Molecular Cloning of Transferrin Receptor 2

Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: α (∼2.9 kilobase pairs), and β (∼2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-α protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-β protein lacked the amino-terminal portion of the TfR2-α protein including the putative transmembrane domain. Northern blot analysis showed that the α transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-α. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tf-bound 55Fe uptake. Taken together, TfR2-α may be a second transferrin receptor that can mediate cellular iron transport. Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: α (∼2.9 kilobase pairs), and β (∼2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-α protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-β protein lacked the amino-terminal portion of the TfR2-α protein including the putative transmembrane domain. Northern blot analysis showed that the α transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-α. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tf-bound 55Fe uptake. Taken together, TfR2-α may be a second transferrin receptor that can mediate cellular iron transport. Iron is essential in a wide variety of cellular processes including oxidative phosphorylation and DNA synthesis. Our knowledge concerning cellular iron transport has been markedly advanced by the recent discoveries of several genes such as HFE, associated with hereditary hemochromatosis (1Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R.J. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E. Meyer N.C. Mintier G.A. Moeller N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3366) Google Scholar), and divalent metal transporter (DMT1/Nramp2), a transmembrane iron transporter (2Fleming M.D. Trenor III, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1025) Google Scholar, 3Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2679) Google Scholar). One of the well-studied key molecules involved in iron uptake is transferrin receptor (TfR) 1The abbreviations used are: TfR, transferrin receptor; RT-PCR, reverse transcription-polymerase chain reaction; Tf, transferrin; Lf, lactoferrin; PSMA, prostate-specific membrane antigen; RACE, rapid amplification of cDNA ends; IRP, iron regulatory protein; kb, kilobase pair(s) (reviewed in Refs. 4Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (597) Google Scholar and 5Testa U. Pelosi E. Peschle C. Crit. Rev. Oncog. 1993; 4: 241-276PubMed Google Scholar). On the cell membrane, the TfR homodimer binds to two diferric transferrin (Tf) molecules, resulting in internalization of the complex. In the endosome, iron is released from Tf in a pH-dependent manner and is transported into the cytosol by DMT1/Nramp2 (6Fleming M.D. Romano M.A. Su M.A. Garrick L.M. Garrick M.D. Andrews N.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1148-1153Crossref PubMed Scopus (811) Google Scholar). The iron is utilized as a cofactor by heme, aconitase, cytochromes (reviewed in Ref. 7Lash A. Saleem A. Ann. Clin. Lab. Sci. 1995; 25: 20-30PubMed Google Scholar), and ribonucleotide reductase (8Kauppi B. Nielsen B.B. Ramaswamy S. Larsen I.K. Thelander M. Thelander L. Eklund H. J. Mol. Biol. 1996; 262: 706-720Crossref PubMed Scopus (129) Google Scholar), or it may be stored in ferritin molecules. The affinity of diferric Tf to TfR is modulated by HFE (9Feder J.N. Penny D.M. Irrinki A. Lee V.K. Lebron J.A. Watson N. Tsuchihashi Z. Sigal E. Bjorkman P.J. Schatzman R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1472-1477Crossref PubMed Scopus (731) Google Scholar). Although TfR-mediated endocytosis is the major pathway for cellular iron uptake, cells can also obtain iron through TfR-independent pathways from iron-bound Tf or from inorganic irons (10Trinder D. Zak O. Aisen P. Hepatology. 