The Cytoplasmic Domain of Transferrin Receptor 2 Dictates Its Stability and Response to Holo-transferrin in Hep3B Cells
2007; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês
10.1074/jbc.m610127200
ISSN1083-351X
Autores Tópico(s)Trace Elements in Health
ResumoTransferrin receptor 2 (TfR2) is a homolog of transferrin receptor 1 (TfR1), the receptor responsible for the uptake of iron-loaded transferrin (holo-Tf) into cells. Unlike the ubiquitous TfR1, TfR2 is predominantly expressed in the liver. Mutations in TfR2 gene cause a rare autosomal recessive form of the iron overload disease, hereditary hemochromatosis. Previous studies demonstrated that holo-Tf increases TfR2 levels by stabilizing TfR2 at the protein level. In this study we constructed two chimeras, one of which had the cytoplasmic domain of TfR2 and the remaining portion of TfR1 and the other with the cytoplasmic and transmembrane domain of TfR1 joined to the ectodomain of TfR2. Similar to TfR2, the levels of the chimera containing only the cytoplasmic domain of TfR2 increased in a time- and dose-dependent manner after the addition of holo-Tf to the medium. The half-life of the chimera increased 2.7-fold in cells exposed to holo-Tf like the endogenous TfR2 in HepG2 cells. Like TfR2 and unlike TfR1, the levels of the chimera did not respond to intracellular iron content. These results suggest that although holo-Tf binding to the ectodomain is necessary, the cytoplasmic domain of TfR2 is largely responsible for its stabilization by holo-Tf. Transferrin receptor 2 (TfR2) is a homolog of transferrin receptor 1 (TfR1), the receptor responsible for the uptake of iron-loaded transferrin (holo-Tf) into cells. Unlike the ubiquitous TfR1, TfR2 is predominantly expressed in the liver. Mutations in TfR2 gene cause a rare autosomal recessive form of the iron overload disease, hereditary hemochromatosis. Previous studies demonstrated that holo-Tf increases TfR2 levels by stabilizing TfR2 at the protein level. In this study we constructed two chimeras, one of which had the cytoplasmic domain of TfR2 and the remaining portion of TfR1 and the other with the cytoplasmic and transmembrane domain of TfR1 joined to the ectodomain of TfR2. Similar to TfR2, the levels of the chimera containing only the cytoplasmic domain of TfR2 increased in a time- and dose-dependent manner after the addition of holo-Tf to the medium. The half-life of the chimera increased 2.7-fold in cells exposed to holo-Tf like the endogenous TfR2 in HepG2 cells. Like TfR2 and unlike TfR1, the levels of the chimera did not respond to intracellular iron content. These results suggest that although holo-Tf binding to the ectodomain is necessary, the cytoplasmic domain of TfR2 is largely responsible for its stabilization by holo-Tf. Transferrin (Tf) 2The abbreviations used are: Tf, transferrin receptor 2; TfR, Tf receptor; holo-Tf, iron-saturated transferrin; apoTf, transferrin lacking iron; aa, amino acid; FLAG epitope tag, DYKDDDDK epitope tag; PBS, phosphate-buffered saline; HH, hereditary hemochromatosis; NTA, nitrilotriacetic acid.2The abbreviations used are: Tf, transferrin receptor 2; TfR, Tf receptor; holo-Tf, iron-saturated transferrin; apoTf, transferrin lacking iron; aa, amino acid; FLAG epitope tag, DYKDDDDK epitope tag; PBS, phosphate-buffered saline; HH, hereditary hemochromatosis; NTA, nitrilotriacetic acid. receptor 2 (TfR2) is a homolog of transferrin receptor 1 (TfR1). TfR1 is ubiquitously expressed in most cell types with the notable exception of mature erythroid cells, whereas TfR2 is predominantly expressed in the liver (1Kawabata H. Nakamaki T. Ikonomi P. Smith R.D. Germain R.S. Koeffler H.P. Blood. 