From the Department of Cell and Developmental
Biology, Oregon Health Sciences University, Portland, Oregon
97201-3098 and ¶ Progenitor Inc.,
Menlo Park, California 94025
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ABSTRACT |
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The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. It was recently found to coprecipitate with the transferrin receptor (Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1472-1477; Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., Fleming, R.E., and Sly, W. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13198-13202) and to decrease the affinity of transferrin for the transferrin receptor (Feder et al.). In this study, HeLa cells were transfected with HFE under the control of the tetracycline-repressible promoter. We demonstrate that HFE and the transferrin receptor are capable of associating with each other within 30 min of their synthesis with pulse-chase experiments. HFE and the transferrin receptor co-immunoprecipitate throughout the biosynthetic pathway. Excess HFE is rapidly degraded, whereas the HFE-transferrin receptor complex is stable. Immunofluorescence experiments indicate that they also endocytose into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking. Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These results implicate HFE further in the modulation of iron levels in the cell.
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INTRODUCTION |
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Hereditary hemochromatosis is an autosomal recessive disease more common in people of Northern European descent than cystic fibrosis, phenylketonuria, and muscular dystrophy combined (1). It is characterized by massive iron overload of the parenchymal tissue of many organs, including the liver, pancreas, heart, and pituitary. Siderosis and cirrhosis of these organs cause liver failure, diabetes mellitus, cardiac arrhythmia, hypogonadism, and other complications leading to premature death (2-4). Over 80% of hereditary hemochromatosis patients have the same mutation in the HFE gene (5).
Positional cloning of HFE from the HLA-linked region of chromosome 6p and its subsequent crystallization (6) have revealed its significant sequence and structural homology (37% identity in the ectodomain) to MHC1 class I proteins. MHC class I molecules are normally involved in antigen peptide presentation. HFE has not been found to bind such peptides. Its crystal structure suggests that the ancestral peptide-binding groove is too narrow for such a function (6). Emerging results suggest that the functional role of HFE may lie in its interaction with the transferrin receptor (TfR) (6-8). HFE is therefore described as a nonclassical MHC class I molecule. MHC class I molecules are capable of functions outside of antigen presentation. Some have been shown to interact with receptors such as the insulin receptor and the epidermal growth factor receptor (9-13). Antibodies against these MHC class I proteins decrease surface binding of insulin or the epidermal growth factor to their respective receptors, suggesting that these MHC class I proteins are involved in the formation of high affinity ligand-binding sites. The neonatal Fc receptor is a nonclassical MHC class I molecule that acts as an antibody transporter. The current model of neonatal Fc receptor function proposes binding of ligand at acidic pH and transport in acidic vesicles through the epithelial monolayer. Further evidence suggests the involvement of the neonatal Fc receptor in IgG homeostasis in the serum (for review, see Ref. 14). Likewise, HFE may be involved in regulated transport of molecules associated with iron homeostasis.
The molecular evidence concerning HFE mediation of iron homeostasis has been converging on its close association with TFR. Northern blot analysis indicated that HFE is widely distributed throughout the body, with more abundant message levels in organs that are major sites of iron metabolism, namely the liver and intestine (5). Its unique pattern of protein expression in the gastrointestinal tract (specifically in the crypt cells of the ileum, the putative site of iron absorption in the intestine (15)) suggests a role for HFE in the sensing of bodily iron levels and the regulation of transport of iron across epithelial layers. HFE expression in the basolateral membrane of the intestinal epithelia and the identification of HFE in the placenta (7) are indicative of a more general association with TfR in regulating iron transport across epithelial barriers. It has long been suspected that individuals with hereditary hemochromatosis have a defect in the transport of iron across the mucosal barrier, i.e. transport into the blood rather than uptake from the lumen (4). The perinuclear staining of HFE in proliferating crypt cells is indicative of co-localization with recycling receptors, such as TfR (16), rather than with the hypothesized iron transporter on the apical surface of these cells. The regulatory influence of HFE on iron transport may therefore be either in internal cellular compartments or at the basolateral cell surface.
