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Am J Physiol Cell Physiol 294: C383-C390, 2008. First published December 19, 2007; doi:10.1152/ajpcell.00492.2007
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Protein and Vesicle Trafficking, Cytoskeleton

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

Daniel F. Wallace, Lesa Summerville, Emily M. Crampton, and V. Nathan Subramaniam

Membrane Transport Laboratory, The Queensland Institute of Medical Research, Brisbane, Queensland, Australia

Submitted 17 October 2007 ; accepted in final form 17 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease-causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the TfR2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, whereas the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations that cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.

iron overload; aberrant localization; endoplasmic reticulum; mislocalization

IRON IS AN ESSENTIAL MINERAL required for numerous metabolic processes. The ability of iron to exist in two redox states underlies its indispensable biological role in many cellular processes and in oxygen transport. The level of iron in the body is tightly regulated, because both an excess and a deficiency can lead to severe consequences for the health of the individual. Hereditary hemochromatosis is an iron overload disorder characterized by excessive absorption and deposition in tissues such as the liver, heart, and pancreas (27). If left untreated, the accumulation of iron in parenchymal cells can lead to tissue damage, including cirrhosis, diabetes mellitus, arthropathy, cardiomyopathy, endocrine abnormalities, and hepatocellular carcinoma. The most common form of hemochromatosis (type 1) is caused by mutations in the major histocompatibility complex class I-like protein HFE. A link between HFE and iron metabolism was established when it was shown that HFE associates with the transferrin receptor (7, 26).

Internalization of transferrin-bound iron by transferrin receptor 1 (TfR1) is one of the major pathways by which cells take up iron. This is one of the best-studied processes in cell biology and has provided a valuable tool to study the endocytic process in mammalian cells. A homologue of TfR1, termed transferrin receptor 2 (TfR2), was identified in 1999 (22). While the exact role of TfR2 in iron metabolism is unknown, it is evident that it plays an important function in maintaining iron homeostasis, because mutations in TfR2 lead to a form of hereditary hemochromatosis (type 3) (3, 11, 14, 24, 29). TfR2 is highly expressed in the liver (22). There is some evidence that it is expressed in early erythroid precursor cells (21); however, this is controversial (1). Our recent studies (35) suggest that liver-expressed TfR2 plays a predominant role in the regulation of iron metabolism. TfR2 differs from TfR1 in that it does not contain any iron-responsive elements in its mRNA, and its mRNA does not appear to be regulated by iron (10, 19). Several studies have suggested that TfR2 is capable of binding transferrin, although with a reduced affinity (20, 22, 37). The role of TfR2 as a substitute for TfR1 in its absence is still elusive. Knockout of TfR1 in mice causes embryonic lethality due to severe iron deficiency (23), whereas TfR2 mutations in humans and targeted mutagenesis or loss of TfR2 in mice result in iron overload (3, 9, 33). These studies suggest that TfR1 and TfR2 have distinct functions and that TfR2 is unable to substitute for the absence of TfR1.

It has been shown by us and others that targeted deletion or mutagenesis of the TfR2 gene in mice recapitulates the human iron overload disorder type 3 hereditary hemochromatosis (9, 33). Despite iron loading, these mice have increased iron absorption and liver iron uptake (6). Recently, we have shown that a liver-specific deletion of the mouse TfR2 gene is sufficient to induce iron overload in mice (35). These and other studies have shown that liver-expressed TfR2 is important for maintaining iron homeostasis, through its regulation of the iron-regulatory hormone hepcidin (6, 18, 33, 35). At a cellular level, however, it is still not clear how TfR2 is involved in the regulation of iron metabolism. It is thought that TfR2 acts as a sensor of body iron levels via an interaction with transferrin, detecting either the level of iron saturation or the amount of diferric transferrin in the blood. This interaction increases the stability of the TfR2 protein (17, 28), enabling signaling, perhaps via the MAPK pathway (2). The net result is upregulation of hepcidin resulting from increased diferric transferrin levels.

