From the Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
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ABSTRACT |
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We recently reported that megalin (gp330), an
endocytic receptor found on the apical surface of thyroid cells, binds
thyroglobulin (Tg) with high affinity in solid phase assays.
Megalin-bound Tg was releasable by heparin. Here we show that Fisher
rat thyroid (FRTL-5) cells, a differentiated rat thyroid cell line, can
bind and endocytose Tg via megalin. We first demonstrated that FRTL-5 cells express megalin in a thyroid-stimulating
hormone-dependent manner. Evidence of Tg binding to megalin
on FRTL-5 cells and on an immortalized rat renal proximal tubule cell
line (IRPT cells), was obtained by incubating the cells with
125I-Tg, followed by chemical cross-linking and
immunoprecipitation of 125I-Tg with antibodies against
megalin. To investigate cell binding further, we developed an assay in
which cells were incubated with unlabeled Tg at 4 °C, followed by
incubation with heparin, which released almost all of the cell-bound Tg
into the medium. In solid phase experiments designed to illuminate the
mechanism of heparin release, we demonstrated that Tg is a
heparin-binding protein, as are several megalin ligands. The amount of
Tg released by heparin from FRTL-5 and IRPT cells, measured by
enzyme-linked immunosorbent assay (ELISA), was markedly reduced by two
megalin competitors, receptor-associated protein (RAP) and 1H2
(monoclonal antibody against megalin), indicating that much of the Tg
released by heparin had been bound to megalin (~60-80%). The amount
inhibited by RAP was considered to represent specific binding to
megalin, which was saturable and of high affinity
(Kd~11.2 nM). Tg endocytosis by
FRTL-5 and IRPT cells was demonstrated in experiments in which cells
were incubated with unlabeled Tg at 37 °C, followed by heparin to
remove cell-bound Tg. The amount of Tg internalized (measured by ELISA
in the cell lysates) was reduced by RAP and 1H2, indicating that Tg
endocytosis is partially mediated by megalin.
Thyroglobulin (Tg)1 is
synthesized in thyrocytes and released into the follicle lumen, where
it is stored as the major component of the colloid (1, 2).
Post-transitional modifications of Tg that occur mainly at the
cell-colloid interface lead to forms that are iodine-rich and that
contain the thyroid hormones T4 and T3 (mature Tg). Hormone secretion
requires uptake of Tg by thyrocytes, with transport to lysosomes, where
proteolytic cleavage leads to release of the hormones from mature Tg
molecules (1). Internalization of Tg may result from pseudopod
ingestion under certain circumstances, such as intense, acute
stimulation by the thyrotropic hormone (TSH), but micropinocytosis
(vesicular internalization) is thought to be the usual route (1-3).
There is evidence that micropinocytosis of Tg can take place both by
nonselective fluid phase uptake and receptor-mediated endocytosis, but
the relative importance of these two mechanisms is uncertain (1).
Although evidence has been obtained of low affinity receptors for Tg on thyroid cells, a receptor capable of mediating Tg endocytosis has not
been fully characterized (1-12).
We have previously obtained evidence suggesting that megalin (gp330)
may function as a receptor for Tg (13). Megalin (gp330) is a member of
the LDL receptor family (14, 15) and has been shown to bind multiple,
unrelated ligands and to mediate endocytosis of ligands via coated
pits, leading to delivery of ligands to lysosomes, where degradation
occurs (16-18). In immunohistochemical studies, megalin has been found
principally on the apical surface of a restricted group of absorptive
epithelial cells, including renal proximal tubule cells, epididymal
cells, type II pneumocytes, and thyroid epithelial cells (19, 20).
Based on the assumption that physiological ligands of megalin may be
identified by consideration of the composition of fluids to which it is
exposed in various organs (21), we postulated that megalin on
thyrocytes serves as a receptor for Tg. In support of this possibility
we demonstrated in solid phase assays that purified rat megalin binds
to rat Tg with high affinity (13). Binding was inhibited by several
known competitors of megalin, including the receptor-associated protein (RAP), antibodies to megalin, and heparin, which in addition
dissociated Tg bound to megalin (13). In the present study we have
investigated FRTL-5 cells to determine whether they express megalin and
if they are capable of binding and internalizing Tg via megalin. FRTL-5
cells, an established rat thyroid cell line, exhibit a number of
thyroid-specific functions in a TSH-dependent manner (22,
23). Here we show that FRTL-5 cells do express megalin and that they
can bind and endocytose Tg via megalin.
Materials--
Tg was purified from rat thyroids by ammonium
sulfate precipitation and column fractionation, as described previously
(24). The Tg preparations were analyzed by Western blotting, using a rabbit anti-human Tg antibody cross-reactive with Tg from other species
(Axle-Westbury, NY). Two bands were seen at about 330 and 660 kDa,
corresponding to the monomeric and the dimeric forms of Tg. The RAP was
used in the form of a glutathione S-transferase (GST) fusion
protein. DH5
A rabbit antibody, designated A55, against immunoaffinity purified
megalin was described previously (28, 29). A previously described mouse
anti-megalin monoclonal antibody, designated 1H2, has been shown to
react with ectodomain epitopes in the second cluster of ligand binding
repeats (29). A rabbit antibody against RAP-GST and a mouse antibody
against the low density lipoprotein receptor-related protein (LRP) were
previously described (19). The above antibodies were used as purified
IgG preparations unless otherwise specified. A goat antibody against
GST was purchased from Amersham Pharmacia Biotech. Alkaline phosphatase
(ALP)-conjugated goat anti-rabbit IgG and horseradish
peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad.
