receptors and ASOR Degradation--
About half of the ASGP receptors
on isolated rat hepatocytes recycle constitutively, whereas the rest
recycle only during endocytosis (18). Reduced surface ASGP receptor
activity and ASGP receptor-dependent endocytosis on
metal-treated hepatocytes may reflect, in part, an interruption in the
return of recycling ASGP receptors back to the cell surface. If metal
treatment interferes with ASGP receptor recycling, then one would
predict that the loss of surface ASGP receptor activity would be more
pronounced if hepatocytes were actively endocytosing ASOR during
treatment with copper or zinc ions. To test for this, hepatocytes were
treated with various concentrations of ZnCl2 or
CuCl2 in the presence or absence of excess ASOR, after
which cells were stripped of surface-bound ASOR and assayed for surface
ASGP receptor activity by 125I-ASOR binding at 4 °C
(Fig. 8). Hepatocytes subjected to an
endocytic load in the absence of metals possessed ~10% fewer active
ASGP receptors than did cells not exposed to ASOR, presumably due to a
minor ASOR-induced shift in the steady state distribution of ASGP
receptors between the cell surface and cell interior. As anticipated,
hepatocytes treated with either metal lost 50-60% of their surface
ASOR binding activity. The ASOR-dependent reduction in
surface ASGP receptor activity was not enhanced when cells were treated
with zinc ions (Fig. 8A). On the other hand, cells treated
with 75 µM CuCl2 showed a significant
ASOR-dependent reduction in surface ASGP receptor activity
(Fig. 8B), suggesting that copper ions, but not zinc ions,
partially blocked the return of recycling ASGP receptors back to the
cell surface. Cells treated with 225 µM CuCl2
(Fig. 8B) in the presence of added ASOR showed no loss of
surface 125I-ASOR binding over and above that observed for
cells treated with copper ions alone. This suggests the possibility
that, at high copper ion concentrations, the ability of cells to
internalize and recycle ASGP receptors is so severely impaired that
ligand-stimulated receptor recycling is not observed.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of endocytic load during metal
treatment on surface ASGP receptor activity. Hepatocytes (2 × 106 cells/ml) in BME-BSA were incubated with or without
ZnCl2 (A) or CuCl2 (B) in
the presence ( ) or absence ( ) of ASOR (2 µg/ml) at 37 °C for
2 h. The cells were chilled at 4 °C, washed in Buffer B at
4 °C to remove surface-bound ASOR, then assayed for
125I-ASOR binding at 4 °C. Symbols represent
means of duplicate samples; error bars reflect
standard deviations of the means.
|
|
We also examined the effects of zinc and copper ions on the lysosomal
delivery and degradation of internalized 125I-ASOR. In this
experiment, hepatocytes were prebound with 125I-ASOR at
4 °C, then incubated at 37 °C. At various times after the
temperature shift, the cells were supplemented with metals and the
37 °C incubation was continued for up to 2 h at which time the
amounts of cell-associated 125I-ASOR and
125I-degradation products released from the cells were
determined (Fig. 9). We found that
neither zinc (Fig. 9A) nor copper (Fig. 9B) ions
significantly altered the amount of 125I-ASOR degraded by
the cells as measured by the release of acid-soluble radioactivity from
the cells, even if metals were added just 10 min following the shift to
37 °C. These results indicate that neither zinc nor copper ions
perturbs the intracellular dissociation of internalized ASGP
receptor-ASOR complexes or the delivery of ASOR to lysosomes and its
subsequent degradation.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of metal treatment on degradation of
internalized 125I-ASOR. Hepatocytes (2 × 106 cells/ml) in BME-BSA were incubated with
125I-ASOR at 4 °C for 1 h, after which they were
washed twice in Buffer A to remove unbound 125I-ASOR. Cells
were resuspended in fresh BME-BSA and incubated at 37 °C. At the
indicated times, cells were supplemented with ZnCl2 (225 µM; A) or CuCl2 (75 µM; B), and the incubation was continued for a
total of 2 h. Samples shown at 120 min were not exposed to added
metals. The cells were chilled at 4 °C. Cell media were collected
and assayed for acid-soluble ( ) and acid-precipitable ( )
radioactivity. Cells were assayed for associated radioactivity ( ).