More specific evidence for the interaction of HFE with TfR is demonstrated by the co-immunoprecipitation of HFE with TfR (7, 8) and more significantly by the ability of HFE to increase the dissociation constant of transferrin (Tf) with TfR (6, 8). The association of HFE with TfR and its yet to be defined role in iron regulation led us to investigate more closely its trafficking in the cell. For this study, we have expressed a FLAG epitope-tagged HFE (fHFE) under the control of the tetracycline-repressible promoter. This system allows us to tightly regulate the expression level of fHFE within the same stable cell line. The results of this study demonstrate that cells expressing HFE have decreased ferritin levels and increased TfR number, implying low intracellular iron levels. We also demonstrate that HFE is able to associate with TfR within 30 min of its synthesis and can associate with TfR in both the biosynthetic and metabolic pathways. HFE pools that are not associated with TfR are rapidly degraded. Immunofluorescent detection of fHFE in HeLa cells indicates that it co-localizes with TfR and Tf on the cell surface and in intracellular compartments. Localization of HFE on the cell surface and in endocytic compartments implicates HFE in the regulation of iron uptake, especially in light of indirect evidence suggesting that cells expressing HFE have decreased intracellular iron stores.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- The plasmid pcDNA3.1+ containing HFE cDNA clone 24 fused to the octapeptide FLAG sequence (Progenitor Inc.) has been previously described (17). The tetracycline-repressible plasmid pUHD10-3 was a kind gift from Drs. M. Gossen and H. Bujard (Zentrum fur Molekulare Biologies, Universitat Heidelberg) (18).
Subcloning-- The HFE coding sequence is contained within an ~3.0-kilobase NheI/XbaI fragment, which was subsequently cloned into the XbaI site of pUHD10-3, resulting in the fWTHFE/pUHD10-3 construct.
Cell Culture--
HeLa cells transfected with the
tetracycline-transactivable plasmid pUHD15-1 were a gift from Dr.
Sandra L. Schmid (Scripps Research Institute). They were cotransfected
with the fWTHFE/pUHD10-3 plasmid and the pBSpac plasmid containing the
puromycin resistance gene (12) using LipofectAMINE (Life Technologies,
Inc.) according to the manufacturer's directions. Colonies selected
with puromycin (200 ng/ml) and recloned to ensure a pure cell line were
screened by SDS-PAGE and Western blotting for expression of HFE using
the 1868 rabbit anti-FLAG (19) and CT1 rabbit anti-HFE (17) antibodies. The clone used for these experiments is named fWTHFE/tTA HeLa. The
resulting fWTHFE/tTA HeLa cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 400 µg/ml
G418 (Geneticin, Calbiochem), and 200 ng/ml puromycin, with
(tet+) or without (tet) 2 µg/ml
tetracycline.
Iodination-- Human holotransferrin was labeled with Na125I using lactoperoxidase and used for Scatchard analysis as described previously (19).
Immunoprecipitation-- fWTHFE/tTA HeLa cells were washed three times with 2 ml of PBS (pH 7.4) and lysed with NET-Triton (150 mM NaCl, 5 mM EDTA, and 10 mM Tris (pH 7.4) with 1% Triton X-100). Cell lysates were preadsorbed for at least 45 min at 4 °C with 50 µl of Pansorbin (Calbiochem) per ~106 cells to reduce precipitation of nonspecific protein. Preadsorbed lysates were incubated for at least 45 min at 4 °C with 30 µl of rabbit anti-mouse antibody (Miles Scientific)-coated Pansorbin and 2 µg of either mouse anti-FLAG antibody (M2, Eastman Kodak Co.) or mouse anti-transferrin receptor antibody (4091 and 4093, gifts from Vonnie Landt, Washington University, St. Louis, MO) or with 50 µl of Pansorbin and 3 µl of sheep anti-hTfR serum per ~106 cells. The Pansorbin pellet was resuspended in 200 µl of NET/Triton and washed through 1 ml of radioimmune precipitation assay buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM Tris, 150 mM NaCl, and 0.2% sodium azide) with 15% sucrose. Samples were eluted in 100 µl of 2× Laemmli buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol) (20) with 10% 2-mercaptoethanol and subjected to SDS-PAGE analysis on a 10% denaturing acrylamide gel.
Pulse-Chase Experiments--
Uninduced (tet+) and
induced (tet) subconfluent fWTHFE/tTA HeLa cells in 35-mm
dishes were washed three times with sterile PBS (pH 7.4) and
preincubated for 15 min at 37 °C in RPMI 1640 medium minus
methionine (RPMI
Met medium; Life Technologies, Inc.) prior to
labeling. Cells were pulsed for 10 min at various time points with 100 µCi or incubated overnight with 10 µCi of
[35S]methionine in RPMI
Met medium with 10% fetal
bovine serum. The cells were then washed three times with PBS and
chased at 30-min intervals between 0 and 120 min with complete medium.