ALP-conjugated mouse anti-goat IgG was obtained from Axle. Horseradish
peroxidase- and ALP-conjugated goat anti-mouse IgG were obtained from
Sigma. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG and goat anti-rabbit IgG were obtained from Cappel (Durham, NC).
Biotin-labeled goat anti-mouse IgG was obtained from Vector (Burlingame, CA).
Radiolabeling of Tg--
200 µg of rat Tg were radiolabeled
with 125I-Na (NEN Life Science Products) using IODO beads
(Pierce), according to the manufacturer's instructions. The specific
activity of the preparations ranged from 1,500 to 7,000 cpm/ng.
Cell Cultures--
Fisher rat thyroid cells (FRTL-5) (American
Type Culture Collection, Manassas, VA) were cultured as described
previously (22, 23) in Coon's F12 medium, supplemented with 5% fetal
calf serum and with a mixture of six hormones, including TSH (10 milliunits/ml). In some experiments the medium was replaced with fresh
medium lacking TSH for 24 h, after which TSH was sometimes
readded. An immortalized rat renal proximal tubule cell line (IRPT),
which expresses megalin but not LRP, was established as described
previously (30, 31). IRPT cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. CHO cells, a
Chinese hamster ovary cell line that lacks megalin and expresses LRP
(32), were cultured in Ham's medium supplemented with 10% fetal
bovine serum.
Evaluation of Megalin and LRP Expression by Flow Cytometry (FACS
Analysis)--
Cells were detached from the plates, washed with Tris
buffered-saline (TBS), and incubated in plastic tubes for 1 h at
4 °C with the mouse monoclonal anti-megalin antibody (1H2), with the rabbit polyclonal anti-megalin antibody (A55), with the anti-LRP antibody (tissue culture supernatant), or, as controls, with purified mouse or rabbit IgG. Anti-megalin antibodies and control IgGs were used
at a concentration of 20 µg/ml in binding buffer (TBS, 5 mM CaCl2, 0.5 mM MgCl2,
5% fetal bovine serum). After washing with TBS, FITC-conjugated rabbit
anti-mouse IgG (1:250) or goat anti-rabbit IgG (1:1500) secondary
antibodies were added for 1 h at 4 °C in binding buffer. Cells
were then washed and analyzed by flow cytometry using a FACSCAN from
Becton Dickinson (Mountain View, CA).
Detection of Megalin by Western Blotting--
Cell extracts were
prepared with 1% Triton X-100, 1% deoxycholate (both from Fisher) and
were briefly sonicated. The extracts were then subjected to
SDS-polyacrylamide gel electrophoresis under nonreducing conditions and
blotted onto nitrocellulose membranes, which were incubated with tissue
culture supernatant containing the mouse monoclonal anti-megalin
antibody (1H2) or with the rabbit polyclonal anti-megalin antibody
(A55), followed by horseradish peroxidase-conjugated goat anti-mouse or
anti-rabbit IgG secondary antibodies. Bands were detected using a
chemiluminescent substrate kit (Kirkegard & Perry Laboratories,
Gaithersburg, MD).
Evaluation of Megalin Expression by Immunoperoxidase
Staining--
Cells were cultured on glass coverslips and fixed with
2% paraformaldehyde-L-lysine-sodium periodate. The
preparations were blocked with avidin D (0.1 mg/ml in PBS) and
incubated with tissue culture supernatant containing the mouse
monoclonal anti-megalin antibody (1H2) followed by biotin-labeled goat
anti-mouse IgG secondary antibody (1:200 in PBS) or with the secondary
antibody alone. Staining was detected using the peroxidase labeled
biotin-avidin complex (Vector). 3,3'-Diaminobenzidine (Sigma) was used
for the color reaction.
Cross-linking Experiments--
Cells were incubated with
serum-free Dulbecco's modified Eagle's medium for 1 h at
37 °C and then blocked in 0.5% ovalbumin (OVA, Sigma), 25 mM Tris, 150 mM NaCl, 5 mM
CaCl2, 0.5 mM MgCl2, pH
8.0, for 1 h at 4 °C. Cells were then incubated
with 10 nM of 125I-Tg for 4 h at 4 °C
in blocking buffer, followed by cross-linking with the homobifunctional
cross-linker 3,3'-dithiobis[sulfosuccinimidyl propionate] (Pierce)
(0.5 mM in PBS) for 30 min at 4 °C. After washing with
TBS, cells were lysed with 1% Triton X-100, 1% deoxycholate and
briefly sonicated. Cell lysates (500 µg) were immunoprecipitated with
the rabbit anti-megalin antibody (A55 anti-serum, 1:200) or with the
rabbit anti-human Tg antibody (1:200), or, as control, with normal
rabbit serum, using protein A beads (Pierce). The precipitates were
separated by 4-16% SDS-polyacrylamide gel electrophoresis under
nonreducing conditions and analyzed by autoradiography. Radioactivity
in the cell lysates and in the precipitates was counted with a Release of Cell Bound Exogenous Tg by Heparin--
Cells were
cultured in 96-well tissue culture plates until 80-100% confluence
was reached. Cells were incubated with unlabeled Tg or, as controls,
with RAP-GST or GST in Coon's F12 medium containing 5 mM
CaCl2, 0.5 mM MgCl2, and 0.5% OVA.