2001; 98: 2714-2719Crossref PubMed Scopus (101) Google Scholar, 2Fleming R.E. Migas M.C. Holden C.C. Waheed A. Britton R.S. Tomatsu S. Bacon B.R. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2214-2219Crossref PubMed Scopus (234) Google Scholar, 3Kawabata H. Germain R.S. Ikezoe T. Tong X. Green E.M. Gombart A.F. Koeffler H.P. Blood. 2001; 98: 1949-1954Crossref PubMed Scopus (120) Google Scholar). Like TfR1, TfR2 binds transferrin (Tf) in a pH-dependent manner but with 25-fold lower affinity and delivers iron to cells (4West Jr., A.P. Bennett M.J. Sellers V.M. Andrews N.C. Enns C.A. Bjorkman P.J. J. Biol. Chem. 2000; 275: 38135-38138Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 5Kawabata H. Yang R. Hirama T. Vuong P.T. Kawano S. Gombart A.F. Koeffler H.P. J. Biol. Chem. 1999; 274: 20826-20832Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). The regulation of TfR1 and TfR2 is distinctly different. TfR1 is inversely regulated at the mRNA level by an intracellular iron pool through interaction of the cytosolic iron regulatory protein with iron-responsive elements in the 3′-untranslated region of TfR1 mRNA (6Klausner R.D. Rouault T.A. Harford J.B. Cell. 1993; 72: 19-28Abstract Full Text PDF PubMed Scopus (1045) Google Scholar). By contrast, TfR2 mRNA is not regulated by this mechanism. In keeping with this finding, no iron-responsive element is discernable in TfR2 mRNA (2Fleming R.E. Migas M.C. Holden C.C. Waheed A. Britton R.S. Tomatsu S. Bacon B.R. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2214-2219Crossref PubMed Scopus (234) Google Scholar, 3Kawabata H. Germain R.S. Ikezoe T. Tong X. Green E.M. Gombart A.F. Koeffler H.P. Blood. 2001; 98: 1949-1954Crossref PubMed Scopus (120) Google Scholar, 5Kawabata H. Yang R. Hirama T. Vuong P.T. Kawano S. Gombart A.F. Koeffler H.P. J. Biol. Chem. 1999; 274: 20826-20832Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 7Kawabata H. Germain R.S. Vuong P.T. Nakamaki T. Said J.W. Koeffler H.P. J. Biol. Chem. 2000; 275: 16618-16625Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). TfR2 is regulated at the level of protein stability by a novel mechanism. Unlike most receptors identified to date, Tf binding to TfR2 stabilizes TfR2 (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar). Most receptors either cycle independently of their ligand and their half-life is not affected by ligand binding as is the case for TfR1 (10Ajioka R.S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6445-6449Crossref PubMed Scopus (58) Google Scholar) or upon ligand binding are targeted for degradation in the lysosome, like the epidermal growth factor (11Carpenter G. Cohen S. J. Cell Biol. 1976; 71: 159-171Crossref PubMed Scopus (857) Google Scholar). In contrast, the half-life of TfR2 increases in response to holo-Tf treatment over a physiological range of holo-Tf concentrations (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar), indicating that TfR2 correlates with changes in Tf saturation. The response of TfR2 to holo-Tf appears to be hepatocyte-specific. Non-hepatic cell lines that either endogenously express TfR2 such as K562 cells or are transfected with a plasmid coding for TfR2 do not show a similar response (7Kawabata H. Germain R.S. Vuong P.T. Nakamaki T. Said J.W. Koeffler H.P. J. Biol. Chem. 2000; 275: 16618-16625Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar, 12Vogt T. Blackwell A. Giannetti A. Bjorkman P. Enns C. Blood. 