At the indicated times, cells were lysed for 5 min on ice in
NET-Triton. Cell extracts were subjected to immunoprecipitation with M2
or 4093 antibody as described above and analyzed by SDS-PAGE on a 10%
denaturing acrylamide gel under reducing conditions. Gels were fixed,
treated with Amplify (Amersham Pharmacia Biotech) for 30 min, dried,
and subjected to PhosphorImager analysis (Molecular Dynamics,
Inc.).
Endo--N-acetylglucosaminidase H
Treatment--
Immunoprecipitated pellets were resuspended in 90 µl
of denaturing buffer (0.5% SDS and 1% 2-mercaptoethanol) and boiled
for 10 min at 95 °C. The samples were iced, and 10 µl of 10×
citrate buffer (500 mM sodium citrate (pH 5.5)) was added
to the pellet. Samples were incubated for at least 2 h at 37 °C
in the presence or absence of
endo-
-N-acetylglucosaminidase H (endo H; 8 units; New
England Biolabs Inc.). Samples were eluted with 50 µl of 2× Laemmli
buffer with 10% 2-mercaptoethanol and subjected to SDS-PAGE analysis
on a 10% denaturing acrylamide gel. Gels were fixed, treated with
Amplify, dried, and subjected to PhosphorImager analysis.
Half-life Experiments--
Subconfluent fWTHFE/tTA HeLa cells
(tet+ and tet) in 35-mm dishes were washed
three times with sterile PBS (pH 7.4) and preincubated for 15 min at
37 °C in RPMI
Met medium prior to labeling. Cells were pulsed for
1 h at various time points with 100 µCi of
[35S]methionine in RPMI
Met medium with 10% fetal
bovine serum. The cells were washed three times with PBS and chased in
2-h increments from 0 to 8 h with complete medium. At the
completion of the chase time point, all cells were lysed for 5 min on
ice in NET-Triton. Cell extracts were subjected to immunoprecipitation
with antibody M2 to immunoprecipitate HFE or with antibody 4091 to
immunoprecipitate TfR as described above and analyzed by SDS-PAGE on a
10% denaturing acrylamide gel under reducing conditions. Gels were
fixed, treated with Amplify, dried, and subjected to PhosphorImager
analysis.
Western Immunodetection-- Cell extracts from ~3 × 105 cells were diluted with 4× Laemmli buffer, or immunoprecipitates were eluted with 2× Laemmli buffer and subjected to electrophoresis on SDS-10 or 12% polyacrylamide gels under reducing conditions. The proteins were transferred to nitrocellulose. Immunoblot analysis was performed using sheep anti-hTfR serum (1:10,000 dilution) (19), mouse anti-FLAG antibody M2 (1:20,00 dilution), and/or sheep anti-human ferritin antibody (1:100 dilution; The Binding Site, Ltd.) followed by the appropriate secondary antibody conjugated to horseradish peroxidase and chemiluminescence (SuperSignal, Pierce) per the manufacturer's directions.
Tunicamycin Treatments--
Subconfluent fWTHFE/tTA HeLa cells
(tet+ and tet) in 35-mm dishes were washed
three times with sterile PBS (pH 7.4) and preincubated for 1 h at
37 °C with 5 µg/ml tunicamycin (Calbiochem) or mock-treated with
Me2SO (final concentration of 0.05%). Cells were washed
three times with sterile PBS (pH 7.4) and incubated overnight at
37 °C with complete medium. The next day, the cells were lysed for 5 min on ice in NET-Triton and subjected to immunoprecipitation with
antibody M2 or sheep anti-hTfR serum as described above. Immunoprecipitates were eluted with 100 µl of 2× Laemmli buffer with
10% 2-mercaptoethanol and subjected to SDS-PAGE on a 10% denaturing
acrylamide gel. Gels were subjected to Western blotting with monoclonal
anti-transferrin receptor antibody (H68.4, Zymed Laboratories, Inc.) as described (19).
Tf-Agarose Precipitation--
Lysates of 106
tet+ or tet fWTHFE/tTA HeLa cells were
preadsorbed for 1 h at 4 °C with 100 µl of bovine serum
albumin covalently linked to agarose (50% suspension in PBS).