For inhibition experiments, FRTL-5 and IRPT cells were incubated with
Tg alone, or in the presence of megalin inhibitors, namely RAP-GST (200 µg/ml) or 1H2 (200 µg/ml) or, as controls, with GST (200 µg/ml)
or normal mouse IgG (200 µg/ml). After 4 h of incubation at
4 °C, the cells were washed twice with ice-cold PBS to remove
nonspecifically bound proteins and then incubated for 1 h at
4 °C with ice-cold heparin (10-100 units/ml in PBS), to release
receptor-bound proteins from the cell surface. In certain experiments,
to measure total cell-bound Tg, cell lysates were prepared by treating
the cells with ice-cold H2O on ice, immediately after
incubation with Tg at 4 °C. The amounts of Tg, RAP-GST or GST in the
heparin wash or in the cell lysates were measured by ELISA. For this
purpose, 96-well microtiter plates were coated overnight at 4 °C
with the heparin wash or with the cell lysates, blocked with bovine
serum albumin (Sigma) and incubated with rabbit anti-human Tg (1:500), rabbit anti-RAP (20 µg/ml), or goat anti-GST (1:1000) antibodies, followed by ALP-conjugated goat anti-rabbit IgG (1:3000) or mouse anti-goat IgG (1:2500) secondary antibodies. After incubation with
p-nitrophenyl-phosphate (Sigma), absorbance at 405 nM was determined with an El-311 ELISA microplate reader.
The amount of Tg, RAP-GST, or GST was calculated using a standard
obtained by coating the wells with 10 µg/ml of purified Tg, RAP-GST,
or GST and was normalized for the total amount of protein in the cell
lysates, measured with a commercial kit (Bio-Rad).
Binding of Tg to Heparin in Solid Phase Assays--
96-well
microtiter plates were coated overnight at 4 °C with rat Tg or, as
control, with OVA, at a concentration of 100 µg/ml in PBS. After
blocking with bovine serum albumin, plates were incubated for 3 h
at room temperature with a biotin-labeled heparin-albumin complex
(Sigma) or, as control, with biotin-labeled albumin (Sigma) (in PBS,
0.05% Tween 20, 0.5% bovine serum albumin), followed by
ALP-conjugated streptavidin (Vector, 1:3000) and
p-nitrophenyl-phosphate. Absorbance was determined at 405 nM. For inhibition experiments, biotin-labeled
heparin-albumin was added to the wells alone or in the presence of
unlabeled heparin (500 units/ml).
Endocytosis Experiments--
Cells were seeded in 96-well tissue
culture plates and cultured until 80-100% confluence was reached.
Cells were then incubated at 37 °C with unlabeled rat Tg or, as
controls, with unlabeled RAP-GST or GST in Coon's F12 medium
containing 5 mM CaCl2, 0.5 mM
MgCl2, and 0.5% OVA. For inhibition experiments, Tg (10 µg/ml) was added to the plates together with one of the following:
RAP-GST (200 µg/ml), 1H2 (200 µg/ml), or, as controls, GST (200 µg/ml) or normal mouse IgG (200 µg/ml). After 6 h of
incubation the cells were washed twice with PBS and incubated with
ice-cold heparin, as described above, to remove cell surface bound
proteins, which were measured by ELISA. The cells were then lysed to
measure internalized Tg, RAP-GST, or GST, using deionized
H2O on ice. The amount of cell protein was measured in an
aliquot of the cell lysate. Internalized Tg, RAP-GST or GST were
measured in the cell lysates by ELISA, as described above.
Expression of Megalin by Cultured Cells--
As shown in Fig.
1A, FRTL-5 cells were found to
express megalin by FACS analysis, using either the mouse monoclonal
(1H2) or the rabbit polyclonal (A55) anti-megalin antibodies. In
addition, immunoperoxidase staining with 1H2 demonstrated surface
megalin on FRTL-5 cells (Fig. 1B). Megalin was also detected
in FRTL-5 cell extracts by Western blotting (Fig. 1C). In
agreement with previous reports (30, 31), IRPT cells were found to
express megalin by FACS analysis, whereas megalin was not detected on CHO cells (not shown). Because LRP binds many of the same ligands as
megalin, we studied its expression on FRTL-5 cells by FACS analysis
and, as shown in Fig. 1D, no LRP was found. In a previous study we showed that IRPT cells do not express surface LRP (30).
Expression of Megalin by FRTL-5 Cells and TSH Dependence--
To
investigate whether megalin expression by FRTL-5 cells is regulated by
TSH, cells were cultured for 24 h in medium lacking TSH, sometimes
followed by 48 h of culture in fresh medium containing TSH. As
shown in Fig. 2, megalin expression was
reduced at 24 h after TSH deprivation, as assessed by FACS
analysis. The staining for megalin ranged from 46 to 69% of the
staining before TSH deprivation, depending on the antibody used.
Readdition of TSH resulted in increased megalin expression, reaching
88-90% of the levels found before TSH deprivation. The results
indicate that TSH is required for high levels of megalin expression on
FRTL-5 cells.
Cross-linking and Immunoprecipitation of 125I-Tg with
Antibodies against Megalin--
In experiments designed to obtain
evidence of Tg binding to cell surface megalin, FRTL-5 and IRPT cells
were incubated with 125I-Tg at 4 °C, followed by
cross-linking and immunoprecipitation with anti-Tg or anti-megalin
antibodies. As shown in Fig.