The effect of disease-causing mutations on TfR2 cellular expression has not been well studied. We have performed a cellular analysis of TfR2 and analyzed the consequences of disease-causing mutations on its expression and localization. Our studies suggest that defective localization of the mutant proteins is one underlying cause for type 3 hereditary hemochromatosis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies, chemicals, and cell lines. Antibodies recognizing the cytoplasmic domain of mouse TfR2 and cellubrevin have previously been described (33, 34). Anti-actin antibodies were obtained from Sigma-Aldrich (Castle Hill, New South Wales, Australia). The generation of sheep antibodies against the extracytoplasmic domain of mouse TfR2 is described below. Anti-calnexin antibodies were a generous gift from Dr. David J. McKean, Mayo Clinic, Rochester, MN (30). The 9E10 hybridoma (American Type Culture Collection, Manassas, VA) was maintained in Xten Hybricell media (Thermo-Trace, Melbourne, Victoria, Australia). Conditioned hybridoma media containing anti-myc antibodies was used in immunoblot and immunofluorescence experiments. Oligonucleotides were obtained from Sigma Genosys (Castle Hill, New South Wales, Australia). All cell culture media, serum, and reagents were purchased from Invitrogen (Mount Waverley, Victoria, Australia). Hybond-C+, glutathione S-transferase (GST)-, and cyanogen bromide-activated Sepharose were obtained from Amersham Biosciences (Castle Hill, New South Wales, Australia). The mouse hepatocyte cell line, Hepa1–6 (CRL-1830), was obtained from American Type Culture Collection. Hepa1–6 cells were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 5% fetal bovine serum (FBS) (D5). Stably transfected cells were cultured in D5 containing 1 µg/ml puromycin.

Generation of recombinant GST-mTfR2-extracytoplasmic region and antibody purification. Oligonucleotide primers pGEX-TfR2-EcoF and pGEX-TfR2-XhoR (Table 1) were used to amplify an 82 amino acid portion of the extracytoplasmic domain of mTfR2 (mTfR2-EC) corresponding to amino acids 103 to 184. The PCR product was ligated into the corresponding sites of pGEX-KG vector (13) and transformed into Escherichia coli BL21 cells. Recombinant GST-mTfR2-EC was purified using GST-Sepharose beads according to the manufacturer's instructions. The recombinant protein was eluted in GST elution buffer (50 mM Tris, pH 8.0, 0.5 mM MgCl2, 6 mg/ml reduced glutathione, and 6.4% glycerol in PBS). Purified protein was used to generate antibodies in sheep, which were then affinity purified by sequential passage of immune serum through a GST- and a GST-mTfR2-EC cyanogen bromide-activated Sepharose column, essentially as described for rabbit antibodies (38). After extensive washing, specific antibodies were eluted with glycine buffer (200 mM glycine, pH 2.0), dialyzed against PBS, and stored at –20°C in 10% glycerol.


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  		Table 1. Primers used for mTfR2 cloning and mutagenesis