Supernatants were incubated for 1 h at 4 °C with 200 µl of
transferrin covalently linked to agarose (50% suspension in PBS). The
pellet was washed two times with NET-Triton and eluted twice with 75 µl of 2× Laemmli buffer with 10% 2-mercaptoethanol and subjected to
SDS-PAGE on a 12% denaturing acrylamide gel as described above. Gels
were transferred to nitrocellulose and subjected to Western blotting as
described above.
PhosphorImager Quantitation-- IP Lab Gel 1.5 (Molecular Dynamics, Inc.) was used to quantitate images by determining the volume within a region of fixed pixel number at each band of interest within the gel. Bands were normalized for methionine numbers within the protein (10 for HFE and 14 for the TfR monomer).
Immunocytochemistry--
Subconfluent fWTHFE/tTA HeLa cells
(tet+ and tet) grown on coverslips were
washed three times with 2 ml of PBS (pH 7.4) and, if applicable,
incubated for 30 min at 37 °C with 4 µg/ml Texas Red-labeled Tf
(Molecular Probes, Inc.) in Dulbecco's modified Eagle's medium with
20 mM HEPES (pH 7.4) and 2 mg/ml ovalbumin. All cells were
washed three times with 2 ml of PBS, fixed for 15 min in 3%
paraformaldehyde at room temperature, and washed an additional two
times with PBS. Cells were blocked in 10% newborn calf serum in PBS
with 0.5% Triton X-100 to permeabilize cells for at least 30 min at
room temperature. The fixed cells were incubated for at least 1 h
at room temperature in 1:50 diluted sheep anti-hTfR serum, 100 µg/ml
anti-TfR antibody 4093, 25 µg/ml anti-FLAG antibody M2, 30 µg/ml
anti-HFE antibody CT1, or 2 µg/ml anti-
2-microglobulin
antibody (Immunotech International). Coverslips were then washed twice
with PBS and incubated again for at least 1 h at room temperature
with a 1:50 dilution of fluorescein isothiocyanate (FITC)-labeled swine
anti-goat antibody (Tago, Inc.), FITC-labeled goat anti-mouse antibody
(Tago, Inc.), or tetramethylrhodamine isothiocyanate-labeled
anti-rabbit antibody (Cappel).
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RESULTS |
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Identification of a Clone That Expresses Epitope-tagged HFE under
Control of the Tetracycline-regulated Promoter--
The
tetracycline-responsive promoter system developed by Gossen and Bujard
(18) was used to create a cell line in which HFE expression could be
tightly controlled. tTA HeLa cells stably express a
tetracycline-transactivable (tTA) fusion protein with the
Escherichia coli tetracycline-responsive element
(tetR)-binding domain and the VP16 activation domain from herpes
simplex virus. In the absence of tetracycline, the fusion protein binds
operator sequences upstream of the fWTHFE/pUHD10-3 multiple cloning
region, promoting expression of fHFE. In the presence of
tetracycline-supplemented (tet+) medium, the tTA fusion
protein binds tetracycline and releases from the
tetracycline-responsive promoter, preventing fHFE transcription. Fig.
1 shows fHFE, TfR, and ferritin (Ft)
expression in fWTHFE/tTA HeLa cells that have been uninduced (tet +) or
induced (tet) for at least 10 days. Western blotting of
lysates of 3 × 105 cells detected no fHFE (~43 kDa)
in tet+ cells with anti-FLAG antibody M2. fHFE was easily
detected in lysates of tet
cells. TfR (94 kDa) levels
increased slightly (~50%) in cells expressing fHFE. A large decrease
(>10-fold) in the levels of Ft (19 and 21 kDa) was seen in cells
expressing fHFE. These results imply that overexpression of HFE reduces
intracellular iron load. The iron regulatory protein modulates changes
in TfR and Ft levels. At low intracellular iron concentrations, the
iron regulatory protein binds to the iron response element stem loop
structure in the 3'-untranslated portion of the TfR mRNA,
stabilizing the message and thus increasing TfR numbers. The same
reduction in intracellular iron load results in the binding of the iron
regulatory protein to the 5'-untranslated stem loop of Ft mRNA,
blocking translation and thus decreasing Ft levels.