Symbols represent the means of duplicate samples;
error bars reflect the standard deviations of the
means. Plotted lines are the best-fit curves calculated by linear
regression (y = mx + b) of data
points.
|
|
Effect of Copper and Zinc on Fluid-phase Pinocytosis and
Transferrin Endocytosis--
The preceding results collectively show
that zinc and copper ions strongly block ASGP receptor-mediated
endocytosis. The question arises as to whether or not the inhibitory
effects of these metal ions are specific for ASGP receptors or whether
other vesicular uptake pathways are also sensitive to the effects of
these metals. In the first series of experiments, we determined if
fluid-phase uptake by hepatocytes was altered by copper or zinc ions.
It has been shown that clathrin-dependent receptor-mediated
endocytosis and pinocytosis can be uncoupled in hepatocytes by
K+ depletion or by hyperosmotic treatment (19). In this
experiment, isolated rat hepatocytes were treated at 37 °C for
2 h with or without ZnCl2 (225 µM) or
CuCl2 (75 µM). After incubation, the cells
were assayed for 125I-ASOR endocytosis, to measure ASGP
receptor activity, and Lucifer Yellow uptake, to measure fluid-phase
pinocytosis. As anticipated, zinc and copper ions inhibited
125I-ASOR total binding and uptake by up to 80% and 85%,
respectively (Fig. 10A).
Under these conditions, however, Lucifer Yellow uptake was largely
unaffected (Fig. 10B). These findings suggest strongly that
neither zinc nor copper ions interrupted clathrin-independent fluid
phase vesicular transport in hepatocytes.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of metal treatment on fluid-phase
uptake. Hepatocytes (4 × 106 cells/ml) in
BME-BSA were incubated in the absence ( ) or presence of
CuCl2 ( ) or ZnCl2 ( ) for 2 h at
37 °C. Cell samples were supplemented with 125I-ASOR (2 µg/ml) and Lucifer Yellow (0.2 mg/ml) and incubated an additional 60 min. The cells were chilled on ice and washed twice with cold BME-BSA
(10 min/wash). Cells were solubilized in HBS supplemented with 0.05%
(v/v) Triton X-100 and BSA (1 mg/ml) for 30 min at 4 °C, after which
insoluble debris was sedimented by centrifugation (13,000 × g, 4 °C, 15 min). Supernatants were assayed for
125I-ASOR (A) and Lucifer Yellow (B).
Symbols represent the mean of duplicate samples;
error bars reflect the standard deviations of
means.
|
|
Finally, we examined the effect of copper or zinc loading on
transferrin binding and endocytosis by hepatocytes. We found that
copper-treated hepatocytes showed no alteration in
125I-transferrin binding at 4 °C or
125I-transferrin binding and internalization at 37 °C
(Fig. 11B) in contrast to
125I-ASOR endocytosis, which was blocked by ~70% (Fig.
11A). Zinc treatment of hepatocytes, however, elicited a
50% reduction in surface 125I-transferrin binding at
4 °C and a 35% reduction in 125I-transferrin
internalization at 37 °C (Fig. 11B). We found that preincubation of 125I-transferrin with ZnCl2
did not alter its ability to bind to hepatocytes
subsequently.2 These data indicate, therefore, that zinc
treatment of hepatocytes partially disrupted their ability to
endocytose transferrin.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of metal treatment on hepatocyte
binding and uptake of transferrin. Cultured adult hepatocytes in
BME-BSA were incubated without or with ZnCl2 (225 µM) or CuCl2 (75 µM) for 2 h at 37 °C. Some cell samples were supplemented with either
125I-ASOR (2 µg/ml; A) or
125I-transferrin (2 µg/ml; B) and incubated at
37 °C for an additional 60 min, whereupon the cells were chilled on
ice and assayed for bound and internalized 125I-ASOR and
125I-transferrin. Other cell samples were chilled on ice
following metal treatment, washed, and assayed for surface binding of
125I-transferrin (B) at 4 °C. Values
represent the means of duplicate samples; error
bars reflect the standard deviations of the means.
|
|
 |
DISCUSSION |
Specificity of Copper and Zinc Inhibition of Endocytosis--
We
are interested in understanding the role metalloproteins play in
vesicular transport and the sensitivity of membrane dynamics toward
metal toxicity. In this study, we have examined the effects of zinc and
copper ion exposure on vesicular transport in isolated rat hepatocytes.