3A, both anti-Tg and
anti-megalin antibodies produced a high molecular mass band at the same
size, indicating Tg bound to megalin on the cell surface. Another band
at approximately 50 kDa was produced by the anti-megalin antibody and
not by the anti-Tg antibody. The identity of this product is
unknown.
As shown in Fig. 3B, the proportion of 125I-Tg
precipitated by the rabbit anti-Tg antibody from FRTL-5 cells was
19.9% of the total amount of 125I-Tg in the cell lysates,
whereas that obtained from IRPT cells was 11.39%. The results are
consistent with the presence of Tg receptors on thyroid cells in
addition to megalin, as suggested by previous studies (1-12). The
proportion of 125I-Tg precipitated by the anti-megalin
antibody was 8.48% from FRTL-5 cells and 11.81% from IRPT cells.
Binding of Tg to Megalin on Cultured Cells, Assessed by Heparin
Release--
We previously showed that heparin dissociates Tg from
megalin in solid phase assays (13). Here we studied the ability of heparin to dissociate Tg bound to megalin on cells, to be used as a
measure of cell bound Tg. Cells were incubated with unlabeled Tg at
4 °C, followed by incubation with heparin. In addition, some cells
were incubated with RAP (used as a RAP-GST fusion protein), as a
positive megalin binding control, or with GST, as a negative control,
before heparin treatment. The amounts of Tg, RAP-GST, and GST in the
heparin wash were measured by ELISA.
As shown in Fig. 4A, heparin
was found to release Tg and RAP-GST from FRTL-5 and IRPT cells.
Although FRTL-5 cells are capable of synthesizing Tg (22, 23), the
amount of Tg released by heparin from FRTL-5 cells incubated in medium
lacking Tg was negligible. RAP-GST was also released from CHO cells,
which express LRP (32) but not megalin. The amount of Tg released by
heparin from CHO cells was negligible as compared with FRTL-5 and IRPT
cells (Fig. 4A). No release of GST was observed from FRTL-5,
IRPT, or CHO cells. The amount of Tg released from FRTL-5 cells by
heparin was dependent on its concentration, with linear increase up to 100 units/ml, the highest concentration used (not shown).
Inhibition experiments showed that much of the exogenous Tg released by
heparin from FRTL-5 and IRPT cells was bound to megalin on the cell
surface. Thus, when the cells were incubated with Tg plus either of two
megalin competitors, namely RAP-GST or 1H2 (monoclonal antibody against
megalin), the amount of Tg released by heparin was markedly reduced
(Fig. 4B). The mean inhibition produced by RAP-GST was 84%
in FRTL-5 cells and 78% in IRPT cells. The mean inhibition produced by
1H2 was 62% in FRTL-5 cells and 73% in IRPT cells. No reduction of
heparin-releasable Tg was produced by co-incubation of the cells with
Tg plus GST or mouse IgG, used as negative controls.
To measure the proportion of total cell surface-bound Tg that was
released by heparin, experiments were performed in which the amount of
Tg released by heparin was compared with the amount of Tg found in cell
lysates from FRTL-5 or IRPT cells that were not subjected to heparin
treatment but immediately lysed after 4 h of incubation with Tg at
4 °C. Because at this temperature no internalization occurs, the
amount of Tg found in the cell lysates represents only exogenous Tg
bound to the cell surface, minus a negligible amount (~1%)
contributed by endogenous Tg, as found in FRTL-5 cells not
incubated with exogenous Tg. This amount was similar to that
found in the heparin wash of FRTL-5 cells incubated with medium
lacking Tg.
As shown in Fig. 5A
(left bars), almost all (~97%) of the total cell-bound Tg
was released by heparin both from FRTL-5 and IRPT cells, indicating
that heparin-releasable Tg can be considered a measure of total cell
surface-bound Tg. Co-incubation of cells with Tg plus the megalin
competitors RAP-GST or 1H2 resulted in reduced amounts of total-cell
bound Tg (Fig. 5A, right bars), showing that much
of the Tg was bound to megalin. No reduction of total cell-bound Tg was
produced by co-incubation of the cells with Tg plus GST or mouse IgG,
used as negative controls. The percentage of inhibition of total
cell-bound Tg obtained with RAP-GST or 1H2 was similar to the
percentage of inhibition of heparin-releasable Tg produced by the same
competitors (Fig. 4B). Therefore, inhibition of
heparin-releasable Tg by megalin competitors can be considered as a
measure of megalin-bound Tg, which ranged from ~60 to ~80%,
depending on the cell type and on the competitor used.
