From the § Departments of Exploratory Sciences,
Structural Informatics, Protein Chemistry, and Transgenics, Biogen,
Inc., Cambridge, Massachusetts 02142, the Institute of
Biochemistry, University of Lausanne, BIL Biomedical Research Center,
Chemin des Boveresses 155, CH-1066, Epalinges, Switzerland, and
¶ MEMOREC Stoffel GmbH, Stoeckheimer Weg 1, D50829 Koeln, Germany
Received for publication, October 21, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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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 NF 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)1 ligand and receptor
superfamily (1). 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 (2, 3),
the BAFF pathway in B-cell development (4), the RANKL pathway in
osteoclastogenesis (5), and the EDA pathway in hair-follicle formation
(6). 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 (7).
TNF receptor family members are characterized by the presence of
cysteine-rich repeats (CRDs) in their extracellular domains (8, 9). 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 (10, 11), 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
(12-15).
Whereas many members of the TNF ligand superfamily demonstrate
one-to-one pairing with their cognate receptors, others exhibit complex
ligand/receptor cross-talks (8). In particular, the TNF ligand TRAIL
has five receptors in human, at least based on in vitro
binding assays (16, 17). 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 (18, 19). As mentioned before,
hTRAILR3 and -R4 are both considered decoy receptors, but their
in vivo function is not clear. The fifth TRAIL-binding TNF
receptor is OPG, which also binds to RANKL (17). 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 (17, 20). Interestingly, only one murine TRAIL
receptor, mTRAILR2/mKiller, has been identified so far (21). 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, -2 and -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.
Reagents--
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.
Cell Lines--
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 (22).
Identification and Cloning of mDcTRAILR-1, mDcTRAILR-2 and
mTNFRH-3 cDNAs--
The cDNA of mTNFRH1/mDcTRAILR1 was
obtained from EST clones (GenBankTM accession numbers
AI156311, 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 × 106
independent 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.
Expression Vector--
The PCR-3 mammalian expression vectors
encoding the various FLAG ligand and receptor:Fc fusion proteins were
generated as described (22), 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).
Northern Analysis--
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 [ Coimmunoprecipitation and ELISA--
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 (23). 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.
Phospholipase C Treatment--
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.
Cytotoxicity Assay--
For mTRAIL-induced Jurkat cell death,
assays were performed as described (22). 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.
Caspase and NF
For NF mTRAIL Binding Assay--
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.
Homology Modeling and Visualization--
Models of
mTRAIL 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 (24)) using the homology modeling
module of the InsightII program (version 2000, Accelrys (25)). The alignment of the receptors used for modeling is shown in Fig. 2A. Multiple models generated for each complex were
validated using ProsaII program (25), and the ones having lowest the z scores were selected for further analysis. The models were visualized in the MOLMOL program (26).
Identification of the Murine TNFRHs Locus on Chromosome
7F4--
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 the
obph1 and car1 genes, the three mTnfrh
genes span roughly 100 kb with no other apparent intervening genes,
based on analysis using GenScan (Fig.
1A). Because of our functional data (see below), we propose to rename the first two Tnfrh genes mDcTrailr1 and -r2.
Cloning, Sequence Analysis, and Expression Pattern of
mTNFRHs--
Upon data base search, we found that a full-length
cDNA sequence containing the predicted cDNA sequence for
mDcTrailr1 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. For
mTnfrh3, 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). For
mDcTrailr2, 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 mDcTrailr2
variants (GenBankTM accession numbers AY165626 and
AY165627) (Fig. 1B).
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. 1B). 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 (8) (Fig. 1C). 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. 1B) (28). 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.
1B), 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. 2A). 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 expressed
mTnfrh3 (Fig. 2B). 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.
2C).
Murine TNFRH1/DcTRAILR1 and TNFRH2/DcTRAILR2
Bind Specifically to the TNF Ligand TRAIL--
The CRDs of TNF
receptors not only provide the overall structural scaffold but also
determine their ligand binding specificity (8). 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. 1C), as suggested by the
crystallographic studies of the hTRAIL-hTRAILR2 complex (24, 29,
30).
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 (22, 23). As shown in Fig.
3A, both mDcTRAILR1:Fc and
mDcTRAILR2L:Fc fusion proteins bound soluble mouse TRAIL.
Interestingly, mDcTRAILR2 also bound soluble human TRAIL but to a
lesser degree, whereas mDcTRAILR1 appeared species-specific and did not
interact at all with human TRAIL. The interactions between mDcTRAILRs
and soluble TRAIL appeared highly specific because no other TNF ligand,
including the closely related RANKL, bound to mDcTRAILR1:Fc and
mDcTRAILR2L:Fc in this assay (Fig. 3A and data
not shown). Although the absolute binding affinities between these
ligand/receptor pairings have not been measured, our observations are
nonetheless suggestive of the possibility that mDcTRAILR1 and -R2 may
compete effectively against muTRAILR2 for their common ligand TRAIL.
