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• W2109587559 abstract "Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFκB activation, we propose that themTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed themmDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event. Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFκB activation, we propose that themTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed themmDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event. In many biological systems, cellular outcomes are often determined by environmental cues delivered through ligand and receptor interactions on the cell surface. One group of ligand/receptor pairings critical to this decision-making process is the tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; CRDs, cysteine-rich domains; TM, transmembrane; GPI, glycosylphosphatidylinositol; PBS, phosphate-buffered saline; TNFR, TNF receptor; mTNFR, murine TNF receptor; PI-PLC, phosphatidylinositol-specific phospholipase C; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; RT, reverse transcriptase; ELISA, enzyme-linked immunosorbent assay; aa, amino acid; FACS, fluorescence-activated cell sorter; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescent protein; BWS, Beckwith-Wiedemann syndrome; 7-AAD, 7-aminoactinomycin D; TRAF, TNF receptor-associated factor ligand and receptor superfamily (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3038) Google Scholar). Upon ligand engagement, TNF receptors trigger intracellular signaling pathways that lead to cell proliferation, differentiation, or apoptosis. The pivotal roles of these TNF ligands and receptors across diverse biological areas are perhaps best illustrated by gene knockout studies demonstrating the essential involvement of the lymphotoxin pathway in lymphoorganogenesis (2Koni P.A. Sacca R. Lawton P. Browning J.L. Ruddle N.H. Flavell R.A. Immunity. 1997; 6: 491-500Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 3Rennert P.D. Browning J.L. Mebius R. Mackay F. Hochman P.S. J. Exp. Med. 1996; 184: 1999-2006Crossref PubMed Scopus (349) Google Scholar), the BAFF pathway in B-cell development (4Schiemann B. Gommerman J.L. Vora K. Cachero T.G. Shulga-Morskaya S. Dobles M. Frew E. Scott M.L. Science. 2001; 293: 2111-2114Crossref PubMed Scopus (906) Google Scholar), the RANKL pathway in osteoclastogenesis (5Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2887) Google Scholar), and the EDA pathway in hair-follicle formation (6Headon D.J. Overbeek P.A. Nat. Genet. 1999; 22: 370-374Crossref PubMed Scopus (312) Google Scholar). The ability of many members of this family to regulate both innate and adaptive immunity also makes them attractive targets for therapeutic intervention of various immune disorders, as exemplified by the success of anti-TNF therapy in treating rheumatoid arthritis and Crohn's disease (7Taylor P.C. Curr. Opin. Rheumatol. 2001; 13: 164-169Crossref PubMed Scopus (93) Google Scholar). TNF receptor family members are characterized by the presence of cysteine-rich repeats (CRDs) in their extracellular domains (8Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar, 9Naismith J.H. Sprang S.R. Trends Biochem. Sci. 1998; 23: 74-79Abstract Full Text PDF PubMed Scopus (188) Google Scholar). A CRD typically contains two structural motifs, called modules, that are stabilized by disulfide bridges formed between the cysteine residues. The linear arrangement of modules creates a scaffold that supports the unusual elongated structures seen in all known crystal structures of TNFR family members. In contrast to the absolute conservation of CRDs, the signaling potentials of TNF receptors vary a great deal. Whereas most TNF receptors, such as TNFR1, CD40, and Fas, have cytoplasmic domains containing well characterized signaling motifs such as TRAF-binding sites and/or death domain (10Inoue J. Ishida T. Tsukamoto N. Kobayashi N. Naito A. Azuma S. Yamamoto T. Exp. Cell Res. 2000; 254: 14-24Crossref PubMed Scopus (374) Google Scholar, 11Hofmann K. Cell. Mol. Life Sci. 1999; 55: 1113-1128Crossref PubMed Scopus (125) Google Scholar), others lack signaling capacity. These non-signaling receptors include soluble receptors