 
Generation of DNA constructs. Wild-type (WT) TfR2 was amplified from an mTfR2-expressed sequence tag clone (GenBank accession number AI663104) using the primers TfR2-XhoF and TfR2-XhoR or pEF-TfR2-NheF and pEF-TfR2-EcoR (Table 1). The mTfR2 gene was cloned into the mammalian expression plasmid pEFIRES-P (15) engineered to contain an amino- or carboxy-terminal double- myc tag (pEFIRES-NH2-DMyc-mTfR2 or pEFIRES-mTfR2-COOH-DMyc). To create the pEFIRES-NH2-DMyc plasmid, the double-myc tag from pDMyc-Neo (32) was excised and subcloned into the NheI and NotI sites of pEFIRES-P. To create the pEFIRES-COOH-DMyc plasmid, the double-myc tag from pEFIRES-NH2-DMyc was PCR amplified using primers NotF-pEF and NotR-pEF (Table 1) and was subcloned into the NotI site of pEFIRES-P. Mouse equivalents of human hemochromatosis type 3-causing mutations were introduced into the mTfR2 cDNA by site-directed mutagenesis or PCR. The following mutations were introduced by site-directed mutagenesis: a methionine to lysine at position 172, M172K (mouse M167K), deletion of an alanine-valine-alanine-glutamine sequence between positions 621 and 624, AVAQ621–624del (mouse AVVQ612–615del or AVAQ616–619del), and a glutamine-to-proline mutation at position 690, Q690P (mouse K685P). The truncation mutation, a tyrosine to stop codon change at position 250, Y250X (mouse Y245X) was introduced by PCR using the primers TfR2-XhoF and TfR2-XbaR (Table 1). Human TfR2 contains two AVAQ sequences next to each other (AVAQAVAQ). A disease-causing mutation deletes one of these AVAQ sequences. The mouse sequence differs by one amino acid (AVVQAVAQ); therefore it was decided to delete both of these sequences separately (AVVQ612–615del or AVAQ616–619del). The glutamine (Q) at position 690 of the human TfR2 sequence is not conserved in the mouse; in its place is a lysine (K685). A disease-causing mutation in humans replaces this glutamine with a proline (Q690P). Because proline residues can often cause kinks in peptide chains, the decision was made to replace the mouse equivalent with a proline also (K685P). Mutagenesis was performed using a pair of primers containing the mutation to be targeted according to the method described by Scott et al. (31). Primers used for mutagenesis are listed in Table 1.

Immunoblot analysis. Samples were separated by 10% SDS-PAGE, transferred onto Hybond-C+ membrane, and blocked in 10% skim milk powder-0.5% Tween 20 in PBS (blocking buffer) overnight at 4°C. The blots were incubated with anti-myc (1:50, 9E10 hybridoma supernatant), anti-actin (0.6 µg/ml), or anti-mTfR2-EC antibody (20 µg/ml) in blocking buffer, washed extensively with 0.1% Tween 20 in PBS, and then incubated with anti-rabbit, anti-mouse, or anti-sheep horseradish peroxidase for 1 h at room temperature. Supersignal West Pico chemiluminescent substrate (Pierce Chemical, Rockford, IL) was applied for 5 min to the blot, which was then exposed to film (Super RX, Fujifilm, Tokyo, Japan).

Transfections. Hepa1–6 cells were cultured in D5 medium in six-well plates. Transfections were performed using Lipofectamine 2000 reagent according to the manufacturer's instructions. Plasmid DNA (4 µg) was complexed with 10 µl Lipofectamine 2000 reagent. The complexes were added to the cells and incubated overnight. Stably transfected cell lines were isolated by selection with 10 µg/ml puromycin for 2 days and were then stably maintained in 1 µg/ml of the antibiotic. Expression of myc-tagged TfR2 in transfectants was confirmed by immunofluorescence and Western blot analysis.

Immunofluorescence analysis. Cultured cells were grown on glass coverslips to a confluency of 50–80%. The cells were washed twice in PBS containing 1 mM CaCl2 and 1 mM MgCl2 (PBSCM), fixed with 3% paraformaldehyde, and permeabilized with 0.1% saponin in PBSCM. Antibodies were diluted in fluorescence dilution buffer (5% FBS, 5% normal goat serum, and 2% bovine serum albumin in PBSCM, pH 7.6) and were incubated with the cells for 1 h at room temperature using the following dilutions: anti-myc (1:4 hybridoma media), anti-cellubrevin (10 µg/ml), and anti-calnexin (1:100 antiserum). Following three washes in 0.1% saponin PBSCM, 10 µg/ml goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 594 secondary antibodies (Invitrogen) diluted in fluorescence dilution buffer were applied to the cells. After three washes in 0.1% saponin PBSCM, the coverslips were mounted onto glass slides using ProLong Gold Antifade mounting medium (Invitrogen). When calnexin antibodies were used, cells were fixed and permeabilized in methanol, and all washes were done with PBSCM. For surface expression analysis, cells on coverslips were cooled to 4°C and washed with cold PBSCM. Cells were incubated with sheep anti-mTfR2-EC antibodies (20 µg/ml) on ice for 30 min to label only protein regions external to the cell surface. The coverslips were rinsed with cold PBSCM and fixed with 4% paraformaldehyde in PBSCM. The cells were then incubated with donkey anti-sheep Alexa 568 secondary antibodies (Invitrogen), washed three times in PBSCM, and mounted. Fluorescent images were viewed and captured using a confocal microscope (Leica TCS SP2) or with a Zeiss Axioskop microscope equipped with epifluorescence optics.

