Hereditary Hemochromatosis Protein, HFE, Interaction with Transferrin Receptor 2 Suggests a Molecular Mechanism for Mammalian Iron Sensing Tapasree Goswami1 and Nancy C. Andrews1,2 From 1Harvard Medical School, Children’s Hospital Boston, Division of Hematology/ Oncology and 2Howard Hughes Medical Institute, Boston Massachusetts, USA

Running Title: HFE Interacts with TFR2 Address correspondence to: Nancy C. Andrews, Children’s Hospital, Karp Family Research Laboratories, RM 8-125, 1, Blackfan Circle, Boston, Massachusetts 02115-5737, USA. Tel. 617 919-2116; Fax: 617 432-3639; E-mail: nancy_andrews@hms.harvard.edu.

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2 and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.

All mammalian cells have an absolute

requirement for iron. Both cellular iron deficiency and iron-overload are pathological and iron concentration in cells and body fluids is tightly regulated. Chronic malaccumulation of iron in tissues results in systemic iron-overload diseases that are collectively called hemochromatosis. Hereditary hemochromatosis in humans is linked to mutations in several genes namely HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV), and hepcidin (HAMP) (reviewed in reference 1).

Hepcidin, a hepatocyte-derived soluble factor, negatively regulates intestinal absorption of dietary iron and the release of recycled iron into

circulation from macrophages (2). Also, hepcidin expression responds to body iron status (3). Hepcidin, therefore, plays a pivotal role as a regulator of whole-body iron homeostasis. Since disruption of HFE, TFR2 or HJV causes decreased hepcidin production these gene products appear to be involved in the upstream regulation of hepcidin (reviewed in reference 4).

HFE, an atypical MHC class I molecule, associates with transferrin receptor 1 (TFR1), a type II transmembrane glycoprotein which is the primary effector of cellular iron uptake (5). TFR2 is a homolog of TFR1 and like TFR1, can bind and internalize diferric-transferrin (Fe2-TF) (6). However, while TFR1 is widely expressed, TFR2 is expressed predominantly in hepatocytes, hematopoietic cells and duodenal crypt cells, overlapping with HFE expression (6, 7). This and other differences in transcriptional regulation, Fe2-TF binding-affinities and gene deletion phenotypes suggest that TFR1 and TFR2 have distinct roles in iron homeostasis. While TFR1 is a key mediator of iron uptake, TFR2 is postulated to play a regulatory role in whole-body iron homeostasis.

Since hepcidin is produced predominantly by hepatocytes, it is likely that these cells express molecular determinants of iron sensing. Serum transferrin deficiency in hpx mice is associated with low hepcidin levels despite parenchymal iron loading, a phenotype corrected by transferrin administration (8). Also, elevated serum Fe2-TF stabilizes TFR2 protein in liver (9), an effect recapitulated in vitro (9, 10). Circulating Fe2-TF is therefore likely to be an iron signal sensed by hepatocyte membrane proteins that regulate hepcidin production and TFR2 may be part of the regulatory system sensing transferrin saturation.

Previous investigators hypothesized that HFE and TFR2 might belong together in such a regulatory pathway. An earlier study, however, demonstrated that soluble, purified ectodomains of HFE and TFR2 do not interact in vitro (11), precluding the possibility that their roles in a convergent iron homeostasis pathway involve formation of an HFE-TFR2 complex. Here we investigate whether HFE and TFR2 interact when expressed in cells. Based on our findings, we propose that HFE plays an important role in iron sensing by conveying the whole-body iron status, reflected by transferrin saturation, from the HFE-

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Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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TFR1 complex to TFR2, resulting in potential downstream signaling events.