2002; 101: 2008-2014Crossref PubMed Scopus (41) Google Scholar). Understanding which domain is important for the stability and trafficking of TfR2 is crucial for understanding its function in iron homeostasis. Although the ectodomains of TfR1 and TfR2 show 45% identity and 66% similarity, their cytoplasmic domains bear no significant similarity. In this study we constructed two chimeras consisting of different domains of TfR1 and TfR2 and investigated their response to holo-Tf to map which domain of TfR2 is responsible for its stabilization in a hepatoma cell line that does not express detectable levels of TfR2. We found that although Tf binding is necessary for the increase in stability, it did not matter which TfR binding domain was utilized. The cytoplasmic domain of TfR2 is largely responsible for its stabilization by holo-Tf. Chimera Plasmid Construction—The templates for PCR amplification of TfR1 and TfR2 are fTfR/pUHD10-3 (13Warren R.A. Green F.A. Enns C.A. J. Biol. Chem. 1997; 272: 2116-2121Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and pcDNA3.1-TfR2, respectively. For the chimera of TfR2-cyto/TfR1-tm-ecto-FLAG, a fragment of TfR2 cytoplasmic domain (amino acids (aa) 1–80) was amplified using primers 5′-CTGGATCCATGGAGCGGCTTTGGGGTCTA-3′ (forward) and 5′-CAGATACTTCCACTACACCTCCGTCCTGCTGCC-3′ (TfR2 aa 75–80 and TfR1 aa 62–63, reverse), a fragment of TfR1 transmembrane and ectodomain (aa 62–760) was amplified using primers 5′-GCAGCAGGACGGAGGTGTAGTGGAAGTATCT-3′ (TfR2 aa 75–80 and TfR1 aa 62–63, forward) and 5′-CTGAATTCTTACTTGTCATCGTCGTC-3′ (reverse), then overlapping PCRs were employed to generate TfR2-cyto/TfR1-tm-ecto-FLAG using primers of 5′-CTGGATCCATGGAGCGGCTTTGGGGTCTA-3′ (forward) and 5′-CTGAATTCTTACTTGTCATCGTCGTC-3′ (reverse). A FLAG epitope tag (DYKDDDDK) was added to the C terminus of TfR1 to distinguish the chimera from endogenous TfR1. For the chimera of TfR1-cyto-tm/TfR2-ecto, a fragment of TfR1 cytoplasmic and transmembrane domain (aa 1–89) was amplified using primers of 5′-CTGGCAGGACCCTCGACAATAGCCCAAGTA-3′ (forward) and 5′-TACTTGGGCTATTGTCGAGGGTCCTGCCAG-3′ (TfR1 aa 85–89, TfR2 aa 105–109, reverse), a fragment of TfR2 ectodomain (aa 105–810) was amplified using primers of 5′-TTGGCTACTTGGGCTATTGTCGAGGGTCCTGCCAGGCGTGC-3′ (TfR1 aa 85–89, TfR2 aa 105–109, forward) and 5′-CTGAATTCTCAGAAGTTGTTATCAATGT-3′ (reverse), then overlapping PCR was employed to generate TfR1-cyto-tm/TfR2-ecto using primers of 5′-CTGGCAGGACCCTCGACAATAGCCCAAGTA-3′ (forward) and 5′-CTGAATTCTCAGAAGTTGTTATCAATGT-3′ (reverse). The PCR product of TfR2-cyto/TfR1-tm-ecto-FLAG was digested with BamHI and EcoRI and inserted into pcDNA3 to generate pcDNA3-TfR2-cyto/TfR1-tm-ecto-FLAG, and the PCR product of TfR1-cyto-tm/TfR2-ecto was digested with HindIII and EcoRI and inserted into pcDNA3 to generate pcDNA3-TfR1-cyto-tm/TfR2-ecto. All constructs were verified by sequencing. Cell Culture—Hep3B cells were maintained in minimal essential medium (Invitrogen) supplemented with 1.0 mm sodium pyruvate, 0.1 mm nonessential amino acids (Invitrogen), and 10% fetal bovine serum. Hep3B cells stably expressing a chimera of TfR2-cyto/TfR1-tm-ecto-FLAG (Hep3B/TfR2CD) and wild type TfR2 (Hep3B/wtTfR2) were maintained in minimal essential medium with 400 μg/ml G418. In all experiments the cells were seeded at 3 × 104 cell/cm2 for 1 day before treatment. Holo-Tf, apoTf, FeNTA, and cycloheximide were added to the culture medium of cells as described in the figure legends. Antibodies—Rabbit anti-human TfR2 serum (16637) was produced at Pocono Rabbit Farm and Laboratory (Canadensis, PA) by immunizing rabbits with purified human TfR2 ectodomain (gift from Pamela Bjorkman, California Institute of Technology). Generation of rabbit anti-FLAG, monoclonal antibodies 3B8 2A1 against the ectodomain of human TfR1, and 9F8 1C11 against the ectodomain of TfR2 were described previously (12Vogt T. Blackwell A. Giannetti A. Bjorkman P. Enns C. Blood. 