1996; 23: 1512-1520Crossref PubMed Google Scholar, 11Sturrock A. Alexander J. Lamb J. Carven C.M. Kaplan J. J. Biol. Chem. 1990; 265: 3139-3145Abstract Full Text PDF PubMed Google Scholar, 12Chan R.Y.Y. Ponka P. Schulman H.M. Exp. Cell Res. 1992; 202: 326-336Crossref PubMed Scopus (44) Google Scholar, 13Levy J.E. Jin O. Fujiwara Y. Kuo F. Andrews N. Nat. Genet. 1999; 21: 396-399Crossref PubMed Scopus (456) Google Scholar). These processes are thought to be through fluid-phase endocytosis, passive perfusion, or other membrane-based transport systems, and no other receptor for Tf has been reported to date. In this study, we describe the cloning of a new TfR-like family member, TfR2, which may mediate the cellular uptake of iron via a new pathway. ML-1, NB4, Kasumi 3 (myeloid leukemia), and both CHO-TRVb (TfR-deficient Chinese hamster ovary) and TRVb-1 (humanTfR stably transfected TRVb) cells were kindly provided by Drs. J. Minowada (14Fukuda M. Koeffler H.P. Minowada J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6299-6303Crossref PubMed Scopus (66) Google Scholar), M. Lanotte (15Lanotte M. Martin-Thouvenin V. Najman S. Balerini P. Valenisi F. Berger R. Blood. 1991; 77: 1080-1086Crossref PubMed Google Scholar), H. Asou (16Asou H. Suzukawa K. Kita K. Nakase K. Ueda H. Morishita K. Kamada N. Jpn. J. Cancer Res. 1996; 87: 269-274Crossref PubMed Scopus (29) Google Scholar), and T. E. McGraw (17McGraw T.E. Greenfield L. Maxfield F.R. J. Cell Biol. 1987; 105: 207-214Crossref PubMed Scopus (163) Google Scholar), respectively. All of the other cell lines were obtained from American Type Culture Collection (Manassas, VA). Human mononuclear cells were isolated from the blood of a normal volunteer by centrifugation on a Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) gradient at 400 × g for 30 min. Informed consent was obtained from the individual. Complementary DNA libraries were constructed from TF-1 (erythroid leukemia) and HL60 (myeloid leukemia) cells using a commercial kit (Marathon cDNA Amplification Kit; CLONTECH, Palo Alto, CA) and used for 5′- and 3′-rapid amplification of cDNA ends (RACE) reactions to obtain a full-length cDNA clone. Primers A (Fig.1 B) and B (5′-CAGTTGCATCATCAGGCCTTCC-3′) were used for 5′- and 3′-RACE, respectively. The products of RACE reactions were subcloned into the pGEM-Teasy vector (Promega, Madison, WI). We isolated two transcripts of 2.9 kb (α transcript) and 2.5 kb (β transcript) from the TF-1 and HL60 cDNA libraries, respectively. Genomic DNA was isolated from a human genomic library (Lambda FIX II Library; Stratagene, La Jolla, CA) using a 2.2-kb fragment of the 3′-end of the TfR2cDNA as a probe (shown asProbe-1 in Fig. 1 A). After restriction enzyme mapping, a 3.85-kb fragment that included exons 4–6 was subcloned into the pBluescript II(+) plasmid (Stratagene). Complementary and genomic DNA sequences were determined using an ABI Prism 373 automated sequencer (Perkin-Elmer). The GeneBridge 4 Radiation Hybrid Panel, RH02 (Research Genetics, Huntsville, AL) was used to determine the chromosomal location of the TfR2gene as described previously (18Yang R. Morosetti R. Koeffler H.P. Cancer Res. 1997; 57: 913-920PubMed Google Scholar). Primers A and C (Fig. 1 B) amplified a 178-base pair fragment located in exon 4. The polymerase chain reaction products were electrophoresed, Southern blotted, and hybridized with a32P-labeled probe of TfR2. Northern blot and RT-PCR analyses were performed as described previously (18Yang R. Morosetti R. Koeffler H.P. Cancer Res. 1997; 57: 913-920PubMed Google Scholar). Human tissue Northern blot membranes and cDNAs were purchased from OriGene (Rockville, MD). For Northern blot analysis, two TfR2cDNA fragments (Probe-1 and Probe-2shown in Fig. 1 A), a human β-actin cDNA fragment (OriGene), and an approximately 300-base pair TfR cDNA fragment were used as probes. For