Several lines of evidence support the view that the effects of copper
and zinc were specific for receptor internalization and recycling and
not for vesicular transport in general. First, neither metal blocked
significantly the transport to and subsequent degradation of
internalized ASOR in lysosomes (Fig. 9), even though both copper and
zinc ions severely reduced continuous ASOR endocytosis (Figs. 1-3 and
11). These data suggest that copper and zinc ions under these
conditions do not block ASGP receptor/ASOR dissociation and segregation
for ASOR en route to lysosomes. Second, copper and zinc ions
did not alter appreciably fluid-phase pinocytosis of Lucifer Yellow by
hepatocytes under conditions where ASOR endocytosis was severely
reduced (Fig. 10). Hepatocytes internalize bulk fluid predominately by
a clathrin-independent pathway such that hyperosmotic disruption of
clathrin-dependent uptake of ASOR by ASGP receptors has
little or no effect on Lucifer Yellow uptake (19). Thus, the molecular
machinery that mediates vesicular budding for this clathrin-independent
pathway apparently is not susceptible to the effects of copper or zinc
ions. Third, transferrin endocytosis in copper-treated hepatocytes was
not reduced, as was ASOR endocytosis (Fig. 11). These data suggest that
the effects of copper ions on ASGP receptor-dependent
uptake were specific for this receptor and not inhibitory for
endocytosis in general. In contrast, zinc ions blocked or reduced
uptake of ASOR and transferrin by hepatocytes, suggesting the
possibility that a mechanism or structural motif necessary for the
proper functioning of these two receptors was sensitive to the effects
of zinc ions.
Although the effects of copper and zinc ion exposure were relatively
specific for ASGP receptor-mediated endocytosis, not all ASGP receptors
responded to metal treatment equally. In several experiments, we found
that only a portion of surface ASGP receptor activity was lost when
cells were exposed to copper or zinc ions (Figs. 5-8). In one
instance, metal-dependent reductions of surface 125I-ASOR binding were evident only when cells were exposed
to metal ions at or above 22 °C (Fig. 7C), and cells
treated with copper ions at temperatures below 33 °C frequently
expressed more surface ASGP receptor activity than did control cells
(Fig. 7C). Copper ion transport in rat cells is
temperature-dependent (20), and the amount of copper or
zinc ions accumulated by hepatocytes necessary to block endocytosis may
be temperature-dependent. Alternatively, it may be that
copper and zinc ions exert their effects only on ASGP receptors
actively moving along the endocytic pathway. Only a subset of ASGP
receptors, termed "state 2" ASGP receptors (21), recycle
constitutively. When isolated rat hepatocytes are incubated at
temperatures below 37 °C, the surface:intracellular ratio of state 2 ASPG receptors reversibly decreases (22). Accordingly, copper-treated
hepatocytes may have consistently expressed higher surface ASGP
receptor activity than did control cells (Fig. 7C) because
copper ions, by inhibiting ASGP receptor internalization (Fig. 5),
blocked the temperature-induced shift of receptors from the cell
surface to the cell interior. Even so, it is expected both "state
1" and state 2 receptor classes are sensitive to these metals under
conditions when both populations are mediating endocytosis of ligand.
Effect of Transition Metals on ASGP Receptor Activity--
Copper
and zinc ions induced cells to accumulate a portion of their ASGP
receptors in an inactive form (Fig. 6). Hepatocytes depleted of ATP or
treated with agents that alkalinize acidic endosomal compartments
(e.g. chloroquine, monensin) reversibly accumulate inactive
ASGP receptors with no loss of total detectable ASGP receptor protein
(22, 23). Weigel and colleagues (24-26) have found a direct
correlation between ASGP receptor ligand binding activity and receptor
palmitoylation (at Cys35 of RHL1) such that deacylated ASGP
receptor subunits do not bind ASOR. In addition, constitutive recycling
of ASGP receptors and receptor inactivation/reactivation are not
necessarily coupled, as monensin treatment (22) and copper treatment
(this report) induce the loss of ligand binding activity of ASGP
receptors at the cell surface with little or no loss of
immunodetectable receptor protein. Because copper treatment blocks ASGP
receptor internalization (Figs. 5 and 8), it is likely that inactive
ASGP receptors are accumulating on the surfaces of copper-treated
cells. On the other hand, zinc-treated hepatocytes lost both ASGP
receptor activity and immunodetectable RHL1 protein from the cell
surface (Figs. 5 and 6), and this lost surface ASGP receptor activity
was not recovered when permeable cells were assayed for
125I-ASOR binding (Fig. 6). It appears, therefore, that
zinc-treated cells accumulated inactive ASGP receptors intracellularly,
as zinc treatment alone did not block internalization of receptors from
the cell surface (Fig. 5).