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HFE Lowers the Affinity of TfR for Tf in fWTHFE/tTA HeLa
Cells--
Scatchard analysis of 125I-Tf surface binding
to tet+ or tet fWTHFE/tTA HeLa cells at
4 °C reflects the ability of HFE to modulate Tf binding to TfR (Fig.
2). The Kd of Tf and
TfR increased from 1.2 to 11 nM Tf with expression of HFE,
indicating that the affinity of Tf for TfR decreases when fHFE is
overexpressed. The linearity of the slope rather than a biphasic slope
of the Scatchard plot shows that fHFE sufficiently saturated TfR such
that no high affinity binding sites were evident. These data also show
that fHFE expression moderately increased the amount of Tf-binding sites (52%), in keeping with the increased expression of TfR on Western blots (Fig. 1).
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Early Association of HFE and TfR--
fHFE associates with TfR at
the cell surface in a manner that lowers the affinity of Tf for TfR
(Ref. 8 and this study). To investigate where this association occurs
in the biosynthetic pathway, we tracked the synthesis and association
of fHFE and TfR via endo H and pulse-chase analysis in fWTHFE/tTA HeLa
cells (Fig. 3). Endo H is an
endoglycosidase that cleaves between the proximal GlcNAc residues of
high mannose and hybrid, but not complex N-linked,
oligosaccharides. Loss of endo H sensitivity indicates that the protein
has exited the endoplasmic reticulum and the cis-Golgi
region of the biosynthetic pathway in pulse-chase experiments. The
peptide sequence of HFE has three consensus sites for glycosylation at
amino acids 110, 130, and 234. Immunoprecipitation of fHFE shortly
after synthesis indicated little association between HFE and TfR (Fig.
3, 0 min chase). TfR was capable of coprecipitating with HFE
within 30 min of chase when a significant portion of TfR was completely
sensitive to endo H (Fig. 3, 30 min chase, lower
bands of doublet at ~87 kDa (Endo
H) and ~80 kDa (+Endo H)). Thus, HFE can
associate with TfR in the endoplasmic reticulum/cis-Golgi compartments. Between 30 and 120 min of chase, the amount of TfR that
coprecipitated with HFE did not change, indicating that HFE does not
gain any more ability to bind HFE over this period of time. In
addition, within the first 120 min of chase, most of the HFE was
rapidly degraded in that the levels of HFE decrease 2.5-fold.
PhosphorImager analysis of the amount of TfR that immunoprecipitated with HFE (taking into consideration the relative abundance of Met in
each protein) indicated that after an overnight label, the ratio of TfR
to HFE was 1.7:1.
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HFE Associated with TfR Is More Stable-- The relative stabilities of HFE and TfR were compared over 8 h (Fig. 4, A and B) to further investigate the turnover of free and complexed HFE. Cells were fully induced to express fHFE by withdrawal of tetracycline from the medium for at least 3 days. Quantitation of the absolute amount of fHFE synthesized indicated that initially four times more fHFE was synthesized than TfR (Fig. 4, A and C, 0 chase after 1-h label). fHFE was rapidly degraded in the first 4 h of pulse-chase (Fig. 4, A and B). Degradation continued at a slower rate over the next 4 h, whereas the amount of TfR that co-immunoprecipitated with fHFE was unchanged (Fig. 4B). The 35S quantitation shown here is specific to the experiment shown and is representative of several experiments.
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HFE Recognizes a Site on TfR Other than That of Tf--
The close
association of fHFE and TfR over time and the lowered affinity of Tf
for TfR in fHFE-expressing cells led us to investigate whether HFE has
the same TfR-binding site as Tf. Earlier studies examining the
association of Tf and TfR indicated that the unglycosylated form of TfR
does not fold correctly and does not bind Tf (23-25). We investigated
whether Asn-linked glycosylation was necessary for the association of
fHFE with TfR. tet+ and tet fWTHFE/tTA HeLa
cells were treated with tunicamycin to inhibit Asn-linked
glycosylation. Western blotting showed co-immunoprecipitation of
unglycosylated TfR (~80 kDa) with HFE (Fig.
5, eighth lane, arrow). The discrepancy in the amount of unglycosylated TfR
in tet+ and tet
cells immunoprecipitated with
sheep anti-hTfR serum was due to the total amount of TfR in the cell.