As shown in Fig. 5B, when FRTL-5 cells were incubated with
increasing concentrations of unlabeled Tg, the total amount of cell
surface-bound Tg released by heparin was saturable, as is typical of
receptor binding. When cells were incubated with increasing concentrations of unlabeled Tg plus a constant concentration of RAP-GST
(200 µg/ml), inhibition of heparin-releasable bound Tg was obtained,
ranging from ~80% at low Tg concentrations to ~60% at high Tg
concentrations. The amount of Tg bound in the presence of RAP-GST was
considered to be unrelated to megalin, and this showed some degree of
saturation, suggesting the presence of Tg receptors, in addition to
megalin, on FRTL-5 cells. The amount of Tg binding that was inhibited
by RAP-GST (obtained by subtracting from the total amount of heparin
releasable Tg the amount released in the presence of RAP-GST) was
considered to be specific binding to megalin, and this was more highly
saturable. The equilibrium dissociation constant
(Kd) of Tg binding to FRTL-5 cells was calculated
according to the method of Furchgott (33), which is based on comparison
between the binding of an unlabeled protein to a receptor in the
presence or in the absence of a known competitor of the receptor, in
this case RAP-GST. The calculated Kd values in the
three experiments performed ranged from 7.8 to 14.3 nM
(mean 11.2 ± 3.0 nM), indicating a high affinity
interaction between megalin and Tg on FRTL-5 cells.
Binding of Heparin to Tg--
To help understand the mechanism by
which heparin competes with megalin for Tg binding, and in view of the
knowledge that megalin does not bind to heparin (26, 34), solid phase
binding assays were performed to determine whether Tg is a
heparin-binding protein. Tg-coated wells were incubated with
biotin-labeled heparin or, as a control, with biotin-labeled albumin.
As shown in Fig. 6, biotin-labeled
heparin bound to Tg-coated wells (Fig. 6, line 1) but not to
ovalbumin-coated wells (Fig. 6, line 4). No binding of
biotin-labeled albumin was observed (Fig. 6, line 3).
Binding of biotin-labeled heparin-albumin to Tg was saturable and was almost completely inhibited by co-incubation with unlabeled heparin (Fig. 6, line 2).
Uptake of Tg by Cultured Cells Is Partially Mediated by
Megalin--
Experiments were performed by incubating cells at
37 °C with unlabeled Tg, or as positive and negative controls with
RAP-GST or GST, followed by heparin treatment, to remove ligands bound to the cell surface. The amounts of Tg, RAP-GST, and GST in the cell
lysates were determined by ELISA as a measure of internalization. As
shown in Fig. 7A, FRTL-5 and
IRPT cells were found to internalize Tg and RAP-GST, whereas no uptake
of GST was observed. The amount of endogenous Tg in FRTL-5 cell
lysates, estimated from cells incubated in medium lacking Tg, was only
10-20% of that found after addition of 10 µg/ml of exogenous Tg. No
appreciable uptake of Tg or GST was observed by CHO cells, whereas
RAP-GST was internalized by these cells.
To investigate the role of megalin in Tg internalization, experiments
were performed by incubating FRTL-5 and IRPT cells with Tg alone or in
the presence of RAP-GST or 1H2. The results show that megalin is
partially responsible for Tg endocytosis. As shown in Fig.
7B, Tg uptake was reduced by RAP-GST (mean inhibition 46.5%
in FRTL-5 cells and 71.2% in IRPT cells) and by 1H2 (mean inhibition
50.3% in FRTL-5 cells and 65.1% in IRPT cells), whereas no
significant inhibition was obtained with GST or normal mouse IgG, used
as negative controls (Fig. 7B).
To obtain quantitative information about Tg uptake, the ratio between
the amount of internalized Tg, measured in the cell lysates, to the
amount of cell surface bound Tg, measured as the amount released by
heparin after incubation with Tg at 37 °C, was calculated. As shown
in Fig. 7C, this ratio was ~0.5 for both FRTL-5 and IRPT
cells, and it was reduced by co-incubation of the cells with Tg plus
RAP-GST or 1H2, confirming a role of megalin in uptake of cell surface
bound Tg.
In the present study we show that megalin is a receptor for Tg
endocytosis on cultured thyroid cells. We first demonstrated that
FRTL-5 cells, a well studied established rat thyroid cell line (22,
23), express megalin when cultured in standard medium containing the
thyrotropic hormone (TSH). As shown by FACS analysis and
immunoperoxidase staining, megalin was present on the cell surface.
FRTL-5 cells are known to maintain several specific thyroid functions,
most of which are TSH-dependent (22, 23). Therefore, the
demonstration that megalin expression by FRTL-5 cells is
TSH-dependent indirectly supports a thyroid-related
function of megalin. In particular, because TSH provides a signal for
Tg internalization (1-3), the finding suggests a role of megalin as an
endocytic receptor.
Direct evidence of binding of Tg to megalin on FRTL-5 cells as well as
on IRPT cells, a rat renal proximal tubule cell line that expresses
abundant megalin (30, 31), was provided by experiments in which cells
were incubated with 125I-labeled Tg followed by
cross-linking and incubation of the cell extracts with antibodies
against megalin, which resulted in co-immunoprecipitation of
125I-Tg. In other experiments, binding of Tg to megalin on
FRTL-5 and IRPT cells was demonstrated by incubation of cells with
unlabeled Tg at 4 °C, followed by treatment with heparin, which
released Tg into the medium, as detected by ELISA. Because almost all
(~97%) of the total cell-bound Tg was released by heparin, the
amount of heparin-releasable Tg can be considered as a measure of cell surface-bound Tg.