2002; 101: 2008-2014Crossref PubMed Scopus (41) Google Scholar). H68.4, which is specific to the cytoplasmic domain of TfR1, M2 anti-FLAG, mouse monoclonal anti-actin, and rabbit anti-ferritin were purchased from Zymed Laboratories Inc. (San, CA), Sigma, Chemicon (Temecula, CA), and DAKO A/S, (Denmark) respectively. Secondary antibodies against rabbit and mouse (IgG) conjugated to horseradish peroxidase were purchased from Chemicon. Fluorescence-labeled AlexaFluor 488, and AlexaFluor 680 goat anti-rabbit IgG and AlexaFluor 546 goat anti-mouse IgG were purchased from Molecular Probes (Eugene, OR), IRDye 800 donkey anti-mouse IgG secondary antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA). Reagent Preparation—Stock solutions of Tf were prepared by dissolving holo-Tf or apoTf (Serologicals, Norcross, GA) in phosphate-buffered saline (PBS; pH 7.4). For FeNTA, stock solutions of 400 mm nitrilotriacetic acid (NTA; Sigma) in PBS (pH 7.4) and 400 mm FeCl3 in 0.1 n HCl were prepared. Before each experiment, FeCl3 and NTA were combined in a 1:40 molar ratio to give a final iron concentration of 10 mm. For the control, NTA was combined with the appropriate volume of 0.1 n HCl. Stock solutions of 10 mg/ml cycloheximide (Sigma) were prepared in PBS. Cell Transfection—For stable transfection, Hep3B cells were transfected on day 1 with pcDNA3-TfR2cyto/TfR1-tm-ecto-FLAG using Lipofectamine (Invitrogen), and on day 3, cells were selected by 400 μg/ml G418 (Geneticin). The stable clone expressing TfR2-cyto/TfR1-tm-ecto-FLAG was recloned to ensure a pure cell line. Selected clones were screened by gel electrophoresis and Western blot analysis and immunofluorescence with M2 anti-FLAG antibody. Transfected cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum and 400 μg/ml G418. For transient transfections, Hep3B cells in 6-well plates were transfected on day 1 with pcDNA3-TfR1-cyto-tm/TfR2-ecto using Lipofectamine. On day 2, transfected cells in 1 well of a 6-well plate were split to 2 wells of a 12-well plate, on day 3, cells were treated with holo-Tf or PBS for 24 h, and on day 4 lysates were collected. Western Blots—Cells were lysed on ice in NET-Triton X-100 buffer (150 mm NaCl, 5 mm EDTA, 10 mm Tris, pH 7.4, 1% Triton X-100) containing 1× Complete Mini Protease Inhibitor Mixture (Roche Diagnostics). Lysates were cleared by centrifugation at 15,000 × g for 10 min. Protein concentration was measured using the BCA Protein Assay (Pierce). Aliquots of lysates containing 60 or 80 μg of total protein were incubated in 3.6× Laemmli buffer (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) for 5 min at 95 °C and subjected to reducing SDS-PAGE on 10% gels for analysis of TfR2-cyto/TfR1-tm-ecto-FLAG, TfR1-cyto-tm/TfR2-ecto, TfR1, TfR2, and actin or 12% gels for analysis of ferritin. After transfer to nitrocellulose, proteins were detected using M2 anti-FLAG (1:4,000), rabbit anti-TfR2 (1:10,000), 3B8 2A1 (1:5,000), H68.4 (1:10,000), mouse anti-actin (1:10,000) or rabbit anti-ferritin (1:2,000) primary antibodies and then horseradish peroxidase-conjugated (1:10,000) or fluorescently labeled (1:5,000) secondary antibodies. Bands were