Surprisingly, mTNFRH3:Fc did not bind to TRAIL, RANKL, or any other
tested TNF ligand, despite its sequence homology to mDcTRAILR1 and -R2
and other TRAIL receptors (Fig. 3A). The ability of
mDcTRAILR1 and -R2 to bind murine TRAIL was further
examined by immunoprecipitation followed by Western blot. We found
that, similar to hTRAILR2:Fc and mTRAILR2:Fc, both mDcTRAILR1:Fc
and mDcTRAILR2L:Fc efficiently precipitated FLAG-tagged murine TRAIL and again demonstrated the strict specificity of mDcTRAILR1 for murine but not human TRAIL (Fig. 3B). The
ability of mDcTRAILR1 and -2 to bind TRAIL was further demonstrated
using a biological assay in which both mDcTRAILR1:Fc and
mDcTRALR2L:Fc inhibited significantly mTRAIL-induced
cytotoxicity in Jurkat cells (Fig. 3C).
We then confirmed the interactions between mTRAIL and mDcTRAILR1
and -R2 by using full-length mDcTRAILR1 and -R2 expressed on the cell
surface (Fig. 3D). We found that mTRAIL readily bound to
293T cells expressing mTRAILR2, mDcTRAILR1, and
mDcTRAILR2L. The failure of soluble mTRAIL to bind 293T
cells transfected with the mDcTRAILR2S-expressing
construct, as shown in Fig. 3D, is not surprising because
mDcTRAILR2S lacks the transmembrane domain and does not
express on the cell surface. To circumvent this problem, we tested the
ability of mDcTRAILR2S:Fc fusion protein to bind 293T cells
expressing full-length mTRAIL on the surface. As shown in Fig.
3E, both mDcTRAILR2S:Fc and
mDcTRAILR2L:Fc could bind surface mTRAIL. Based on these
results, we conclude that mDcTRAILR1, mDcTRAILR2L,
and mDcTRAILR2S are indeed previously unknown murine TRAIL receptors.
Murine TNFRH-1/DcTRAILR-1 and
TNFRH-2/DcTRAILR-2 Are Putative Murine Decoy TRAIL
Receptors--
Whereas both mDcTRAILR1 and mDcTRAILR2L
appear to be surface receptors for TRAIL, sequence analysis also
suggests that they anchor in the cytoplasmic membrane by different
mechanisms. The C-terminal sequence of mDcTRAILR1 is a predicted
glycosylphosphatidylinositol (GPI) anchor addition signal,
whereas mDcTRAILR2L seems to contain a conventional
transmembrane domain with an extremely short cytoplasmic domain. In
both cases, the receptors do not seem to possess the ability, upon
ligand engagement, to trigger the various intracellular signal
transduction pathways involved in TRAIL-mediated cytotoxicity. In
contrast, mDcTRAILR2S is a secreted soluble receptor.
To demonstrate formally that mDcTRAILR1, but not
mDcTRAILR2L, is a GPI-anchored receptor, we employed a well
established method for assaying GPI anchorage of receptors by measuring
the sensitivity of surface receptors to phosphatidylinositol-specific
phospholipase C (PI-PLC) (31). As expected, surface expression of
mDcTRAILR2L, as determined by mTRAIL staining, was not
altered following PI-PLC treatment, indicating that
mDcTRAILR2L anchors in the plasmic membrane through its
conventional TM region (Fig.
4A). On the when other hand, an obvious decrease in mTRAIL staining could be
detected in mDcTRAILR1-expressing 293 cells treated with PI-PLC (Fig.
4A), consistent with our hypothesis that mDcTRAILR1 is a GPI-linked surface TRAIL receptor.
Signaling TRAIL receptors hTRAILR1, hTRAILR2, and mTRAILR2 all contain
the death domain motif in their cytoplasmic domains that is responsible
for TRAIL-induced cytotoxicity, and the decoy receptors do not.
Overexpression of these death domain-containing receptors can induce
apoptosis in a number of cell lines such as 293 and NIH3T3, likely
through self-oligomerization of the receptors. We therefore tested the
effect of overexpression of mDcTRAILR1, mDcTRAILR2L, and
-R2S, on 293T survival. As demonstrated in Fig.