EXPERIMENTAL PROCEDURES Cell Culture and Transfection — All cell

culture media were supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin and 10 mM glutamine (Invitrogen) unless otherwise noted. AML12, a differentiated, non-transformed mouse hepatocyte cell line (12) was maintained in 1:1 Dulbecco's modified Eagle's medium (DMEM)/ Ham’s nutrient F12 mixture (F12) supplemented with 10% fetal bovine serum (FBS, ATCC), 5 µg/ml human insulin (Sigma), 5 µg/ml human transferrin (Roche), 5 ng/ml sodium selenite (Sigma), 40 ng/ml dexamethasone (Sigma) and 5 mM sodium pyruvate. CHO-TRVb-0 (TFR1-deficient Chinese hamster ovary cells) and TRVb-1 (human TFR1 stably transfected TRVb-0) cells (13) were cultured in Ham’s F12 supplemented with 5% FBS and 0.2% glucose. Human embryonic kidney 293T (HEK293T) cells were grown in DMEM containing 10% FBS. Total amounts of DNA transfected in each experiment were kept equal in all samples by adding appropriate vector DNA. AML12, TRVb-0 and TRVb-1 cells were transfected using LipofectAMINE 2000 (Invitrogen) in antibiotic-free growth medium using methods described by the manufacturer. HEK293T cells were transfected with DNA:calcium phosphate co-precipitates using a HEPES-buffered calcium phosphate method (14). Transfection medium was replaced with fresh culture medium 12 h post-transfection and cells were harvested and lysed 48 h post-transfection.

Expression Plasmids and Mutagenesis — 1 µg of mouse liver RNA was used in reverse transcription reactions using Superscript II (Invitrogen) following protocols described by the manufacturer. Specific oligonucleotide primers were used to amplify mouse HFE, TFR1 and TFR2 cDNAs. Myc epitope-tagged TFR1 and TFR2 cDNAs were constructed by replacing the respective stop codons with an XhoI site and cloning into pcDNA3.1 myc-His plasmid (Invitrogen). HFE cDNA was first cloned into pcDNA3.1 (Invitrogen). This parent HFE cDNA was mutagenized using the QuikChange mutagenesis kit (Stratagene) to insert sequence

coding for a FLAG epitope downstream of the HFE signal peptide. Mutagenesis of the mouse cDNAs to produce HFE and TFR2 mutants (HFEH67D, TFR2M167K, TFR2Y245X and TFR2K685P ) corresponding to human hemochromatosis associated HFE and TFR2 mutations (HFEH63D, TFR2M172K, TFR2Y250X and TFR2Q690P respectively) were also performed using the QuikChange mutagenesis protocol. The TFR2Y245X mutant was generated by deletion of the sequences coding for amino-acids 246 to 798 from the parent TFR2-myc construct. The resulting construct thus encoded amino-acids 1 to 245 of mouse TFR2 followed directly by an in-frame myc epitope tag. The conserved RGD motif in mouse TFR2 (spanning amino-acids 673 – 675) was mutated to GGD. All plasmids were subjected to sequence analysis to confirm integrity. The T91-Display construct (encoding FSTL3-myc in pDisplay, Invitrogen) was a kind gift from Dr. Y. Sidis (MGH, Harvard Medical School) and is described in reference 15.

Antibodies — Primary antibodies, anti-TFR1 monoclonal antibody (Zymed), anti-TFR2 rabbit polyclonal antibody (Alpha Diagnostics), anti-FLAG M2 monoclonal antibody and agarose conjuguated M2 antibody (Sigma) and anti-myc monoclonal antibody (Upstate Biotechnology) were purchased. Protein G conjugated agarose beads and Protein A conjugated sepharose beads were purchased from Roche and Sigma respectively. Horseradish peroxidase conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from Amersham. Mouse TrueBlot and Rabbit TrueBlot secondary antibodies which do not recognize denatured IgG were purchased from eBiosciences.

Preparation of Cell Lysates — Cells were washed once in ice-cold Dulbecco’s phosphate buffered saline (PBS) and harvested. Cell pellets were lysed in ice-cold Triton X-100 lysis buffer (1% Triton X-100 (v/v), 50 mM Tris-HCl, pH 8.0, 10 mM KCl, 0.15 M NaCl, 20 mM NaF, 10 mM Na2P2O7, 1 mM Na3VO4) supplemented with a cocktail of protease inhibitors (Complete Mini, Roche). After mixing on ice for 15 mins, cell lysates were centrifuged at 8000 x g for 5 min in a cooled tabletop microcentrifuge to sediment nuclei and debris and supernatants were collected and used in subsequent experiments.