visualized by chemiluminescence (SuperSignal WestPico; Pierce) or were visualized and quantified by fluorescence imaging (Odyssey Infrared Imaging System; Li-Cor, Lincoln, NB). Rabbit anti-human TfR2 was incubated with Alexa 680 goat anti-rabbit fluorescent secondary antibody. All mouse monoclonal primary antibodies (M2 anti-FLAG, anti-actin, 3B8 2A1, and H68.4) were incubated with the IRDye 800 donkey anti-mouse fluorescent secondary antibodies. The fluorescent bands were converted to black and white images in the figures. Immunofluorescence—Hep3B/TfR2CD cells and Hep3B cells transiently transfected with TfR1-cyto-tm/TfR2-ecto growing on coverslips were washed twice with PBS, fixed for 15 min with 4% (v/v) paraformaldehyde in PBS, then washed 3 times with PBS, and blocked with 10% fetal bovine serum in PBS for 60 min at room temperature. Cells were incubated in primary antibodies diluted in PBS containing 5% fetal bovine serum for 60 min, washed 3 × 5 min with PBS, incubated with secondary antibodies diluted in PBS for 60 min, and washed 3 × 5 min with PBS, and coverslips were mounted in ProLong Gold anti-fade reagent (Molecular Probes/Invitrogen). Images were acquired by laser-scanning confocal microscopy using a Plan-Apochromat 20× objective on a Zeiss LSM 5 Pascal confocal inverted microscope. AlexaFluor 546 and AlexaFluor 488 signals were excited with helium neon (543 nm) and argon (488 nm) lasers, respectively, and obtained using the single-tracking function. Cells expressing TfR2-cyto/TfR1-tm-ecto-FLAG or TfR1-cyto-tm/TfR2-ecto were labeled with either 25 μg/ml M2 anti-FLAG or 8 μg/ml purified IgG fraction of the 16637 rabbit anti-TfR2 polyclonal anti-serum, respectively and visualized with either goat anti-mouse AlexaFluor 546 (1:500) or goat anti-rabbit AlexaFluor 488 (1:500), respectively. Immunoprecipitation—Hep3B/TfR2CD (250 μg) and Hep3B (250 μg) (as a control) cell lysates were precleared with protein A or protein G for 30 min at 4 °C. The cell lysates were incubated overnight at 4 °C with either 2 μl of rabbit anti-FLAG to immunoprecipitate the chimera containing the FLAG epitope tag or in separate experiments with 2 μl of H68.4 to immunoprecipitate endogenous TfR1. After centrifugation, the pellets were washed 3 times for 5 min with NET-1% Triton X-100, and protein was eluted by boiling in SDS loading buffer at 95 °C for 5 min. The supernatant of immunoprecipitation with H68.4 was re-immunoprecipitated with 2 μl of H68.4 to determine how much endogenous TfR1 was left after the first immunoprecipitation. The majority (88%) of TfR1 was immunoprecipitated in the first round of immunoprecipitation, and the remaining 12% was immunoprecipitated in the second round. The first immunoprecipitation with rabbit anti-FLAG was split to two equal aliquots to detect the chimera with M2 anti-FLAG and endogenous TfR1 with H68.4, respectively. The sum of first and second immunoprecipitation of H68.4 detected with H68.4 is the total endogenous TfR1 level. All of these samples were subjected to 10% SDS-PAGE, transferred to nitrocellulose, and immuno-detected by H68.4 and M2 anti-FLAG and quantified by the IRDye 800 donkey anti-mouse fluorescent secondary antibodies using a Licor fluorimeter. The Cytoplasmic Domain of TfR2 Is Largely Responsible for the Stabilization of TfR2 by Holo-Tf—To determine which domains are responsible for stabilization of TfR2 by holo-Tf, chimeras consisting of different combinations of the ecto-, transmembrane, and cytoplasmic domains of TfR1 and TfR2 were generated by overlapping PCR (Fig. 1A). Like