Unglycosylated TfR from tet+ cells is detectable at a
longer exposure (data not shown). Since TfR co-immunoprecipitation with
HFE is independent of glycosylation, the structural elements necessary
for fHFE binding to TfR are different than for Tf binding to
TfR.
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HFE Co-localizes with TfR-- Immunocytochemical staining of fHFE and TfR was performed to determine their intracellular localization. HFE synthesis was turned off in fWTHFE/tTA HeLa cells grown in the presence of tetracycline (tet+). Immunocytochemical staining of TfR in cells grown for >2 weeks in tet+ medium exhibited punctate cytoplasmic staining and localization to a perinuclear compartment (Fig. 7A). TfR is a marker for recycling endosomes, as 75-80% of TfR recycles through the endosomes, whereas only 20-25% is found on the surface at any point in time (26). fHFE levels were undetectable in cells maintained in tet+ medium (Fig. 7B). The staining pattern of TfR did not change in fWTHFE/tTA HeLa cells induced for fHFE expression (Fig. 7C). fHFE localized to the same perinuclear compartment (Fig. 7D) and exhibited complete co-localization with TfR. These results demonstrate that HFE does not drastically alter the distribution of TfR.
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HFE Co-localizes with Tf-positive Vesicles--
To determine
whether fHFE co-internalizes with TfR, fWTHFE/tTA HeLa cells were
allowed to take up Texas Red-labeled Tf for 30 min at 37 °C. The
cells were washed with PBS, fixed, and stained as described under
"Experimental Procedures." tet+ cells showed
co-localization of TfR and Tf (Fig. 8,
A and B, respectively). TfR and Tf
co-localization was not changed in cells expressing fHFE (Fig. 8,
C and D, respectively). tet cells
also exhibited co-localization of fHFE and Tf (Fig. 8, E and
F, respectively). Combined with results from Fig. 7, Tf, TfR, and HFE all appear to traffic through the same endocytic perinuclear compartment.
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DISCUSSION |
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To continue to define the relationship among iron, Tf, TfR, and HFE, we have investigated the biosynthesis and trafficking of fHFE in relation to TfR and have shown that HFE may regulate intracellular iron levels since its expression greatly decreases the levels of ferritin and modestly increases the levels of TfR. Both Ft and TfR are encoded by mRNAs containing iron response elements in their untranslated regions (for a review, see Ref. 27). TfR mRNA is stabilized by iron regulatory protein binding in a low cytoplasmic iron environment, whereas the translation of Ft mRNA is blocked. Modulation of TfR and Ft by HFE in this manner implicates HFE in the regulation of intracellular iron load.
Scatchard analysis of 125I-Tf binding to tet+
and tet cell lines shows that Tf-binding sites increase
from 1.8 × 105/uninduced cell to 2.8 × 105/induced cell, confirming the elevated level of TfR seen
on Western blotting. The Scatchard data also show that fHFE decreases
TfR affinity for Tf as seen in the increase in Kd
from 1.2 to 11 nM. This is comparable to the 5-75
nM increase in Kd upon addition of 1 mM soluble HFE·
2-microglobulin heterodimer to HeLa cells reported earlier (8). The previous report showed no
significant increase in TfR number because HeLa cells were only exposed
to soluble HFE for the length of the experiment, not for days in
culture as in the experiments reported here. The tetracycline-repressible system has the added benefit of comparing HFE-positive and -negative states within the same clonal cell line,
circumventing discrepancies that may arise due to clonal variation.
Our studies on the synthesis of fHFE and TfR and susceptibility
to endo H digestion demonstrate that these proteins are capable of
associating with each other within 30 min of synthesis while the
proteins are in the endoplasmic reticulum/cis-Golgi
compartment. The pulse-chase analysis also indicated that a significant
amount of fHFE is degraded between 90 and 120 min, the remainder of
which is further processed by addition of complex carbohydrates less sensitive to endo H digestion. This pool of higher molecular mass fHFE
is protected from degradation by its association with TfR and/or
2-microglobulin.
The kinetics of degradation of the fHFE pool associated with TfR are much slower than the degradation rate of the non-TfR-associated pool. Approximately 50% of the fHFE expressed in our system was degraded within 4 h of synthesis, whereas the amount of TfR remained steady. The pool of fHFE that associated with the TfR, however, did not change significantly over the course of 8 h. These results suggest that the complex is much more stable than HFE alone. Quantitation of the steady-state amount of fHFE co-immunoprecipitated with TfR yielded a stoichiometry of two TfRs for every fHFE, or one transferrin receptor dimer for every fHFE. The same stoichiometry has been reported by Lebron et al. (6) using gel filtration.