Flow cytometry analysis of mTfR2. Untransfected and stably transfected Hepa1–6 cells were detached by incubation with trypsin. Cells were then washed with PBS plus 2% FBS and processed for flow cytometry. To detect membrane-bound TfR2, 1.5 x 106 cells were resuspended in 100 µl of PBS plus 2% FBS in the presence of 2 µg sheep anti-mTfR2-EC antibodies (20 µg/ml) and were incubated on ice for 1 h. Following two washes, the cells were incubated with 10 µg/ml rabbit anti-goat Alexa 488 (Molecular Probes) on ice for 20 min. Cells were analyzed with FACScan (Becton Dickinson). Statistical analysis was performed with Student's paired t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mutations of TfR2 result in intracellular retention. We have previously shown that both a total or liver-specific deletion of the mouse TfR2 gene in mice recapitulates the iron overload observed in subjects with type 3 hereditary hemochromatosis (33, 35). To define the nature of the molecular and cellular defect, mouse orthologues of several naturally occurring mutations in human TfR2 were introduced into mTfR2 and cloned into the mammalian expression vector, pEF-IRES. Double-myc epitope tags were placed on either the amino (NH2-DMyc-mTfR2) or carboxy (mTfR2-COOH-DMyc) terminus of the receptor. Constructs were transfected into the mouse hepatocyte cell line, Hepa1–6, and stable transfectants were analyzed by indirect immunofluorescence and confocal microscopy. All experiments were performed at least three times. Wild-type NH2-DMyc-mTfR2 in Hepa1–6 cells localized to endosomal-like structures and colocalized to some extent with cellubrevin, a marker of the endosomal pathway (5), whereas the Y245X mutant did not (Fig. 1A). The NH2-DMyc-mTfR2 mutants M167K, Y245X, AVVQ612–615del, AVAQ616–619del, and K685P were exclusively retained in structures reminiscent of the endoplasmic reticulum (ER), and they overlapped significantly with the ER chaperone calnexin (Fig. 1, B and C), whereas no overlap was observed with WT NH2-DMyc-mTfR2 (Fig. 1B). The positioning of the double-myc tag at the COOH terminus of the protein gave similar results (data not shown).


Figure 1
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  		Fig. 1. Expression of wild-type (WT) and mutant amino-terminal double-myc-tagged mouse transferrin receptor 2 (TfR2) in Hepa1–6 cells. Mutations were introduced into double-myc-tagged mouse TfR2 and transfected into Hepa1–6 cells. Stably transfected cells were selected using puromycin. The localization of NH2-DMyc-mTfR2 wild type (WT-mTfR2) and mutants M167K, AVVQ612–615del (AVVQdel), AVAQ616–619del (AVAQdel) and K685P and Y245X was determined by immunofluorescence and confocal microscopy. WT-mTfR2 and mutant TfR2 (red) labeled with myc antibodies (1:4 hybridoma media) were costained with the endosome marker cellubrevin (10 µg/ml) (A) or the endoplasmic reticulum (ER) marker calnexin (green, 1:100 antiserum) (B and C). WT-mTfR2 partially colocalized with cellubrevin but not with calnexin; the mutant versions of TfR2 did not colocalize with cellubrevin (shown for Y245X), and they colocalized almost completely with calnexin, suggesting retention and localization in the ER. Scale bar is 10 µm.

 
Mislocalization of transfected proteins is sometimes associated with increased overexpression. We analyzed TfR2 protein levels in WT and mutant transfected Hepa1–6 cells. The results are shown in Fig. 2A as a representative Western blot. Quantitation of TfR2 expression in these blots (relative to β-actin) indicated that WT TfR2 was expressed at 50% higher levels than the M167K and AVAQdel mutants and at similar levels to the other mutant proteins. Importantly, none of the mutant proteins were expressed at significantly higher levels than the WT mTfR2 protein, indicating that the mislocalization of the mutant proteins to the ER is not due to their overexpression.