RT-PCR, the α form-specific primers (primers A and D) and the β form-specific primers (primers C and E) were used (Fig. 1 B). Conditions for amplification were 35 cycles of 94 °C for 30 s, 56 °C for 40 s, and 72 °C for 1 min. As a control, glyceraldehyde-3-phosphate dehydrogenase was amplified in a separate reaction using primers, 5′-CCATGGAGAAGGCTGGGG-3′ and 5′-CAAAGTTGTCATGGATGACC-3′ for 27 cycles. CHO-TRVb cells were maintained in F12-nutrient mixture (Life Technologies, Inc.) supplemented with 5% fetal bovine serum. An amino-terminal FLAG-tagged TfR2-α cDNA was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). This plasmid (10 μg) was transfected into CHO-TRVb cells using Lipofectin (Life Technologies, Inc.). For transient expression, cells were harvested 48 h after the transfection. We also isolated a stably expressed clone using G418 (200 μg/ml) selection and a standard limiting dilution method. The protein expression was confirmed by immunoblotting using anti-FLAG (M5) antibody (Eastman Kodak, New Haven, CT). Immunoblot analysis was performed as described previously (19Chumakov A.M. Grillier I. Chumakova E. Chih D. Slater J. Koeffler H.P. Mol. Cell. Biol. 1997; 17: 1375-1386Crossref PubMed Google Scholar). Approximately 3 × 105 cells were incubated with 5 μg/ml biotinylated human holo-Tf (Sigma) in 500 μl of minimum Eagle's medium α (Life Technologies, Inc.) in either the presence or absence of nonlabeled human holo-Tf (Sigma) or human lactoferrin (Lf) (Calbiochem, San Diego, CA) for 30 min on ice. After two washes with phosphate-buffered saline supplemented with 0.1% bovine serum albumin, the cells were incubated with streptavidin-phycoerythrin (DAKO). The cells were washed twice and subsequently analyzed by flow cytometry. 55Fe-Tf was prepared by the method described previously (20Eriksson L.C. Torndal U. Andersson G.N. Carcinogenesis. 1986; 7: 1467-1474Crossref PubMed Scopus (29) Google Scholar), except that we used 0.4 mCi of 55FeCl3 (NEN Life Science Products, Boston, MA) instead of 0.1 mCi of 59FeCl3. A specific activity of 27,000 cpm/μg was obtained. Cells were incubated with 55Fe-Tf in minimum Eagle's medium α in either the presence or absence of a 200-fold excess of nonlabeled holo-Tf at 37 °C with 5% CO2. After washing with phosphate-buffered saline, the cells were lysed with 0.1 nNaOH, and the radioactivity was counted using a liquid scintillation counter. During 5′-RACE, while attempting to isolate genes encoding new transcriptional factors, we serendipitously cloned an 831-base pair human cDNA fragment that had significant amino acid homology to the middle portion of the TfR protein from the TF-1 cDNA library. We obtained an approximately 2.9-kb cDNA sequence for TfR2from this library (α form; GenBankTMaccession number AF067864). A cDNA clone encompassing the putative full-length coding sequence was created by polymerase chain reaction using 5′ and 3′ gene-specific primers. When we used a HL60 cDNA library for cloning TfR2, the 5′-RACE products were shorter than those from the TF-1 library, and the sequences around the 5′-end were different (β form; Fig.1 B). According to the radiation hybrid panel analysis, TfR2mapped on chromosome 7q22, between the D7S651and WI-5853markers. The restriction enzyme mapping and partial sequencing of a 16-kb genomic DNA clone and comparison with the genomic sequence in GenBankTM (accession number AP053356) deposited by Gleockner et al. (21Gleockner G. Scherer S. Schattevoy R. Boright A. Weber J. Tsui L.C. Rosenthal A. Genome Res. 1998; 8: 1060-1073Crossref PubMed Scopus (54) Google Scholar) revealed that the α form consisted of 18 exons (Fig. 1). However, some differences between their predicted exon-intron borders and ours were noted. Our DNA sequence of theTfR2-α transcript contained an additional 81 nucleotides in exon 8 (nucleotides 1053–1133 in the TfR2-α) and