OPG and DcR3, the GPI-anchored human TRAILR3, and human TRAILR4 that contains a defective signaling cytoplasmic tail. The biological function of these so-called “decoy receptors” is likely to antagonize the pairing between ligands and their signaling receptor counterparts, providing a critical mechanism for ligand desensitization (12Marsters S.A. Sheridan J.P. Pitti R.M. Huang A. Skubatch M. Baldwin D. Yuan J. Gurney A. Goddard A.D. Godowski P. Ashkenazi A. Curr. Biol. 1997; 7: 1003-1006Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 13Pitti R.M. Marsters S.A. Lawrence D.A. Roy M. Kischkel F.C. Dowd P. Huang A. Donahue C.J. Sherwood S.W. Baldwin D.T. Godowski P.J. Wood W.I. Gurney A.L. Hillan K.J. Cohen R.L. Goddard A.D. Botstein D. Ashkenazi A. Nature. 1998; 396: 699-703Crossref PubMed Scopus (684) Google Scholar, 14Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1533) Google Scholar, 15Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Boyle W.J. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4374) Google Scholar). Whereas many members of the TNF ligand superfamily demonstrate one-to-one pairing with their cognate receptors, others exhibit complex ligand/receptor cross-talks (8Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). In particular, the TNF ligand TRAIL has five receptors in human, at least based on in vitrobinding assays (16Griffith T.S. Lynch D.H. Curr. Opin. Immunol. 1998; 10: 559-563Crossref PubMed Scopus (441) Google Scholar, 17Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar). Among the hTRAIL receptors, hTRAILR1 and -R2 each contain a death domain in the cytoplasmic region, and as a result hTRAIL can efficiently induce caspase-dependent apoptosis in cell lines expressing these receptors (18Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1562) Google Scholar, 19Pan G. Ni J. Wei Y.F. Yu G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1383) Google Scholar). As mentioned before, hTRAILR3 and -R4 are both considered decoy receptors, but theirin vivo function is not clear. The fifth TRAIL-binding TNF receptor is OPG, which also binds to RANKL (17Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar). Whereas studies of OPG knockout mice have clearly demonstrated OPG as a decoy receptor for RANKL, the in vivo relevance of OPG to TRAIL biology remains to be established (17Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar, 20Bucay N. Sarosi I. Dunstan C.R. Morony S. Tarpley J. Capparelli C. Scully S. Tan H.L. Xu W. Lacey D.L. Boyle W.J. Simonet W.S. Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2144) Google Scholar). Interestingly, only one murine TRAIL receptor, mTRAILR2/mKiller, has been identified so far (21Wu G.S. Burns T.F. Zhan Y. Alnemri E.S. El-Deiry W.S. Cancer Res. 1999; 59: 2770-2775PubMed Google Scholar). Similar to hTRAILR1 and -R2, mTRAILR2 contains a death domain motif and induces apoptosis when overexpressed or engaged by TRAIL. Known as the TNF receptor “signature,” the uniquely spaced cysteine residues found in these receptors allows identification of potential new family members from unprocessed genomic sequences by bioinformatic means. In this study, we describe the identification through genome mining of three new TNFR family members closely clustered on mouse chromosome 7. All three genes, named mTNFRH1, -2and -3, encode proteins containing classic TNF receptor-like CRDs. We also demonstrate that, whereas mTNFRH3 remains an orphan receptor, mTNFRH1 and two splice variants of mTNFRH2 can specifically bind murine TRAIL, but not the closely related RANKL nor any other ligand we have tested. Both sequence analysis and transient overexpression studies, however, suggest that mTNFRH1 and -2 are not signaling TRAIL receptors but rather the previously unknown murine TRAIL “decoy” receptors. Given their low overall sequence homology to hTRAILR3 and hTRAILR4, we propose that mTNFRH1 and -2 belong to a new class of TRAIL decoy receptors and thus named them mDcTRAILR1 and -R2, respectively. The identification of these two murine TRAIL decoy receptors will likely facilitate our understanding of the complex biology underlying TRAIL ligand/receptor interactions through the generation of mice deficient in these receptors. Anti-FLAG M2 monoclonal antibody, M2-agarose, and Biot-M2 were purchased from Sigma. PI-PLC from Bacillus thuringiensis was purchased from ICN Biochemicals (Aurora, OH). hTRAIL, hRANKL, hEDA, hTRAILR1:Fc, hTRAILR2:Fc, hTRAILR3:Fc, hTRAILR4:Fc, hOPG, and hEDAR:Fc were from Apotech (www.apotech.com). muOPG:Fc was purchased from R & D Systems (www.rndsystems.com). Cell culture reagents were from Invitrogen. 