Figure 2
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  		Fig. 2. Immunoblotting of TfR2 in transfected cells. A: cell lysates from Hepa1–6 cells expressing amino-terminal tagged WT and mutant TfR2 were Western blotted with myc antibodies (1:4 hybridoma media). β-actin (0.3 µg/ml) is shown as a loading control. The NH2-DMyc-mTfR2 WT (WT-mTfR2) and mutant M167K, AVVQ612–615del (AVVQdel), AVAQ616–619del (AVAQdel), and K685P cell lysates were run together on one gel, whereas the Y245X was run on a separate gel because of its smaller size. B: antibodies specific to an 82 amino acid portion of the extracellular domain of mouse TfR2 (anti-mTfR2-EC) were raised in a sheep. These antibodies (20 µg/ml) detect a single band of ~105 kDa in Hepa1–6 cells stably transfected with amino-terminal double-myc-tagged TfR2 (WT-mTfR2) but not in untransfected cells as does the anti-myc antibody (1:4 hybridoma media). β-actin (0.3 µg/ml) is shown as a loading control.

 
Absence of surface expression of mutant TfR2. To confirm the absence of mutant forms of TfR2 on the plasma membrane, we analyzed the cell surface expression of WT and mutant TfR2 proteins, using a sheep antibody specific to an 82 amino acid sequence from the extracytoplasmic domain of mouse TfR2 (corresponding to amino acids 103 to 184). Of the 82 amino acids, 77 are identical in rat TfR2 and 66 in the human protein. This antibody detects a single band, by immunoblotting, in Hepa1–6 cells transfected with amino-terminal myc-tagged WT mTfR2 (Fig. 2B), with no expression detected in untransfected cells. Wild-type mTfR2 tagged at either the amino or carboxy terminus was detected at the cell surface by immunofluorescence analysis. In contrast, none of the mutant mTfR2 proteins were detected at the cell surface (Fig. 3). These results suggest that the naturally occurring disease-causing mutations result in the intracellular retention of TfR2 in the ER and affect its transport to the cell surface.


Figure 3
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  		Fig. 3. Surface labeling of WT and mutant TfR2 using antibodies specific to the extracellular domain of mTfR2. Cells were incubated with anti-mTfR2-EC antibodies (20 µg/ml) on ice, before fixation and immunofluorescence analysis, to label only protein present on the external surface of the cell. Surface expression of TfR2 was detected only in cells expressing the WT protein (B and C), tagged at either the carboxy (B) or amino (C) terminus. No surface expression of TfR2 was detected in untransfected cells (A) or in cells expressing mutant forms of TfR2 (D: M167K; E: Y245X; F: AVVQ612–615del; G: AVAQ616–619del; and H: K685P).

 
With the use of the sheep anti-mTfR2-EC antibody, the expression of WT and mutant NH2-DMyc-mTfR2 at the plasma membrane in nonpermeabilized Hepa1–6 cells was analyzed by flow cytometry (Fig. 4). The amount of each mutant TfR2 (M167K, 4.9 ± 1.8%; Y245X, 7.2 ± 2.2%; AVVQ612–615del, 7.5 ± 3.4%; AVAQ616–619del, 8.3 ± 3.0%; K685P, 7.9 ± 3.6%; mean ± SD, n = 4) being expressed at the cell surface was significantly lower compared with WT TfR2 (37.4 ± 4.0%; P < 0.01, n = 4) (Fig. 4A). The amount of signal detected at the cell surface of mutant TfR2 expressing cells was similar to the background signal detected in untransfected Hepa1–6 cells (6.6 ± 0.3%; P > 0.05, n = 4). The difference in surface expression of TfR2 in WT and mutant TfR2-expressing cells is shown in the overlay of histograms for detection of surface TfR2 (Fig. 4B). The histogram for WT TfR2 is shifted to the right, away from background signal and expression of TfR2 in mutant expressing cell lines. These data indicate an impairment of proper trafficking and sorting of mutant TfR2 to the cell surface and confirm the confocal microscopy analysis.