lacked 18 nucleotides in exon 18 (between nucleotides 2163 and 2164) as compared with their predicted This in a acid and a acid for predicted TfR2-α Also, sequence contained an additional nucleotides in the (nucleotides The β which may be an of or lacked exons and and its exon 4 of the α had an additional at the 5′-end (Fig. 1). was in the of either of the A. Y. Res. 1990; PubMed Scopus Google Scholar). The predicted amino acid sequence of TfR2-α is in The of from 81 to a pair of the predicted transmembrane domain. is located to the amino to the transmembrane of human TfR and prostate-specific membrane in Fig. C. D. Nature. PubMed Scopus Google Scholar, T. Cancer Res. 1993; Google Scholar). to TfR and PSMA, TfR2-α is a type II membrane of TfR2-α may be the and may be the extracellular domain. In the extracellular amino acid sequence between TfR2-α and either TfR or were The extracellular domain of TfR2-α was 45% and 66% with that of TfR. PSMA, the identity was and the similarity was The at and of TfR form resulting in Two at and in TfR2-α are located in an and may a In addition, TfR2-α the in the middle of the which may function as an internalization to the in TfR (Fig. M. V. S. J.A. Cell. 1990; Full Text PDF Scopus Google Scholar, T. K. C. J. Cell Biol. 1990; PubMed Scopus Google Scholar, R. J. S. Maxfield F.R. McGraw T.E. J. Cell Biol. 1996; PubMed Scopus Google Scholar). The β transcript exons which the transmembrane and as as a of the extracellular domain including the two at and The additional in exon 4 not an at the located at which is in with the α transcript The predicted is in Fig. 1 4 and Fig. as respectively. This a at and that it is an for M. Res. 1981; PubMed Scopus Google Scholar). the predicted protein of the β transcript both a transmembrane domain and resulting in a Northern blot analysis of from human showed that a 2.9-kb for expressed predominantly in the and to a in the A). This with the of TfR2-α cDNA isolated from TF-1 In addition, at 4 kb and kb and were These may the presence of additional of Northern blot analysis of of cell lines revealed high expression of TfR2-α in cells and cells B). The expression of TfR2-α were not with those of TfR (Fig. 3 B). transcripts to TfR2-β were by Northern blot To the expression of the α and β RT-PCR was performed using specific primers for a human tissue cDNA panel as a the expression of the α form was to the and blood mononuclear cells A). On the other expression of the β form occurred in of the human Human cell lines from were for expression of the two of the cell lines expressed both two cell and lacked the β transcript (Fig. 4 B). To analyze the function of we stably transfected CHO-TRVb which TfR, with FLAG-tagged TfR2-α. The cell Tf binding was using biotinylated Tf and flow cytometry. cells were for the cell Tf binding (Fig. 5 human TfR stably transfected was for cell Tf which was competed by nonlabeled Tf, but not by (Fig. 5 In the CHO-TRVb cells stably the of cell Tf binding was than that of the cells (Fig. 5 In a excess of nonlabeled Tf the binding of biotinylated Tf, but a excess of not the binding Tf binding to the TfR2-α cells was also in a transient expression using CHO-TRVb and the of Tf binding to the cell were as TfR cells TfR2-α cells pcDNA3 cells not Human TfR and TfR2-α stably transfected CHO-TRVb cells were incubated with uptake was CHO-TRVb cells were used as 55Fe uptake by the TfR2-α cells was to that by TfR and both were than that by cells (Fig. 5 B). by a 200-fold excess of nonlabeled Tf 55Fe in these cell lines after a (Fig. 5 B). the absence of TfR, 55Fe uptake was also in the cells to a as reported previously by et al. R.Y.Y. Ponka P. Schulman H.M. Exp. Cell Res. 1992; 202: 326-336Crossref PubMed Scopus (44) Google Scholar). Cell from the cells transfected with the FLAG-tagged TfR2-α plasmid were by immunoblotting using anti-FLAG antibody