4B, overexpression of death domain-containing receptors TRAMP/DR3, hTRAILR2, or mTRAILR2, but not the human decoy receptors hTRAILR3 and -4, resulted in significant cell death in 293T cell as
measured by 7-AAD uptake. Transient transfection of mDcTRAILR1 and
either the long or short form of mDcTRAILR2 did not have any noticeable
effect on the survival of 293T cells, suggesting that mDcTRAILR1 and
-R2 do not possess death-signaling capability. Consistent with this
hypothesis, overexpression of mDcTRAILR1 and -R2 did not induce caspase
activation that is characteristic of death receptor signaling, as
demonstrated in Fig. 4C.
Many TNF receptor family members can also activate the NF
Previous studies (32) have suggested that decoy receptors could
potentially inhibit ligand-induced cytotoxicity by directly competing
for the ligand and/or disrupting the proper assembly of the signaling
receptors. We have shown in Fig. 3C that apoptosis of
Jurkat cells triggered by mTRAIL can indeed be efficiently blocked by
both mDcTRAILR1:Fc and mDcTRALR2L:Fc, supporting our hypothesis that mDcTRAILR1 and mDcTRAILR2 are likely decoy
receptors that could function through ligand competition. To examine
the latter possibility, we took advantage of the fact that
receptor signaling induced by the overexpression of mTRAILR2 mimics
that triggered by engagement of mTRAILR2 with TRAIL ligand. Our data clearly indicate that the presence of mDcTRAILR1 or
mDcTRAILR2L has no apparent effect on 293T cell death
induced by overexpression of mTRAILR2, suggesting that
neither mDcTRAILR1 nor mDcTRAILR2L interferes with the
proper assembly of mTRAILR2 that is required for transducing the
apoptotic signaling (Fig. 4D).
In this study, we described the identification and
characterization of a new TNF receptor locus where three TNF
receptor-like genes, Tnfrh1, -2 and
-3, are located in tandem in the distal region of mouse
chromosome 7. The close proximity of these three TNF receptor genes is
not surprising as clusters of TNF family ligands and receptors can be
found in many parts of the mammalian genome. For example, TNF We also demonstrated experimentally that mTNFRH1 and -2, but not
mTNFRH3, are receptors for TRAIL. Based on the sequence characteristics and a number of in vitro studies, we further propose that
mTNFRH1 and -2 are in fact murine decoy receptors for mTRAIL, and we
renamed them mDcTRAILR1 and mDcTRAILR2, respectively. Although whether these two murine TRAIL receptors function as decoy receptors in vivo remains to be formally established, several lines of evidence presented in this study, in particular their lack of both death signaling and NF The divergence between mDcTRAILR1/R2 and hTRAILR3/R4 is
also underscored by the potential differences in the mechanisms by which they inhibit TRAIL cytotoxicity. It has been reported previously (33) that, under physiological temperature, the affinities of hTRAILR3
and -R4 for hTRAIL are about 50-100-fold lower than those between
hTRAIL and its cognate receptors hTRAILR1 and -R2. The observation
suggests that the hTRAILR3 and -R4 would be very poor competitors for
hTRAIL against hTRAILR1 and -R2, leading to the hypothesis that the
human decoy TRAIL receptors might inhibit hTRAIL-induced cytotoxicity
through some other mechanism(s), such as by disrupting the proper
assembly of the trimeric cognate receptors (33). In our studies,
however, we found that coexpression of mDcTRAILR1 or -R2 has no effect
on cell death induced by mTRAILR2, arguing against such a scenario. It
is therefore possible that the murine decoy receptors may function by a
different mechanism than their human counterparts. Whether they can do
so through direct competition for the ligand requires further
investigation of the relative affinities of all murine TRAIL
receptors for the TRAIL ligand.
The inability of mTNFRH3 to bind TRAIL is somewhat surprising given its
sequence homology to mDcTRAILR1 and -R2. To understand the molecular
basis of ligand selectivity exhibited by these receptors, we performed
molecular modeling of the trimeric ligand/receptor interfaces between
mTRAIL and various mTNFRHs based on the crystal structure of human
TRAIL-TRAILR2 complex (Protein Data Bank code 1D4V) (24). As expected
from the significant sequence homology in the CRD regions between
hTRAILR2 and mDcTRAILR1 and -R2, the predicted interactions between
mTRAIL and mDcTRAILR1 or -R2 mirror those between hTRAIL and hTRAILR2
to a large degree (Fig. 5A and data not shown).
B activation, we propose that the
mTnfrh1 and mTnfrh2 genes encode the first
described murine decoy receptors for TRAIL, and we renamed them
mDcTrailr1 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
B Activity Assays--
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 mM
MgCl2, 5 mM of EGTA, 150 mM
NaCl, 10 mM Tris-HCl, pH 7.4, + 21 µl of 0.1 M 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.