Figure 4
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  		Fig. 4. Analysis of cell surface WT and mutant TfR2 by flow cytometry. Untransfected Hepa1–6 cells and cells transfected with WT and mutant amino-terminal tagged mTfR2 [M167K, Y245X, AVVQ612–615del (AVVQdel), AVAQ616–619del (AVAQdel), and K685P] were analyzed by flow cytometry using anti-mTfR2-EC antibodies (20 µg/ml). Unstained cells, cells stained with the secondary antibody only (10 µg/ml; rabbit anti-goat Alexa 488), and untransfected cells were used as negative controls. A: representative dot plots and table showing the percentages of positive TfR2-stained cells. Surface expression of TfR2 is reduced in mutant TfR2 compared with WT-expressing cells (P < 0.01; n = 4). The surface expression of TfR2 in mutant cell lines is no different from TfR2 expression in untransfected Hepa1–6 cells (P > 0.05; n = 4). The x-axis indicates the physical parameter of the cells (SSC-H, side scatter height); the y-axis indicates the fluorescence intensity (FL1 Alexa 488). Positive cells are indicated by region R1. The percentages of positive cells are given (means ± SD; n = 4). B: an overlay of a representative histogram analysis for unstained Hepa1–6 cells, Hepa1–6 cells stained with the secondary antibody only, untransfected Hepa1–6 cells, and cells transfected with WT or mutant mTfR2. Surface expression of TfR2 is detected in WT TfR2-expressing cells. The surface expression in mutant TfR2 cell lines is unchanged from surface-expressed TfR2 detected in unstransfected Hepa1–6 cells. The x-axis indicates the fluorescence intensity (FL1, Alexa 488); the y-axis indicates the number of positive cells detected (counts).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
TfR1 expression is intimately related to the iron status of the cell, corresponding with its importance as the premier means of iron entry. With its five iron-responsive elements, the level of expression of TfR1 is adjusted to match the iron requirements of the cell. TfR2 does not possess these iron-responsive elements and thus does not appear to be regulated in a similar fashion. TfR1 is essential to the cell; knockout of the gene in mice results in embryonic lethality (23), and no mutations in TfR1 in humans have been described. Mutations in TfR2 have been shown to cause type 3 hemochromatosis in humans (3), and targeted mutagenesis (9) or deletion (33, 35) of TfR2 in mice results in iron overload with a similar phenotype. Thus, while TfR2 has a significant role in iron metabolism, it does not appear to be essential. Several lines of evidence suggest that TfR1 and TfR2 play different roles in the regulation of iron metabolism.

While the main role of TfR2 may not be directly involved in the transport of iron, it appears to have an important role in its regulation. Several mutations that occur naturally in humans were introduced into mouse TfR2, and their expression was studied by immunofluorescence and confocal microscopy. We show that the majority of mutations in TfR2 result in the retention of the mutant protein in the ER. Fluorescence-activated cell sorting analysis confirmed the loss of cell surface expression of the mutant proteins. Thus, mutant TfR2 is unable to perform its putative function at the cell surface and/or endosomes. The mutations likely result in a disordered secondary structure of the molecule. This may affect residues critical for the trafficking of the protein or residues required for its interaction with a chaperone or other proteins important in the proper trafficking of TfR2. Alternatively, the mutations may affect the structure of the protein, resulting in the quality control system of the cell recognizing it as a misfolded protein, retaining it, and eventually degrading it.

Several diseases have been shown to result from the mislocalization and aberrant trafficking of proteins. These include type 1 hemochromatosis, in which the C282Y mutation in the HFE protein affects secondary structure, resulting in its inability to bind to β2-microglobulin, causing retention in the ER and loss of expression at the cell surface (8). The majority of mutations in the cystic fibrosis transmembrane conductance regulator prevent its trafficking to the cell surface and affect its ability to regulate chloride transport (36). Similarly, our results demonstrate for the first time that the majority of disease-causing mutations of TfR2 are a result of the mislocalization of the protein.