Two of were When was from the the of but a protein of of and were also The of the TfR2-α protein from its is to that of TfR Also, showed that TfR2-α had a function to TfR with to Tf binding and iron uptake. However, the that expression of and TfR may be of the TfR protein are through in its to which iron regulatory protein and can In cells to of TfR and these In the presence of excess are to of the In the and The may TfR protein expression K. A. R. 1999; PubMed Scopus Google Scholar). the the of the have a that may The of the FLAG-tagged TfR2-α expressed in cells is in the presence of a and is in the absence of a (Fig. 5 of TfR2-α through The of FLAG-tagged TfR2-α is than that from the amino acid sequence This may of the protein such as putative and in the TfR2-α the of in Fig. 5 C may be to different of In addition, of and the of and were (Fig. 5 These may the of TfR2-α with a protein through Northern blot analysis using normal human tissue showed that the was the tissue that expressed TfR2-α (Fig. 3 A). was expressed in the cell which is of (Fig. 3 B). This that cells may high of TfR2-α. The major of blood cells is which and TfR2-α is involved in iron it be to be expressed in these In et al. T. T. G. Blood. PubMed Google Scholar) predicted the presence of an form of TfR using a of TfR. may be to TfR2-α. We cloned two different of transcripts from the α and Two different transcripts are also expressed from the human T. Cancer Res. 1993; Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), the of TfR. the expression of is high in the antibody was for as an to 1998; 16: Google Scholar). The shorter form of the 5′-end encoding the transmembrane domain Cancer Res. 1995; Google Scholar), to the β form of TfR2. a in the of expression of the and the shorter form of has been reported of with the shorter form in normal and the form in the cells T. Res. 1998; Google Scholar). the RT-PCR we expression of the α and β of the The expression of the α form was in the blood mononuclear cells and human cell lines from (Fig. The β form was All of the human and of the human cell lines expressed this (Fig. We mapped chromosome or of of this chromosomal has been reported in several including as as and E. Rev. 1997; PubMed Scopus Google Scholar, H. J. L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, G. G. A. J. V. S. Res. 1997; 17: Google Scholar, J. J.A. T. M. E. B. G. 1996; 13: Scholar, A. G. G. P. M. A. Cancer Res. 1996; Google Scholar). are to determine in these To the function of TfR2, both Tf and were as of is Tf family The CHO-TRVb cells transfected with FLAG-tagged TfR2-α showed of Tf binding to the cell than the cells (Fig. 5 A). This that FLAG-tagged TfR2-α was expressed on the cell and was by This binding was competed by nonlabeled Tf but not by A). This that Tf can to TfR2-α than can In addition, iron uptake cells was than that of cells (Fig. 5 B). These that may be involved in iron transport which is not to that of TfR. However, the for TfR2-α is Tf, and the function of TfR2-α is cellular iron uptake, the cells have two different for TfR2-α may be transferrin receptor with a different The of the on the cell may be different from that of the complex. The putative internalization of TfR2-α is not to that of TfR, and a of the internalization may in different of the R. J. S. Maxfield F.R. McGraw T.E. J. Cell Biol. 1996; PubMed Scopus Google Scholar). the that TfR2-α has specific other than We have the from and that it is homologous to human in both and protein sequences but from R. S. and H. P. in with and J.E. Jin O. Fujiwara Y. Kuo F. Andrews N. Nat. Genet. 1999; 21: 396-399Crossref PubMed Scopus (456) Google Scholar). This that for the of the function of TfR. TfR2-α to HFE, which a with TfR on the cell TfR2-α form a with of the role of may an for the and the of cellular iron uptake. We are to E. McGraw for CHO-TRVb and TRVb-1