Not only does HFE have a different stoichiometry of binding to TfR, but
it binds to a different region of TfR than Tf. Tunicamycin-treated tet fWTHFE/tTA HeLa cells are capable of associating
unglycosylated TfR and fHFE, as shown by the co-immunoprecipitation of
TfR with fHFE. Tf binding to TfR is glycosylation-dependent
because Tf does not show measurable binding to unglycosylated TfR (23, 24). Tf and HFE binding is not mutually exclusive since fHFE coprecipitates with TfR bound to Tf-agarose. The original observation (8) that HFE decreases Tf binding affinity for TfR, but not the number
of binding sites, is also in agreement with the present results.
Immunocytochemical staining of fHFE in tet fWTHFE/tTA
HeLa cells is punctate and perinuclear and exhibits co-localization with TfR. The pattern is similar to TfR immunostaining in
tet+ fWTHFE/tTA HeLa cells that do not express fHFE. These
results show that HFE does not drastically alter the trafficking of
TfR. TfR is, traditionally, an excellent marker for recycling
endosomes. Once a clathrin-coated vesicle containing TfR pinches off
the plasma membrane, it loses its clathrin coat and lowers its internal pH. In these low pH endosomes, iron is released from Tf. TfR and apotransferrin remain bound to each other and recycle to the cell surface. At neutral pH, apotransferrin is released, and TfR is free to
bind more diferric Tf (for further discussion, see review in Ref. 28).
HFE has no identifiable internalization motif in its cytoplasmic
domain, yet fHFE co-localizes with Tf and TfR in perinuclear and
cytoplasmic intracellular vesicles. The intracellular location of HFE
may therefore be dependent on its association and endocytosis with TfR.
The co-trafficking of HFE and TfR into endocytic compartments suggests
that HFE may alter iron uptake inside the cell.
In this report, we conclude that TfR and HFE co-localize to endocytic compartments in a pattern typical of TfR. This finding is important in that it indicates that the function of HFE may not be limited to its alteration of the Tf-TfR interaction on the cell surface, but that HFE may also play a role in the cellular uptake of iron from the endosome. The expression of HFE in HeLa cells results in lower intracellular iron levels as reflected by the specific alteration of TfR and Ft levels in cells expressing fHFE. fHFE localization to Tf-positive vesicles suggests that regulation of internal iron stores by HFE may occur within the endosome.
Hereditary hemochromatosis is a disease characterized by an increase in the set point for bodily iron load. Hereditary hemochromatosis patients have elevated levels of Tf iron saturation. The degree of Tf saturation is an indicator of bodily iron load. The association of HFE with Tf may be critical for its role as a sensor of bodily iron load. With the expanding knowledge of novel functions of nonclassical MHC class I molecules, HFE has the potential to regulate iron homeostasis in a number of ways. HFE may act by itself, regulate other proteins involved in iron metabolism, or regulate the level of iron transport through the endosomal membrane or alter the kinetics of TfR cycling in a subtle manner. It will be important to characterize differences between regulation of Tf and iron uptake at the cellular level and in organs such as the liver, pancreas, pituitary, and heart that are damaged by iron overload in hereditary hemochromatosis patients. It will also be important to compare such regulation with what occurs in the cells of the intestinal mucosa that are anomalously iron-depleted in hemochromatosis patients.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK 40608.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.
§ Supported by NHLBI Training Program in Molecular Hematology Grant T32-HL00781 from the National Institutes of Health.
To whom correspondence should be addressed: Dept. of Cell and
Developmental Biology, L215, Oregon Health Sciences University, Portland, OR 97201-3098. Tel.: 503-494-5845; Fax: 503-494-4253; E-mail:
ennsca{at}ohsu.edu.
The abbreviations used are:
MHC, major
histocompatibility complex; Tf, transferrin; TfR, transferrin receptor; hTfR, human TfR; fHFE, FLAG epitope-tagged HFE; PAGE, polyacrylamide
gel electrophoresis; tet, tetracycline; PBS, phosphate-buffered saline; endo H, endo--N-acetylglucosaminidase HFITC, fluorescein isothiocyanateFt, ferritin.
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