TfR1, TfR2 is a type II transmembrane protein consisting of an N-terminal cytoplasmic domain, a single transmembrane domain, and a C-terminal ectodomain. A FLAG epitope tag was added to the C terminus of TfR1 in chimera TfR2cyto/TfR1-tm-ecto-FLAG to distinguish from endogenous TfR1 (Fig. 1A). Hep3B cells, which express endogenous TfR1 but no detectable TfR2, were used to express the chimera with the cytoplasmic domain of TfR2 and the transmembrane and ectodomains of TfR1. The chimera was stably expressed in Hep3B cells (Hep3B/TfR2CD). This chimera formed heterodimers with endogenous TfR1. Cell lysates of Hep3B/TfR2CD were immunoprecipitated with anti-FLAG antibody to isolate the TfR2cyto/TfR1-tm-ecto-FLAG chimera and TfR1 associated with the chimera. The amount of TfR1 co-precipitating with the chimera was compared with the total TfR1 immunoprecipitated (Fig. 1B). Quantitation of the relative amounts of TfR1 immunoprecipitated demonstrated that ∼7% of TfR1 formed heterodimers with TfR2cyto/TfR1-tm-ecto-FLAG. Thus, only a small fraction of TfR1 associated with this chimera. TfR2cyto/TfR1-tm-ecto-FLAG trafficked to the cell surface and bound holo-Tf, indicating that it was not malfolded and retained in the endoplasmic reticulum (Fig. 1C). 3J. Chen and C. A. Enns, unpublished results. To test whether the cytoplasmic domain was sufficient for the increase in the steady state levels of TfR2 by holo-Tf, Hep3B/TfR2CD cells were treated with 25 μm (2 mg/ml) holo-Tf for 24 h, and protein levels were examined by Western blot detection with M2 anti-FLAG antibody. The levels of the chimera increased by about 2.6-fold (Fig. 1D). This is the same -fold increase that was seen in HepG2 cells that endogenously express TfR2 (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar) and Hep3B cells stably transfected with TfR2 (15Johnson M.B. Chen J. Murchison N. Green F. Enns C.A. Mol. Biol. Cell. 2007; (in press)Google Scholar), indicating that the cytoplasmic domain of TfR2 has an important role in its response to holo-Tf. Similar to TfR2, which was able to internalize anti-TfR2 antibody and partially localize in EEA1 (early endosome antigen 1)-positive compartments (supplemental Figs. 1–3), the chimera was capable of internalizing M2 anti-FLAG antibody (supplemental Fig. 4). Hep3B cells were transiently transfected with a chimera consisting of the ectodomain of TfR2 and the remaining domains of TfR1 (TfR1cyto-tm/TfR2-ecto) to examine whether the ectodomain of TfR2 also plays a role in response to holo-Tf. This chimera also trafficked to the cell surface and bound holo-Tf (Fig. 1E), 3J. Chen and C. A. Enns, unpublished results. indicating that it was not malfolded. Cells were treated with 25 μm holo-Tf for 24 h, and protein levels were examined by Western blot analysis with rabbit anti-TfR2. The chimera with the ectodomain of TfR2 and the remaining domains of TfR1 did not respond to the addition of holo-Tf (Fig. 1F). Thus, the cytoplasmic domain of TfR2 is largely responsible for the stabilization of TfR2 by holo-Tf. Holo-Tf Is Required for the Increased Level of TfR2cyto/TfR1-tm-ecto-FLAG—Holo-Tf supplies cells with both Tf and iron. In HepG2 cells that endogenously express TfR2, the binding of holo-Tf to TfR2 appeared to be necessary for the stabilization of TfR2 (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 15Johnson M.B. Chen J. Murchison N. Green F. Enns C.A. Mol. Biol. Cell. 2007; (in press)Google Scholar). Neither the addition of apoTf, which does not bind to TfR2 appreciably at neutral pH, nor iron to cells