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Crude membrane preparations were isolated from cell pellets using a hypotonic lysis method as described before (14). Prior to experiments crude membrane preparations were resuspended in 1% Triton X-100 lysis buffer.

Immunoprecipitation, Gel Electrophoresis, and Western Blot Analysis — Total protein amounts in lysates were measured using the Bio-Rad protein assay and equivalent amounts of protein were used for immunoprecipitation. Immunoprecipitations were performed by first incubating cell lysates with the indicated primary antibodies overnight at 4°C. Immune complexes were then precipitated by incubating reactions for an additional 2 hrs at 4°C with either sepharose-conjugated protein A or agarose-conjugated protein G as appropriate. For FLAG immunoprecipitations, anti-FLAG M2 antibody conjugated to agarose beads were used and additional protein G incubations omitted. Immunoprecipitates were washed four times with 1% Triton X-100 lysis buffer and were resuspended in SDS-PAGE sample buffer for western blotting.

Lysates and immunoprecipitates were separated by SDS-PAGE using 10% polyacrylamide gels and the proteins were transferred to pure nitrocellulose filters (Amersham). The filters were stained with Ponceau-S to confirm transfer and blocked with either 3% non-fat dry milk or 3% bovine serum albumin in Tris-buffered saline (TBS) containing 0.05% Tween 20 for 1 h at room temperature. Primary antibody incubations were performed overnight at 4°C. Filters were washed three times with TBS containing 0.05% Tween 20 (TBST) and then incubated with appropriate secondary antibodies. The filters were again washed four times and immune complexes formed on the blot were visualized by enhanced chemiluminescence (ECL, Amersham). Immunoreactive protein bands were analyzed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

RESULTS AND DISCUSSION First, we transfected HEK293T cells with N-

terminal FLAG epitope-tagged HFE (FLAG-HFE) cDNA together with C-terminal myc epitope-tagged TFR2 (TFR2-myc) cDNA. In both cases

epitope tags were in the extracellular domains. Lysates from transfected cells were used in immunoprecipitation analyses with anti-FLAG or anti-myc antibodies (Fig. 1A, B and E). TFR2-myc was co-immunoprecipitated by anti-FLAG antibody when co-expressed with FLAG-HFE. Singly transfected TFR2-myc was not immunoprecipitated by the anti-FLAG antibody. In reciprocal anti-myc immunoprecipitations FLAG-HFE was co-immunoprecipitated when co-transfected with TFR2-myc. Hence, we demonstrate that HFE and TFR2 interact when expressed in cells. In similar experiments, when co-expressed with FLAG-HFE, TFR1-myc co-immunoprecipitated with FLAG-HFE while an unrelated cell surface expressed C-terminal myc-tagged protein, T91-Display, did not (Fig. 1A and E). Hence, HFE interacts specifically with TFR1 and TFR2.

To extend our findings in a relevant cell-type, we assessed HFE-TFR2 interaction in crude membrane fractions of transfected AML12 mouse hepatocytes and confirmed TFR2-myc interaction with FLAG-HFE in these cells (Fig. 1C). To eliminate the possibility that HFE-TFR2 interaction involves TFR1 (by TFR1-TFR2 heterodimerization) we examined HFE-TFR2 interaction in CHO-TRVb-0 cells, which lack TFR1 expression. Again, TFR2-myc was immunoprecipitated by anti-FLAG antibody in lysates from cells expressing both TFR2-myc and FLAG-HFE (Fig. 1D). To test whether addition or chelation of non-transferrin-bound iron influences the interaction we repeated the co-immunoprecipitation analyses after treating transfected HEK293T cells with or without iron ammonium citrate or desferrioxamine. We found that neither treatment affected binding of FLAG-HFE to TFR1-myc or TFR2-myc (Fig. 1E).