293T cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS). Jurkat cells were maintained in RPMI + 10% FCS and HEK-293 cells in DMEM:F12 + 2% FCS. All media contained 10 μg/ml each penicillin and streptomycin. All the cell lines used for Northern analysis were purchased from ATCC and grown in recommended culture media. Transient transfections in 293T cells for the production of soluble proteins in serum-free Opti-MEM and establishment of stable transfectants in HEK-293 cells were performed as described previously (22Schneider P. Methods Enzymol. 2000; 322: 325-345Crossref PubMed Google Scholar). The cDNA of mTNFRH1/mDcTRAILR1 was obtained from EST clones (GenBankTM accession numbersAI156311, AI747041, and BG077775). A full-length coding cDNA was generated from these clones by a PCR-based method and cloned into the PCR-3 mammalian expression vector (Invitrogen). The full-length cDNA clone of mTNFRH3 was obtained by screening a mouse spleen phage cDNA library (Stratagene) using a partial cDNA probe amplified from the E14 ES cell line. The screening was performed according to recommended protocol from Stratagene and resulted in one cDNA clone from about 1 × 106independent clones. The cDNAs of both splicing variants of mTNFRH2/mDcTRAILR2 were obtained by RT-PCR using primer sequences designed on genomic sequences. Briefly, total RNA was isolated from NIH3T3 cells using TRIzol (Invitrogen) followed by first strand synthesis using Superscript II (Invitrogen). PCR was performed using the Touchdown protocol. The cDNA of mTRAILR2/mKiller was obtained similarly using RNA from the J1 ES cell line. The PCR-3 mammalian expression vectors encoding the various FLAG ligand and receptor:Fc fusion proteins were generated as described (22Schneider P. Methods Enzymol. 2000; 322: 325-345Crossref PubMed Google Scholar), using cDNA sequences encoding the following amino acid residues: mDcTRAILR1 (aa 1–158), mDcTRAILR2L (aa 1–171), mDcTRAILR2S (aa 1–180), mTNFRH3 (aa 1–162), muTRAILR2 (aa 1–177), muRANK (aa 1–200), muTRAIL (aa 120–291), and muRANKL (aa 157–316). Tissue expression patterns were done using premade Northern blots from Ambion. For expression patterns in various murine cell lines, total RNA was isolated using TRIzol reagent (Invitrogen), and 20 μg of total RNA was loaded for each lane. Probes for each gene were generated using PCR amplification of the coding regions and labeled with [α-32P]dCTP using Ready-To-Go DNA labeling beads (Amersham Biosciences). Blots were hybridized and washed in ExpressHyb solution (Clontech) according to the manufacturer's protocol. Receptors:Fc (∼500 ng) mixed with FLAG ligands (200 ng) in 1 ml of PBS were incubated for 2 h at 4 °C on a wheel with 2.5 μl of protein A-Sepharose (Amersham Biosciences). Beads were harvested, loaded in empty mini-columns, washed 3× with 100 volumes of PBS, eluted with 15 μl of 0.1 m citrate NaOH, pH 2.7, neutralized, and analyzed by Western blotting with anti-FLAG M2 antibody. Membranes were subsequently reprobed with goat anti-human IgG antibodies. The interaction between receptor:Fc and FLAG ligands by ELISA was performed as described previously (23Thompson J.S. Bixler S.A. Qian F. Vora K. Scott M.L. Cachero T.G. Hession C. Schneider P. Sizing I.D. Mullen C. Strauch K. Zafari M. Benjamin C.D. Tschopp J. Browning J.L. Ambrose C. Science. 