increased TfR2 levels (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar). To test whether the same was true in cells expressing the chimera with the cytoplasmic domain of TfR2, Hep3B/TfR2CD cells were cultured in 25 μm apoTf for 24 h, and the levels of TfR2-cyto/TfR1-tm-ecto-FLAG were quantified by Western blot detection with fluorescence-labeled secondary antibodies. Similar to TfR2 (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar), TfR2-cyto/TfR1-tm-ecto-FLAG increased by about 2.6-fold in response to holo-Tf but remained unaltered in response to apoTf (Fig. 2A). TfR1 and ferritin, which are post-transcriptionally regulated by the iron-responsive element/iron regulatory protein system in response to changes of intracellular iron, served as positive controls to show that the cells were being loaded with iron. As expected, TfR1 decreased by about 25% (Fig. 2B), ferritin levels increased (Fig. 2C) markedly, and neither protein changes after the addition of apoTf. The increase of the chimera containing the cytoplasmic domain of TfR2 in response to holo-Tf and lack of the response to apoTf is indistinguishable from wild type TfR2 as reported previously (9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar). These results demonstrate that the Tf binding to the chimera containing the ectodomain and transmembrane domain of TfR1 and the cytoplasmic domain of TfR2 responds similarly as endogenous TfR2 in HepG2 cells. Because holo-Tf supplies cells with iron, we wanted to determine whether the increase in TfR2-cyto/TfR1-tm-ecto-FLAG results from elevated cellular iron levels. FeNTA (100 μm), a non-Tf-bound iron source, was added to the culture medium for 24 h, and protein levels were examined by Western blot. As expected, TfR1 levels decreased by 17% (Fig. 3B), and ferritin levels increased remarkably in cells treated with FeNTA (Fig. 3C). However, TfR2-cyto/TfR1-tm-ecto-FLAG levels remained unchanged (Fig. 3A). Therefore, in contrast to TfR1 and ferritin but similar to TfR2, the levels of the chimera are not controlled by intracellular iron. The Effect of Holo-Tf on TfR2-cyto/TfR1-tm-ecto-FLAG Is Time- and Dose-dependent—Hep3B/TfR2CD cells were cultured in medium with 25 μm holo-Tf for 0–72 h, and protein levels were examined by Western blot analysis to test time dependence of holo-Tf on stabilization of TfR2-cyto/TfR1-tmecto-FLAG. After the addition of 25 μm Tf, TfR2-cyto/TfR1-tm-ecto-FLAG levels increased visibly within 4 h and reached a maximum at 48 h (Fig. 4A). Control cells that were not treated with holo-Tf did not show significant changes in TfR2-cyto/TfR1-tm-ecto-FLAG (Fig. 4B). By comparison, in HepG2 cells, which endogenously express TfR2, increases in TfR2 can also be detected within 4 h after holo-Tf treatment and reach maximal levels at 48 h (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar). The dose response of TfR2-cyto/TfR1-tm-ecto-FLAG to holo-Tf was examined by adding 0–30 μm (0–2.4 mg/ml) holo-Tf to the medium for 24 h. Quantitative Western blot analysis showed that TfR2-cyto/TfR1-tm-ecto-FLAG increased as the concentration of holo-Tf increased from 0 to 30 μm (Fig. 5A). The increase in TfR2-cyto/TfR1-tm-ecto-FLAG was half-maximal when the concentration of holo-Tf was ∼3.1 μm (Fig. 5B), which was consistent with the observations of Johnson and Enns (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar) and Robb and Wessling-Resnick (9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar), indicating the chimera acts in the same way as TfR2. The chimera was stabilized maximally