These findings contrast the lack of interaction previously reported between purified soluble ectodomains of HFE and TFR2 (11). It is possible that truncated HFE and TFR2 have altered binding characteristics compared to the native proteins. Also, expression in cell membranes might promote structural conformations or provide other molecular components that are required for, or stabilize, HFE-TFR2 interaction, while the ectodomain structures alone are sufficient for HFE-TFR1 interaction. Additionally, although residues in TFR1 essential for HFE-TFR1

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interaction are not conserved in TFR2 (11), it appears that they are not necessary for HFE-TFR2 interaction. Indeed, while mutation of a conserved RGD motif in TFR1 abrogates its interaction with both HFE and transferrin (16), we demonstrate (Fig. 1A) that this mutation in TFR2 does not affect binding of TFR2 to HFE.

To address whether hemochromatosis-associated mutations (HFEH63D, TFR2M172K, TFR2Y250X and TFR2Q690P) affect HFE-TFR2 interaction we introduced corresponding mutations (HFEH67D, TFR2M167K, TFR2Y245X and TFR2K685P) into the mouse proteins. We show that wildtype FLAG-HFE interacts with all three TFR2 mutants tested (Fig. 1A). Most notably the TFR2Y245X

mutant that lacks 553 amino-acids of the extracellular domain retains ability to interact with HFE suggesting that known disease-associated mutations downstream of Y250X (17) also do not influence HFE-TFR2 binding. Similarly, the HFEH67D mutation does not abrogate HFE-TFR2 binding (Fig. 1B). Since iron homeostasis is compromised by these mutations in humans, our observations imply that in disease states, HFE and TFR2 interact but subsequent signal transduction leading to hepcidin production is impaired. This in turn suggests that signal transduction initiated by the HFE-TFR2 interaction may require other molecular components that together with HFE and TFR2 form an iron sensor and signal transduction effector.

Fe2-TF competes with HFE for binding to TFR1

(16) displacing HFE from the HFE-TFR1 complex. Also, increased serum transferrin saturation (9) and treatment of cells with Fe2-TF (9, 10) enhances TFR2 protein stability. It is possible that when serum Fe2-TF is elevated, increased cellular TFR2 competes with TFR1 for binding to HFE in hepatocytes. To test whether HFE-TFR1 interaction is competed by TFR2, CHO-TRVb-1 cells, stably over-expressing TFR1 but not expressing TFR2, were transfected with either FLAG-HFE alone or with added increasing amounts of TFR2-myc cDNA. Lysates were assayed for the co-immunoprecipitation of FLAG-HFE and TFR2-myc or endogenous TFR1. Increasing TFR2 expression reduced the amount of TFR1 co-immunoprecipitated with HFE, as determined after normalization to total cellular TFR1 (Fig. 2A and B). The expression of T91-Display did not alter HFE-TFR1 co-

immunoprecipitation. Additionally, as expected, treatment with Fe2-TF, reflecting competition of HFE-TFR1 interaction by transferrin, reduced co-immunoprecipitation of TFR1 with HFE. Taken together, these results strongly suggest that TFR2 competes with TFR1 for binding to HFE irrespective of Fe2-TF levels. Maximum competition of HFE-TFR1 interaction was seen, however, in the presence of both Fe2-TF and highest TFR2 expression. Elevated serum Fe2-TF increases TFR2 expression (9, 10) with concomitant reduction of TFR1 (18), in hepatocytes. These changes would favorably bias the formation of an HFE-TFR2 complex over an HFE-TFR1 complex. Thus emerges an iron status sensing role for HFE and the HFE-TFR2 interaction.