We previously showed in solid phase experiments that heparin
dissociates Tg from purified megalin (13). Goldstein and colleagues (27) had shown earlier that heparin releases the LDL from its receptor
on cultured fibroblasts and used this finding to measure the amount of
binding of LDL to the LDL receptor. However, it is known that heparin
can release molecules not only from members of the LDL receptor family
but also from heparan sulfate proteoglycans (35), which are expressed
in many cell types, including FRTL-5 cells (36). Nevertheless, in our
experiments with FRTL-5 cells we obtained compelling evidence that most
of the Tg released by heparin had been bound to megalin. Thus, when
cells were incubated with Tg plus the monoclonal anti-megalin antibody
1H2 or RAP, there was roughly a 60-80% reduction in heparin
releasable Tg as well as in total cell-bound Tg, which represents the
proportion of megalin-bound Tg. 1H2 is entirely specific for megalin
(29), and, although RAP binds to certain other members of the LDL
receptor family, notably LRP and the very low density lipoprotein
receptor (16, 19, 37-39), these receptors are not expressed by thyroid epithelial cells or by renal proximal tubule cells in vivo
(19, 40, 41). Furthermore, LRP is not expressed on FRTL-5 cells as
shown here, nor as previously reported on IRPT cells (30). In addition,
RAP does not bind appreciably to heparan sulfate proteoglycans nor
inhibit ligand binding to heparan sulfate proteoglycans (42). Thus, in
dealing with FRTL-5 or IRPT cells, inhibitory effects of RAP can be
considered specific for megalin. Based on this, we showed that Tg
binding to megalin on FRTL-5 cells is saturable and of high affinity
(Kd = ~11.2 nM). However, the finding
that binding not related to megalin (noninhibitable by RAP) also showed
some degree of saturation supports the existence, in addition to
megalin, of other Tg receptors on thyroid cells, as suggested in other
studies (1-12). This possibility is also supported by the finding that
in co-immunoprecipitation experiments a higher proportion of Tg was
precipitated by the anti-Tg antibody from FRTL-5 cells than from IRPT cells.
Based on the finding that heparin dissociates Tg from megalin, we
postulated that Tg is a heparin-binding protein, because megalin itself
does not bind to heparin (26, 34) and because several megalin ligands
are heparin-binding proteins (26). Indeed, this prediction was
confirmed by solid phase assays, which showed specific binding of Tg to
heparin. This observation suggests that regions rich in positively
charged amino acid residues (arginine and lysine) in the Tg molecule
may contribute to its binding to megalin, as has been demonstrated for
binding of certain other megalin ligands, including aprotinin and
polybasic drugs (43).
Experiments in which FRTL-5 and IRPT cells were incubated with
unlabeled Tg at 37 °C, followed by heparin treatment to remove cell-bound Tg, showed that megalin can mediate Tg endocytosis. The
detection of Tg in FRTL-5 cell lysates clearly showed that exogenous Tg
had been internalized, because Tg of endogenous origin was considerably
lower, as found in lysates from cells incubated in medium lacking Tg.
Furthermore, the demonstration that almost all of the cell
surface-bound Tg was released by heparin provides evidence that the
amount of Tg found in the cell lysates represented only Tg that had
been internalized. Moreover, because Tg was measured by ELISA, the
amount of Tg internalized may have been underestimated, because Tg
degradation during the course of the incubation should cause some loss
of immunoreactivity.
Inhibition experiments provided evidence that a certain amount of Tg
uptake is mediated by megalin. Thus, internalization of Tg by FRTL-5
and IRPT cells was appreciably reduced when cells were co-incubated
with exogenous Tg plus RAP or 1H2. Furthermore, we obtained evidence
against the possibility that reduction of Tg uptake in FRTL-5 and IRPT
cells by megalin competitors resulted merely from lowering the amount
of Tg bound to the cells. Thus, the ratio of internalized Tg to cell
surface bound Tg was reduced by RAP and 1H2.
The inhibition of Tg uptake produced by megalin competitors in FRTL-5
cells was not complete (~50%), suggesting that, in addition to
megalin, other mechanisms are responsible for Tg endocytosis, as
suggested by previous studies (1-12, 44). The finding that less
inhibition of uptake was produced by megalin competitors in FRTL-5
cells than in IRPT cells (~70%) suggests that the contribution of
megalin to Tg uptake is greater in IRPT cells.
As noted earlier, there is evidence that micropinocytosis of Tg by
thyrocytes occurs both through fluid phase uptake and receptor-mediated endocytosis (1-12). However, despite extensive investigations, a
specific receptor shown to have a major role in Tg uptake by thyrocytes
has not previously been characterized. Consiglio et al. (4,
5) showed the existence of a specific binding site for asialogalacto-Tg
in thyroid membrane preparations as well as in cultured thyrocytes, and
this was confirmed by others (6). The receptor was identified in
porcine thyroid membranes as a 45-kDa protein, which was suggested to
be involved mainly in Tg recycling (7). More recently, Lemansky and
Herzog (11) performed a study using porcine thyroid follicles, designed
to investigate the role of mannose-6-phosphate receptors in Tg
endocytosis through interaction with mannose-6 recognition markers on
Tg N-linked glycans. Although they failed to show that
mannose-6-phosphate receptors are responsible for Tg endocytosis, they
did obtain evidence of specific low affinity binding sites on the
apical surface of thyrocytes involved in Tg endocytosis. However, the responsible receptor was not identified. In another study, Giraud et al. (10) obtained evidence of selective, moderately high affinity binding of Tg to thyrocytes in cultured "inside-out" porcine follicles, as well as to cultured CHO and Madin-Darby canine
kidney cells from other tissues. The receptor was not isolated, but its
pH dependence, its presence on CHO and Madin-Darby canine kidney cells,
and its apparent recognition of anionic charges indicate that it is not
megalin. One reason why megalin has not been found in thyroid cells in
previous studies may be related to the fact that in most of those
studies primary cultures of thyroid cells or tissues were used. In this
regard, we have observed that primary cultures of rat renal proximal
tubule cells cease to express megalin after several days (30).