2001; 293: 2108-2111Crossref PubMed Scopus (777) Google Scholar). Briefly, ELISA plates were coated with mouse anti-human IgG and then sequentially incubated at 37 °C with receptor:Fc, FLAG ligands, biotinylated M2, and horseradish peroxidase-coupled streptavidin. The binding of receptor:Fc was also probed directly with a horseradish peroxidase-coupled rabbit anti-human IgG polyclonal antibody. Parental HEK-293 cells (2 × 105) and stable transfectants expressing full-length mDcTRAILR1 or full-length mDcTRAILR2L were incubated for 1 h at 37 °C in 100 μl of DMEM + 10% FCS containing or not 0.05 unit of PI-PLC from B. thuringiensis. After transfer on ice, cells were washed and sequentially stained with 50 μl of FLAG-muTRAIL (100 ng/ml), 50 μl of biotinylated M2 (1:500), and 50 μl of phycoerythrin-coupled streptavidin (1:500), and submitted to FACS analysis. For mTRAIL-induced Jurkat cell death, assays were performed as described (22Schneider P. Methods Enzymol. 2000; 322: 325-345Crossref PubMed Google Scholar). Briefly, the TRAIL-sensitive Jurkat cells were incubated for 16 h with the indicated amount of FLAG-hTRAIL or FLAG-mTRAIL in the presence of 2 μg/ml anti-FLAG M2 cross-linking antibody. In other experiments, cell death induced by a fixed dose of FLAG-mTRAIL (50 ng/ml) + M2 antibody (2 μg/ml) was inhibited with the indicated amount of mDcTRAILR1:Fc, mDcTRAILR2:Fc, or hTRAILR2:Fc. Cell viability was measured by the phenazine methosulfate/3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay (Promega). For 293T cell death induced by expression of various TNF receptor family members, both adherent and floating cells, were collected 24–48 h post-transfection and stained with annexin V and 7-AAD (Pharmingen) according to the manufacturer's recommended protocol. Transfected cells were identified as cells expressing GFP. Dead cells were quantified as the percentage of GFP-positive cells that were also positive for 7-AAD staining. For caspase activity assay, 293T cells (90-mm dish) were transfected with 7 μg of indicated plasmids. Cells were washed and harvested 24 h post-transfection and lysed in 70 μl of lysis buffer (0.2% Nonidet P-40, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10% glycerol) for 10 min on ice. Caspase activity was determined by loading 20 μl (triplicate) of lysates in black 96-well plate followed by addition of 100 μl of DEVDase buffer (0.1% CHAPS, 2 mmMgCl2, 5 mm of EGTA, 150 mmNaCl, 10 mm Tris-HCl, pH 7.4, + 21 μl of 0.1m dithiothreitol + 15 μl of DEVD-AMC 5 mm in Me2SO). Fluorescence was then measured (excitation 355 nm, emission 460 nm) at different time points, and 5-h time point values are shown. For NFκB assay, 2 × 105 293T cells were plated in each well of 24-well plates overnight and then transfected with various amounts of the indicated TNF receptor expressing vectors in triplicate together with 40 ng of an NFκB luciferase reporter construct. Luciferase activity was measured 24 h post-transfection using the LucLite luciferase reporter gene assay kit (PerkinElmer Life Sciences). 293T cells were transfected with various TNF receptor family members using the Polyfect Tranfection (Qiagen) protocol. Briefly, cells were plated at 5 × 105 cells/well in 6-well plates for 24 h and then cotransfected with 500 ng of a GFP expression construct (AN050) and various concentrations of expression constructs containing the individual TNF receptor family members. After 24–48 h, cells were harvested and sequentially stained with FLAG-tagged mTRAIL, an anti-FLAG M2 monoclonal antibody (Sigma, 1:2000), and phycoerythrin-conjugated anti-murine IgG (Jackson ImmunoResearch, 1:200) each for 30 min at 4 °C. All cell samples were analyzed on the BD Biosciences FACSCalibur. To detect the binding of mTRAIL expressed on the cell surface, 293T cells were transfected with either mock vector or full-length murine TRAIL using LipofectAMINE 2000 (Invitrogen) according to the recommended protocol. The cells were harvested 24 h later with 5 mm EDTA in PBS and incubated for 1 h at room temperature with the following murine receptor Fc fusion proteins diluted in FACS buffer: DcTRAILR2L:Fc (10 μg/ml), BCMA:Fc (10 μg/ml), TRAILR2:Fc at (10 μg/ml), and DcTRAILR2S:Fc (100 μl of tissue culture supernatant). After washing, the cells were incubated with phycoerythrin-labeled goat anti-human IgG:Fc (Jackson ImmunoResearch) at 1:200 dilution for 30 min at room temperature. After final washes, cells were resuspended in 1% paraformaldehyde/PBS and analyzed using the BD Biosciences FACSCalibur. Models ofmTRAIL trimer (residues 125–291) complexed to a single subunit of mTNFRH3 (residues 41–148), mDcTRAILR1(residues 52–160), and