Our previous observations, which showed a correlation between
iron-dependent inactivation of ASGP receptors and formation of disulfide-bonded RHL1 multimers, suggested the possibility that
iron-dependent oxidation of Cys35 could
preclude RHL1 acylation and thereby promote receptor inactivation (8,
9). We have found that hepatocytes treated with copper or zinc ions
lose
90% of reduced glutathione content,2 suggesting
that both metals induce a shift in the oxidative status of the cells.
Under these conditions, we frequently (but not always) detect RHL1
dimers formed to a limited extent in hepatocytes treated with copper
ions; we have never detected RHL1 multimers on zinc ion-treated
cells.2 Thus, copper and zinc ions may induce ASGP receptor
inactivation by disrupting receptor reacylation via a mechanism other
than oxidation of Cys35.
Nature of Copper- and Zinc-sensitive Sites--
The nature of
copper or zinc ion inhibition of ASGP receptor-mediated endocytosis is
unclear. Both Cu2+ and Zn2+ are borderline
metals and show increased binding strength to organic ligands compared
with class A metals (e.g. K+, Na+,
Mg2+, and Ca2+)(10). Zinc ions typically
function by conferring specific structures to proteins they bind
(e.g. zinc-finger proteins), whereas copper ions perform
redox reactions in copper ion-bound proteins (e.g. superoxide dismutase). One possible mode of endocytic interruption is
that OH· generated by copper ion-mediated Fenton reaction would
oxidatively damage lipids and proteins crucial for normal uptake and
recycling of ASGP receptors (1). Evidence arguing against this
possibility includes the following. (i) The copper ion inhibition of
ASOR binding and endocytosis were largely reversed following incubation of copper-treated cells in the absence of copper ions (Fig. 4). Protein
and lipid damage mediated by reactive oxygen species is essentially
irreversible with the exception of disulfide bond formation, which can
be reversed by glutathionered-mediated reduction (27). Our
results, however, do not rule out the possibility that some recovery
was due to de novo synthesis of ASGP receptors. (ii) Copper
ions did not alter significantly bulk-phase pinocytosis of Lucifer
Yellow or transferrin endocytosis by treated hepatocytes. Because
transferrin and ASOR are internalized by the same set of endocytic
machinery, inactivation or damage exerted on this machinery by reactive
oxygen species would block uptake of both ligands. (iii) We did not
observe significant diminishment of cell viability as a result of
copper ion treatment. (iv) Exposure of hepatocytes with up to 220 µM MnCl2 had no detectable effect on ASGP
receptor-mediated endocytosis. This finding is significant because
manganese ions have been shown to facilitate generation of reactive
oxygen species (27). Because zinc ions are not redox active and do not
mediate production of reactive oxygen species, these findings taken
together suggest that reactive oxygen species were not responsible for
the inhibitory effects of these metal ions on ASGP receptor-mediated endocytosis.
An alternate hypothesis is that copper and zinc ions bind ASGP
receptors and interfere directly with their function. RHL1 does not
contain canonical copper or zinc ion binding domains (e.g.
MXM, MXXM, CC, CXC, or
CXXC) present in metallothioneins (28) or FYVE-finger
proteins (29). Even so, copper and zinc ions likely coordinate with a
common set of histidyl and carboxyl groups on the RHL1 cytoplasmic
domain, some of which are located near the internalization signal YQDF
(amino acids 5-8) (30). Nonetheless, we observed significant
differences in the effects of the two metals. First, unlike copper
ions, zinc ions did not block internalization of receptor-ligand
complexes from the cell surface (Fig. 5). Second, ASOR binding to ASGP
receptors was itself partially altered by zinc ions but not by copper
ions (Table II). Regardless, the extent of zinc-dependent
inhibition of endocytosis and accumulation of inactive ASGP receptors
cannot be attributed solely to the direct inhibition of ASOR binding by
zinc ions (<30% reduction, Table II). Third, zinc ions, but not
copper ions blocked at least partially transferrin endocytosis (Fig.
11). Fourth, inhibition of ASOR endocytosis required higher zinc ion
concentrations compared with copper ions (Figs. 1, 5). Thus, copper and
zinc ions may bind to elements of the cytoplasmic tail of ASGP receptor
subunits which directly or indirectly interferes with the
internalization signal (copper ions) or the trafficking of ASGP
receptors (and transferrin receptors in the case of zinc ions) back to
the cell surface, thereby, diminishing sustained endocytosis.
Our findings here provide evidence that transition metal toxicity on
cells can be highly specific toward the function of membrane proteins.
We are currently investigating whether or not iron, copper, and zinc
interact with the cytoplasmic tail of RHL1 subunits, and if so,
determine the specific amino acids involved in that interaction.