Exactly how TfR2 is involved in the regulation of iron homeostasis is still unclear. It appears that the hepatocytes are the primary site for TfR2 expression and activity, because hepatocyte-specific deletion of TfR2 in mice leads to a similar iron overload phenotype as global deletion (17, 18, 25, 28, 33, 35). TfR2 is also involved in the regulation of hepcidin (18, 25, 33), and it is likely that TfR2 activity is modulated by an interaction with transferrin. Two studies have shown that the TfR2 protein is stabilized by diferric transferrin (17, 28). The cytoplasmic domain of TfR2 appears to be responsible for this stabilization, because a chimeric receptor containing the cytoplasmic domain of TfR2 fused to the transmembrane and ectodomain of TfR1 retained the ability to be stabilized by diferric transferrin (4). Other experiments have shown that diferric transferrin increases the fraction of TfR2 being targeted to the recycling pathway and decreases the amount being targeted to the late endosomes and lysosomes for degradation (16). The YQRV endocytosis motif in the cytoplasmic domain is important for directing this process (16). The stabilization of TfR2 by diferric transferrin may facilitate the activation of a signaling pathway resulting in the induction of hepcidin. Another study has shown that TfR2 is localized in lipid rafts and coimmunoprecipitates with caveolin-1. It was also shown that TfR2 stimulated by either TfR2 monoclonal antibody or transferrin was able to activate the ERK/MAPK signaling pathway, and the localization of TfR2 in lipid rafts was important for signaling (2). This study was performed in the erythroleukemic cell line K562. Whether this signaling pathway is functional in hepatocytes and responsible for the downstream induction of hepcidin remains to be determined. The retention of mutant TfR2 in the ER as observed in the present study would result in no TfR2 being present on the plasma membrane or lipid raft microdomains. This would result in the protein being insensitive to diferric transferrin levels and unable to facilitate signaling, leading to loss of hepcidin induction. A recent study suggested that TfR2 interacts with HFE in an iron-sensing complex (12). However, an earlier study found no interaction between the two soluble forms of the proteins (37). The interaction between TfR2 and HFE was unaffected by the disease-causing mutations of TfR2 studied here, including the truncated Y245X (12). This suggests that any interaction must involve a region in the amino-terminal third of the TfR2 protein. The intracellular localization of the TfR2 mutants was not reported in the study by Goswami and Andrews (12). In the present study, we have shown that all the mutants analyzed cause ER retention of TfR2. Therefore, given the localization of the mutants, any interaction between TfR2 and HFE must be occurring in the ER. This would seem an unlikely location for any functional interaction to occur, and further studies will be required to characterize any interaction between TfR2 and HFE in vivo.

In conclusion, we have shown that wild-type TfR2 is present on the plasma membrane and in an endosomal compartment. Disease-causing mutations of TfR2 result in the retention of the mutant protein in the ER and no expression at the cell surface. In type 3 hemochromatosis, the lack of surface expression of TfR2 would result in an inability to interact with diferric transferrin and in impaired signaling, leading to a loss of hepcidin induction. Low levels of circulating hepcidin would result in progressive iron loading, consistent with the phenotype of type 3 hemochromatosis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a Program Grant from the National Health and Medical Research Council (NHMRC) of Australia (339400) to V. N. Subramaniam. D. F. Wallace is the recipient of a NHMRC R. D. Wright Career Development Award. V. N. Subramaniam is the Janssen-Cilag/Gastroenterological Society of Australia Senior Research Fellow.


   ACKNOWLEDGMENTS
 
We thank Uda Ho and Edgar Benko for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. N. Subramaniam, Membrane Transport Laboratory, The Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia (e-mail: nathan.subramaniam{at}qimr.edu.au)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
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S. Pelucchi, R. Mariani, P. Trombini, S. Coletti, M. Pozzi, V. Paolini, D. Barisani, and A. Piperno
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