mDcTRAILR2 (residues 62–170) were built based on the crystal structure of human TRAIL/DR5 (TRAILR2) complex (Protein Data Bank code 1D4V (24Mongkolsapaya J. Grimes J.M. Chen N. Xu X.N. Stuart D.I. Jones E.Y. Screaton G.R. Nat. Struct. Biol. 1999; 6: 1048-1053Crossref PubMed Scopus (232) Google Scholar)) using the homology modeling module of the InsightII program (version 2000, Accelrys (25Šali A. MODELLER: Implementing 3D Protein Modeling, Version 5.0. MC2 Molecular Simulations Inc., Burlington, MA1995Google Scholar)). The alignment of the receptors used for modeling is shown in Fig. 2 A. Multiple models generated for each complex were validated using ProsaII program (25Šali A. MODELLER: Implementing 3D Protein Modeling, Version 5.0. MC2 Molecular Simulations Inc., Burlington, MA1995Google Scholar), and the ones having lowest the z scores were selected for further analysis. The models were visualized in the MOLMOL program (26Koradi R. Billeter M. Wuthrich K. J. Mol. Graphics. 1996; 14 (29–32): 51-55Crossref PubMed Scopus (6498) Google Scholar, 27Engemann S. Strodicke M. Paulsen M. Franck O. Reinhardt R. Lane N. Reik W. Walter J. Hum. Mol. Genet. 2000; 9: 2691-2706Crossref PubMed Scopus (130) Google Scholar). To identify potential new TNF receptor family members, we used a TNFR signature profile (Prf:TNFR_NGFR_2 at www.expasy.ch/cgi-bin/nicedoc.pl?PDOC00561) to search a data base generated by the Swiss Institute of Bioinformatics that predicts proteins from the public genomic data bases. Our initial screening resulted in one TNFR signature-containing hit from the mouse genomic BAC clone RP23-6I17 (GenBankTM accession number AC068006). By using RT-PCR, we were able to confirm that this TNF receptor-like gene was indeed expressed (data not shown). Subsequent determination of its genomic localization revealed a tight linkage to two potentially novel TNF receptor homologous genes, Tnfrh1 and-2, predicted from the genomic sequencing effort on the distal region of the mouse chromosome 7, the murine syntenic region of the Beckwith-Wiedemann syndrome (BWS) region in human ((27) GenBankTM accession number of the full genomic locus,AJ276505). Because the TNF receptor-like gene we identified is located to the immediate 3′ of the predicted Tnfrh1 and-2 genes, we named the gene Tnfrh3. Based on the above information, we hypothesized that there exists a previously unknown TNF receptor cluster on the distal region of mouse chromosome 7. We then proceeded to obtain the full-length coding regions of all three Tnfrh genes (see below), and we identified several BAC clones (Resgen) containing the entire Tnfrhs locus. Upon extensive sequencing efforts, we concluded that, sandwiched between theobph1 and car1 genes, the three mTnfrhgenes span roughly 100 kb with no other apparent intervening genes, based on analysis using GenScan (Fig. 1 A). Because of our functional data (see below), we propose to rename the first two Tnfrh genesmDcTrailr1 and -r2. Upon data base search, we found that a full-length cDNA sequence containing the predicted cDNA sequence formDcTrailr1 had been isolated previously and reported in a patent filing (international publication number, WO 98/43998). We then employed two different approaches to obtain the complete coding sequences for mDcTrailr2 and mTnfrh3. FormTnfrh3, partial cDNA fragment amplified by RT-PCR from total RNA prepared from the mouse ES cell line E14 was used to probe a Stratagene mouse spleen cDNA phage library, resulting in the isolation of a single mTnfrh3 full-length cDNA clone (GenBankTM accession number AY165628). FormDcTrailr2, the full coding region was obtained by a combination of computer-assisted exon prediction and PCR amplification from 1st strand DNA synthesized from NIH3T3 total RNA, which revealed the presence of two alternatively spliced mDcTrailr2variants (GenBankTM accession numbers AY165626 andAY165627) (Fig. 1 B). As expected, each mTNFRH contains a signal peptide sequence at the N terminus followed by TNFR-like cysteine-rich repeats, consistent with the typical type I membrane protein topology observed in most TNFR family members (Fig. 1 B). The structures of their CRDs are remarkably similar, with three tandem A1/B2 domains in both mDcTRAILR1 and mDcTRAILR2, whereas mTNFRH3 has two A1/B2 followed by one A2/B2 domain (8Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar) (Fig. 1 C). The C-terminal portions of the mTNFRHs, however, are surprisingly divergent. Whereas mTNFRH3 contains a typical transmembrane domain (TM), the C terminus of mDcTRAILR1 instead exhibits structural features of a GPI anchor addition signal (Fig. 1 B) (28Udenfriend S. Kodukula K. Methods Enzymol. 