when the holo-Tf concentration was 30 μm. The affinity of TfR1 for holo-Tf is much higher than that of TfR2; however, the addition of the FLAG epitope tag to the C terminus of TfR1 decreased the affinity of Tf for TfR1 by about 10-fold (13Warren R.A. Green F.A. Enns C.A. J. Biol. Chem. 1997; 272: 2116-2121Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and made the chimera responsive to changes in holo-Tf over the same range in concentration as TfR2 (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar). Holo-Tf Increases TfR2-cyto/TfR1-tm-ecto-FLAG Protein Stability—The elevation in TfR2 is due to an increase of its half-life in cells exposed to holo-Tf (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar, 9Robb A. Wessling-Resnick M. Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (166) Google Scholar). To investigate whether the elevation in TfR2-cyto/TfR1-tm-ecto-FLAG also results from an increase in stability of the chimera, we measured its half-life in Hep3B/TfR2CD cells. After culturing for 24 h in the medium without (0 μm) or with 25 μm holo-Tf, 100 μg/ml cycloheximide was added to the medium to inhibit protein synthesis. The levels of TfR2-cyto/TfR1-tm-ecto-FLAG were quantified by Western blot detection with fluorescence-labeled secondary antibodies. In the absence of Tf, the chimeric TfR2 decreased to half the original level after 8 h of treatment with cycloheximide; however, in holo-Tf-treated cells, the chimera was stabilized (Fig. 6). The half-life of TfR2-cyto/TfR1-tm-ecto-FLAG calculated from 5 independent experiments was 8.1 and 21.8 h in untreated and holo-Tf-treated cells, respectively. The 2.7-fold increase in the stability of the chimera with the TfR2 cytoplasmic domain after the addition of holo-Tf is similar to that of TfR2 endogenously expressed in HepG2 cells (8Johnson M.B. Enns C.A. Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (186) Google Scholar). Thus, the cytoplasmic domain plays the predominant role in the trafficking of TfR2 in response to holo-Tf treatment of cells. A Monoclonal Antibody to the Ectodomain of TfR2 Down-regulates rather than Stabilizes TfR2—To test whether binding of another protein to the ectodomain could also stabilize TfR2, we treated Hep3B cells stably expressing wild type TfR2 with a monoclonal antibody to the ectodomain of TfR2 (9F8 1C11) for 4 h and then examined TfR2 levels by Western blot analysis. In contrast to holo-Tf treatment (Fig. 7B), TfR2 levels in antibody-treated cells decreased by 53% (Fig. 7A). This antibody binds TfR2 with similar affinity as Tf (Ka ∼ 15 nm) but does not compete with Tf binding (results not shown). These results indicate that the binding of holo-Tf to the TfR1 or TfR2 ectodomains transmits a signal to the cytoplasmic domain or that the complex interacts with another protein(s) to alter its trafficking away from a degradation pathway. Iron is an essential but potentially noxious metal for almost all organisms (16Syed B.A. Sargent P.J. Farnaud S. Evans R.W. Hemoglobin. 2006; 30: 69-80Crossref PubMed Scopus (11) Google Scholar). Therefore, iron uptake into cells and body is tightly regulated. In humans, iron deficiency results in anemia, whereas excess iron results in hemochromatosis. The consequences of iron overload include liver cirrhosis, hepatocellular carcinoma, diabetes, heart failure, arthritis, and hypogonadism (17Andrews N.C. Curr. Opin. Pediatr. 2000; 12: 596-602Crossref PubMed Scopus (11) Google Scholar, 18Cartwright G.E. Edwards C.Q. Kravitz K. 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