Furthermore, we have found that primary cultures of porcine thyroid
cells or follicles cease to express megalin after a few days, although
megalin is normally expressed on pig thyroid
cells.2
In summary, evidence obtained in our previous (13) and present studies
supports the conclusion that megalin can function as a receptor on
thyrocytes capable of mediating binding and uptake of Tg. Receptors
other than megalin probably contribute to this process and fluid phase
pinocytosis may play a major role (1-12, 44). In any case, the ability
of megalin to bind Tg with high affinity raises interesting questions
about its function in vivo. High affinity receptors serve to
mediate endocytosis of ligands that are generally present in low
concentrations in extracellular fluids and thus serve to compete
effectively with fluid phase uptake. However, Tg in the colloid is very
highly concentrated (100-200 mg/ml), which is consistent with the
notion that nonspecific fluid phase uptake is the major mechanism for
Tg endocytosis and hormone release (1). The function of a high affinity
receptor for Tg on thyroid cells may therefore be to regulate the
extent of endocytosis only under special circumstances. High affinity receptor binding should lead to an increase of Tg endocytosis, which
would be expected to result in delivery to lysosomes, with hormone
release. However, it is also possible that the receptor could divert Tg
from the usual endocytic pathway, as through recycling or transcytosis.
Further studies are needed to define the role of megalin in thyroid
hormone release.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
bacteria harboring the pGEX-RAP expression construct
were kindly provided by Dr. Joachim Herz (University of Texas Southern
Medical Center, Dallas, TX). The production of RAP-GST and GST was
performed as described (25). Heparin (Sigma) was used because it
inhibits Tg binding to megalin in solid phase assays (13), as occurs
for binding of other megalin ligands (26). Furthermore, heparin
dissociates Tg bound to megalin (13) and releases ligands that are
bound to members of the LDL receptor family (27).
counter.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, detection of megalin on FRTL-5 cells
by FACS analysis. Cells were detached from the plates and incubated at
4 °C with the rabbit (A55) or with the mouse (1H2) anti-megalin
antibodies, followed by secondary antibodies (FITC-conjugated goat
anti-rabbit or anti-mouse IgG). Control, mouse IgG. The
results are representative of one of three experiments performed.
B, detection of megalin on FRTL-5 cells by immunoperoxidase
staining. Cells were cultured on glass coverslips, fixed and blocked
with avidin D, and then incubated with 1H2 followed by a biotin-labeled
goat anti-mouse IgG secondary antibody, or with the secondary antibody
alone (Control). Staining was detected by the
peroxidase-labeled biotin-avidin complex. Surface staining is produced
by 1H2 on most cells and some are intensely stained, with a punctate
pattern. X100. C, expression of megalin by FRTL-5 cells,
detected by Western blotting. Cell extracts were subjected to
SDS-polyacrylamide gel electrophoresis under nonreducing conditions and
blotted onto nitrocellulose membranes, which were incubated with 1H2,
followed by horseradish peroxidase-conjugated goat anti-mouse IgG
secondary antibody. The arrow indicates the only band
observed, which corresponded to megalin. D, absence of LRP
on FRTL-5 cells, assessed by FACS analysis. Cells were incubated with
the anti-LRP antibody or, as a control, with purified mouse IgG,
followed by FITC-conjugated anti-mouse IgG secondary antibody.
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Fig. 2.
TSH-dependent expression of
megalin by FRTL-5 cells. Cells were incubated with the rabbit
polyclonal anti-megalin antibody (A55) or with the mouse monoclonal
anti-megalin antibody (1H2) followed by FITC-conjugated secondary
antibodies. 1, cells cultured in medium containing TSH (10 milliunits/ml). 2, cells cultured for 24 h in medium
lacking TSH. 3, cells cultured in medium without TSH for
24 h followed by 48 h in fresh medium with TSH. Values are
expressed as the mean fluorescence index (MFI) ± S.E.
obtained in three experiments.
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Fig. 3.
Cross-linking and co-immunoprecipitation of
125I-Tg with megalin on FRTL-5 and IRPT cells. Cells
were incubated with 125I-Tg at 4 °C, followed by
cross-linking and lysis. The cell lysates were subjected to
immunoprecipitation with the rabbit anti-megalin antibody (A55) or with
the anti-human Tg antibody. A, SDS-polyacrylamide gel
electrophoresis and autoradiography of immunoprecipitates from FRTL-5
cells. Both A55 and anti-Tg antibodies precipitated a very high
molecular mass material. The figure is representative of one of three
experiments performed. B, proportion of 125I-Tg
precipitated by anti-Tg or A55 antibodies from FRTL-5 or IRPT cells.
Radioactivity in the cell lysates and in the precipitates was measured
with a counter, and the percentage of 125I-Tg
precipitated was calculated.
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Fig. 4.