B 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 NF
B luciferase reporter construct. Luciferase activity was measured 24 h post-transfection using the
LucLite luciferase reporter gene assay kit (PerkinElmer Life Sciences).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, genomic structure of the new
murine TNF receptor locus on chromosome 7. Three TNF receptor family
genes, mTnfrh1/mDcTrailr1,
mTnfrh2/mDcTrailr2, and mTnfrh3,
are clustered within 100 kb between Obph1 and Cars. The intron and
intergenic sequences are shown at a 1:10 scale compared with exons.
M, initiation codon for methionine; S, stop
codon. There are two mRNA variants for
mTnfrh2/mDcTrailr2 due to alternative splicing of
exon 6. B, alignment of the deduced amino acid
sequences of mTNFRH1/mDcTRAILR1, mTNFRH2/mDcTRAILR1, and mTNFRH3 based
on sequence homology. Identical residues are
dark-shaded, and residues with similar properties are
light-shaded. Exon-intron boundaries are indicated by
crosses above the alignment. The open
arrows indicates the predicted processing site of the signal
peptide (SP). The cysteine-rich domains (CRD1, -2, and -3),
the glycosylphosphatidylinositol addition signal (GPI signal), and the
transmembrane domains (TM) are boxed. C,
alignment of the CRD regions of mTNFRH1/mDcTRAILR1, mTNFRH2/mDcTRAILR1,
mTNFRH3, and all other known TRAIL binding TNF receptors. The positions
of disulfide bonds are marked by squared brackets
above (or below) the sequences. The dotted
bracket indicates the position of an additional disulfide bridge
present in mTNFRH3 only. Black, gray, and
white dots above the alignment indicate residues
of muDcTRAILR1, muDcTRAILR2, and hTRAILR2, respectively, that interact
with TRAIL (in the crystal structure of hTRAILR2 complexed with hTRAIL
or in the model of muDcTRAILR1 and muDcTRAILR2 complexed with
muTRAIL).
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Fig. 2.
A, tissue expression pattern of
mTNFRH3 by Northern blot (Ambion). Relatively high expression of
mTNFRH3 was detected in both thymus and spleen. Low but detectable
expression was seen in the lung as well. B, expression
pattern of mTNFRH3 in various murine cell lines by Northern blot. The
expression of mTNFRH3 can be detected in all lymphoid cell lines as
well as the colon carcinoma cell line Colon26. RNA from the human
B-cell line Raji was loaded as a negative control. 20 µg of RNA was
loaded for each cell line. C, combined expression
pattern of mTNFRH1/mDcTRAILR1 and mTNFRH2/mDcTRAILR1 in murine cell
lines by Northern blot. Several mRNA species can be detected in
various cell lines. RNA from the human B-cell line Raji was loaded as a
negative control. 20 µg of RNA was loaded for each cell line.
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Fig. 3.
mDcTRAILR1 and -R2 are receptors for mTRAIL
in both in vitro and cell-based assays.
A, receptor and species specificity of TRAIL and RANKL,
as measured by ELISA. Receptor:Fc fusion proteins were allowed to
interact with the indicated FLAG-tagged ligands at 37 °C. Efficient
coating of the receptor:Fc fusion proteins was assessed with a goat
anti-human antibody. The ectodysplasin-A-ectodysplasin-A
receptor (EDA-EDAR) pair of ligand and receptor was used as a
specificity control. B, receptor and species
specificity of TRAIL, as measured by coimmunoprecipitation. FLAG-tagged
hTRAIL, muTRAIL, and hEDA were incubated with the indicated receptor:Fc
fusion proteins. Following immunoprecipitation (IP) with
protein A-Sepharose, coimmunoprecipitated ligands were detected by
anti-FLAG Western blot (WB) (lower panels). The
blot was subsequently reprobed with an anti-human Ig antibody
(upper panels). C, inhibition of
mTRAIL-induced Jurkat cytotoxicity by mDcTRAILR1:Fc and R2:Fc.