HFE, TFR2 and HJV proteins appear to be upstream regulators of hepcidin production (reviewed in reference 4) and Fe2-TF is likely the iron status signal (8, 19). Since serum Fe2-TF (20)

and TFR2 expression (9, 10) are increased and Hjv and Hamp are intact in Hfe-/- mice that show dysregulated iron homeostasis (20), it appears that HFE is integral to the signal transduction mechanism that senses increased Fe2-TF and produces a cellular response in terms of regulated hepcidin expression. We therefore propose a new model for iron homeostasis (Fig. 3). Under basal transferrin saturation, HFE remains complexed with TFR1. When serum Fe2-TF increases, HFE is competed by Fe2-TF from TFR1, and thus dissociated, HFE constitutes a molecular sensor for serum transferrin saturation. HFE conveys the elevated Fe2-TF status by interaction with TFR2, leading to the formation of an initial iron sensor and signal transduction effector complex. Downstream signaling would then lead to the production of hepcidin that contributes to the regulation of whole-body iron homeostasis. In HFE or TFR2 dependent human hemochromatosis and related mouse models, this sensor and signal transduction effector complex would not be functional, hepcidin production would not be induced by high Fe2-TF, thereby causing dysregulation of whole-body iron homeostasis and iron loading in parenchymal cells.

In conclusion, HFE and TFR2 interact in cells including hepatocytes and this interaction may form the basis of a systemic iron sensor in hepatocytes.

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Acknowledgments — We thank Dr. Y. Sidis for providing the T91-Display-myc cDNA and are indebted to Drs A. Mukherjee and B. Ballif for invaluable discussion and advice. T.G. was supported by a research fellowship from the Cooley’s Anemia Foundation. This study was funded by the Howard Hughes Medical Institute.

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FIGURE LEGENDS FIGURE 1. HFE and TFR2 interact. HEK293T (A, B, E), AML12 (C) or CHO-TRVb-0 (D) cells were transfected with FLAG-HFE, TFR2-myc and TFR1-myc cDNAs as indicated. Cell lysates (A, B, D, E) or crude membrane preparations (C) were used for immunoprecipitation (IP) and subsequent western blot analysis (WB) using indicated antibodies. Mouse homologs of hemochromatosis-associated mutations in TFR2 (A) and HFE (B) were tested in co-immunoprecipitation analyses as indicated. E, Cells were either untreated or treated with ferric ammonium citrate (20 µg/ml) or desferrioxamine (20 µM) for 24 hours before lysis. Vector or T91-Display-myc transfected cells served as controls. Molecular mass standards (–) and immunoreactive bands (←) are indicated. FIGURE 2. TFR2 competes with TFR1 for binding to HFE. A, CHO-TRVb-1 cells, stably over-expressing TFR1, were transfected with FLAG-HFE either alone or with increasing amounts of TFR2-myc, T91-Display-myc or vector as indicated. Cells were either treated with Fe2-Transferrin (75 µg/ml) for 1 hour or left untreated. Cell lysates were used for western blot analysis (WB) or immunoprecipitation (IP) followed by WB using indicated antibodies. Specific immunoreactive bands are indicated by arrows. (*) Only the 37 – 50 kDa region of the anti-myc WB is shown to demonstrate T91-Display-myc expression. TFR2-myc was also identified in lanes 3, 4, 8 and 9 (not shown). B, Bar graph showing the ratio between TFR1 co-immunoprecipitated (Co-IP) with FLAG-HFE and total TFR1 present in whole cell lysates as quantified from A above, using NIH Image. FIGURE 3. A new model for serum iron sensing: a role for HFE and the HFE-TFR2 interaction. HFE and TFR1 exist as a complex at the plasma membrane during low or basal serum iron conditions. Serum diferric-transferrin (Fe2-TF) competes with HFE for binding to TFR1. Increased serum transferrin saturation results in the dissociation of HFE from TFR1. HFE, acting as an iron sensor, then binds TFR2 and thus conveys the Fe2-TF status to a putative iron sensor and signal transduction effector complex. Downstream induction of hepcidin production contributes to whole-body iron homeostasis. Mutation or absence of HFE or TFR2, prevents formation of the iron sensor and signal transduction effector complex leading to dysregulation of systemic iron homeostasis as evidenced in human hereditary hemochromatosis and mouse models of these diseases.

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Tapasree Goswami and Nancy C. Andrewssuggests a molecular mechanism for mammalian iron sensing

Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2

published online August 7, 2006J. Biol. Chem. 

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