A, heparin-release of exogenous Tg and
RAP-GST from FRTL-5, IRPT, and CHO cells. Cells were incubated with
unlabeled Tg or RAP-GST or, as a control, with GST for 4 h at
4 °C at a concentration of 10 µg/ml. Heparin (100 units/ml) was
then added to release ligands bound to the cell surface. The released
Tg or RAP-GST were measured by ELISA in the heparin wash. Values were
normalized for the amount of protein in the cell lysates. The amount of
Tg in the heparin wash of FRTL-5 cells incubated in medium lacking Tg
was subtracted, as background. Results are expressed as the means ± S.E. obtained in three experiments. B, inhibition of Tg
release from FRTL-5 and IRPT cells by megalin competitors. Cells were
incubated at 4 °C with Tg alone or in the presence of RAP-GST or
mouse monoclonal anti-megalin antibody (1H2). GST and normal mouse IgG
were used as controls. Results are expressed as the mean percentage of
inhibition ± S.E. obtained in three experiments.
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Fig. 5.
A, comparison of total cell
surface-bound Tg with heparin-releasable Tg from FRTL-5 and IRPT cells
and proportion of megalin-bound Tg. Cells were incubated for 4 h
at 4 °C, with unlabeled Tg (10 µg/ml), alone or in the presence of
RAP-GST or mouse monoclonal anti-megalin antibody (1H2). GST and normal
mouse IgG were used as controls. Total cell-bound Tg was measured by
ELISA in cell lysates obtained by treating the cells with ice-cold
H2O immediately after incubation with Tg at 4 °C. Tg
released by heparin was measured in the heparin wash after treating the
cells with heparin (100 units/ml). Values were normalized for the
amount of protein in the cell lysates. Similar amounts of Tg were found
in the cell lysate and in the heparin wash of FRTL-5 cells incubated
with medium lacking Tg, which were negligible and were subtracted as
background. Results are expressed as the means + S.E. obtained in three
experiments. B, saturation of Tg release by heparin from
FRTL-5 cells. Cells were incubated with Tg at various concentrations,
alone or in the presence of RAP-GST, followed by heparin (100 units/ml). The amount of Tg released by heparin that was inhibited by
RAP-GST was considered as a measure of specific binding of Tg to
megalin and was calculated by subtracting from total Tg binding in the
absence of RAP-GST the Tg binding obtained in the presence of RAP-GST.
The amount of Tg in the heparin wash of FRTL-5 cells incubated in
medium lacking Tg was subtracted, as background. The figure is
representative of one of three experiments performed.
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Fig. 6.
Binding of heparin to Tg. 96-well
microtiter plates were coated overnight at 4 °C with purified rat Tg
or, as a control, with OVA and incubated with biotin-labeled heparin
or, as a control, with biotin-labeled albumin, followed by
ALP-conjugated streptavidin. Absorbance (OD) was determined
at 405 nM. 1, binding of biotin-labeled heparin
to Tg-coated wells. 2, binding of biotin-labeled heparin to
Tg-coated wells in the presence of 500 units/ml of unlabeled heparin.
3, binding of biotin-labeled albumin to Tg-coated wells.
4, binding of biotin-labeled heparin to OVA-coated wells.
The figure is representative of one of three experiments
performed.
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Fig. 7.
A, uptake of Tg and RAP-GST by FRTL-5,
IRPT, and CHO cells. Cells were incubated with unlabeled Tg or RAP-GST
or, as a control, with GST at a concentration of 10 µg/ml for 6 h at 37 °C. Heparin (100 units/ml) was then added to remove ligands
bound to the cell surface. Tg or RAP-GST internalized by the cells were
measured in the cell lysates by ELISA. Values were normalized for the
amount of protein in the cell lysates. The amount of Tg in cell lysates
from FRTL-5 cells incubated in medium lacking Tg was subtracted, as
background. Results are expressed as the means ± S.E. obtained in
three experiments. B, inhibition of Tg uptake by megalin
inhibitors. FRTL-5 and IRPT cells were incubated with 10 µg/ml of
unlabeled Tg, alone or in the presence of RAP-GST or mouse monoclonal
anti-megalin antibody (1H2). GST and normal mouse IgG (MIgG) were used
as controls. Results are expressed as the mean percentages of
inhibition ± S.E. obtained in three experiments. C,
ratio between uptake and binding of Tg. Cells were incubated at
37 °C with unlabeled Tg, alone or in the presence of RAP-GST or 1H2,
followed by heparin treatment. Bound Tg was measured in the heparin
wash, and internalized Tg was measured in the cell lysates. Results are
expressed as the means ± S.E. obtained in three
experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Ivan Stamenkovic and Dr. David Andrews for their critical reading of the manuscript and for helpful discussions.
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FOOTNOTES |
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* This work was supported by NIDDK, National Institutes of Health Grant 46301.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Scholar of The Department of Endocrinology, University of Pisa, Italy.
§ To whom correspondence should be addressed: Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-5690; Fax: 617-726-5684; E-mail: mccluskey.robert{at}mgh.harvard.edu.
2 M. Marinò, G. Zheng, and R. T. McCluskey, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Tg, thyroglobulin; LDL, low density lipoprotein; TSH, thyroid-stimulating hormone; RAP, receptor-associated protein; GST, glutathione S-transferase; FRTL-5 cells, Fisher rat thyroid cells; CHO cells, Chinese hamster ovary cells; IRPT cells, immortalized rat proximal tubule cells; OVA, ovalbumin; LRP, low density lipoprotein receptor-related protein; ELISA, enzyme-linked immunosorbent assay; ALP, alkaline phosphatase; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; TBS, Tris-buffered saline.
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