1995; 250: 571-582Crossref PubMed Scopus (81) Google Scholar). The two splice variants of mDcTRAILR2 also diverge in their C termini. The cDNA spanning exons 1–7 contains a stop codon within exon 6 and encodes a secreted soluble receptor (mDcTRAILR2S), whereas the splice variant lacking exon 6 encodes a longer isoform (mDcTRAILR2L) containing a TM region and a short intracellular domain. Finally, mDcTRAILR1 and -R2 are highly homologous with 71% identity at the amino acid level (Fig. 1 B), indicating a recent gene duplication event at this locus. We next examined the expression patterns of mTNFRHs in both mouse tissues and cell lines. Based on Northern blot analysis, the expression of mTnfrh3 is primarily restricted to lymphoid tissues with a single ∼3-kb message detected in both the thymus and spleen and at low but detectable levels in the lung (Fig. 2 A). The lymphoid-specific expression pattern of mTnfrh3 was largely confirmed in Northern analysis of a number of murine cell lines, with its expression detected almost exclusively in lymphoid cell lines, including both the T and B lineages. The only noticeable exception is Colon 26, a colorectal adenocarcinoma cell line that also expressedmTnfrh3 (Fig. 2 B). The analysis of expression patterns of mDcTrailr1 and mDcTrailr2, however, is complicated by the extremely high homology between these two genes. Although we could confirm by RT-PCR that both mDcTrailr1 and-2 are indeed expressed genes (data not shown), we were unable to distinguish their individual expression patterns by Northern analysis due to cross-hybridization of the probe to both mRNAs. Instead, the combined expression of mDcTrailr1 and mDcTrailr2, as determined by the presence of at least one of the mRNA species, could be detected at low levels in all the mouse tissues upon prolonged exposure (data not shown). In contrast, the levels of expression were considerably higher in murine cell lines, and various levels of combined expression can be detected in most of mouse lines we have tested so far without obvious patterns in the tissue/organ origins of the positive cell lines (Fig. 2 C). The CRDs of TNF receptors not only provide the overall structural scaffold but also determine their ligand binding specificity (8Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). In an attempt to “deorphanize” mTNFRHs, we performed sequence alignments of their CRD regions with those of the other known TNF receptors. Our analysis revealed significant homologies between mTNFRHs and TRAIL receptors, particularly in regions of the TRAIL receptors that are involved in binding to TRAIL (Fig. 1 C), as suggested by the crystallographic studies of the hTRAIL-hTRAILR2 complex (24Mongkolsapaya J. Grimes J.M. Chen N. Xu X.N. Stuart D.I. Jones E.Y. Screaton G.R. Nat. Struct. Biol. 1999; 6: 1048-1053Crossref PubMed Scopus (232) Google Scholar, 29Hymowitz S.G. Christinger H.W. Fuh G. Ultsch M. O'Connell M. Kelley R.F. Ashkenazi A. de Vos A.M. Mol. Cell. 1999; 4: 563-571Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar,30Cha S.S. Sung B.J. Kim Y.A. Song Y.L. Kim H.J. Kim S. Lee M.S. Oh B.H. J. Biol. Chem. 2000; 275: 31171-31177Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). To examine experimentally whether these novel TNF receptors can indeed bind to the TNF ligand TRAIL, we first used an ELISA-based screening assay that has been optimized for the detection of interactions between TNF family ligands and receptors (22Schneider P. Methods Enzymol. 2000; 322: 325-345Crossref PubMed Google Scholar, 23Thompson J.S. Bixler S.A. Qian F. Vora K. Scott M.L. Cachero T.G. Hession C. Schneider P. Sizing I.D. Mullen C. Strauch K. Zafari M. Benjamin C.D. Tschopp J. Browni" @default.
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