Left panel, titration of the cytotoxic activity of
recombinant human and murine TRAIL on Jurkat cells. Right
panel, Jurkat cell were treated for 16 h with a constant
amount of muTRAIL (50 ng/ml) and in the presence of the indicating
amounts of hTRAILR2:Fc, mDcTRAILR1:Fc, or mDcTRAILR2:Fc. Cell viability
was measured by the phenazine
methosulfate/3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
assay. In this setting, untreated cells and cells treated with a lethal
concentration of FasL gave A490 nm of 1.4 and
0.27, respectively. D, FLAG-tagged soluble mTRAIL bound
to 293T cells transiently transfected with full-length mTRAILR2,
mDcTRAILR1, or mDcTRAILR2L but not mDcTRAILR2S
which presumably is not expressed on the cell surface. All cells were
also cotransfected with a GFP-expressing construct. The levels of
binding, as measured by the percentage of GFP-positive cells that bind
soluble mTRAIL by FACS analysis, correlated with the amounts of DNAs
transfected. E, FACS analysis of binding of various
receptor:Fc fusion proteins to mTRAIL expressed on the surface of 293T
cells. 293T cells were transfected with a full-length mTRAIL-expressing
construct and stained 24 h post-transfection with the following
receptor:Fc proteins: BCMA:Fc, mDcTRAILR1:Fc, and
mDcTRAILR2L:Fc were all at 10 µg/ml and 100 µl of
tissue culture supernatant from 293T cells transfected with an
mDcTRAILR2S:Fc-expressing construct.
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Fig. 4.
A, PI-PLC sensitivity of mDcTRAILR1
and -R2. HEK-293 control cells and HEK-293 cells stably expressing
mDcTRAILR1 or mDcTRAILR2 were treated with or without PI-PLC. Cells
were subsequently stained with FLAG-tagged mTRAIL and analyzed by FACS.
A control without TRAIL staining is shown in the upper panel.
B, mDcTRAILR1, mDcTRAILR2L, and
mDcTRAILR2S do not induce apoptosis overexpressed in 293T cells. 293T cells were cotransfected with
either 2 or 1 µg of indicated TNF receptor expression vectors and 0.5 µg of GFP expression vector. Cells were harvested 48 h later and
analyzed by FACS for cell death based on annexin V and 7-AAD staining.
The data shown are representative of three separate experiments.
C, transient transfection of mDcTRAILR1 and
mDcTRAILR2L do not induce caspase activation in 293T cells.
293T cells were transfected with indicated TNF receptor expression
vectors. Cells were harvested 24 h later, and cellular caspase
activity was determined using the fluorogenic caspase substrate
DEVD-AMC. The values shown are average of duplicate plates.
D, mDcTRAILR1, mDcTRAILR2L, and
mDcTRAILR2S do not trigger NF B activation when
overexpressed in 293T cells. 293T cells were cotransfected with
indicated amount of various TNF receptor expression vectors and 40 ng
of an NF
B luciferase reporter construct. Cells were harvested
24 h later for luciferase activity assay, and the values shown are
average of triplicate wells. E, cotransfection of
mDcTRAILR1, mDcTRAILR2L, and mDcTRAILR2S does
not interfere with mTRAILR2-induced cell death in 293T cells. 293 cells
were cotransfected with 1 µg of mTRAILR2 expression vector, 0.5 µg
of GFP expression vector, and either with 2, 1, or 0.5 µg of
indicated mDcTRAILRs. Cells were harvested 48 h later, and cell
death was analyzed as described under "Experimental Procedures."
The percentages of cell death are normalized against the absolute
percentage of cell death induced by transfection of mTRAILR2
alone.
B pathway,
through direct or indirect association with the TNF receptor-associated factor (TRAF) family members. Although mDcTRAILR1 and -R2 do not seem
to possess the TRAF-binding motif according to sequence analysis, we
nonetheless examined their ability to trigger indirectly NF
B activation (Fig. 4D). As reported previously (21),
transient transfection of mTRAILR2 induced considerable NF
B
activation in a dose-dependent manner as measured by
cotransfection of an NF
B luciferase reporter construct.
Overexpression of mDcTRAILR1 and both the long and short forms
of mDcTRAILR2, however, failed to induce NF
B activity above that of
the empty vector control level, again in agreement with the notion that
both mTRAILR1 and -2 lack signaling capability and are likely murine
TRAIL decoy receptors.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
LT
, and LT
are all tightly clustered within the major
histocompatibility region on chromosome 17 (mouse) and 6 (human).
Similarly, two recently identified TNF ligands Tweak and April are also
closely linked on human chromosome 17p13, whereas TL1A and CD30L are on
9q32-33. On the receptor side, TNF-RI, LT
-R, and CD27 are
located at 12p13 (human), whereas TNFR-RII, HVEM, CD30, and OX40 are
clustered at 1p36 (human). Even more strikingly, all four known human
receptors for TRAIL are found at 8p21-22. The presence of these
clusters has led to the suggestion that the expanding TNF family has
evolved from a limited number of ancestor molecules by means of gene
duplication (1). Our identification of the Tnfrhs locus thus
presents yet another example of TNF family clustering. Perhaps more
interestingly, the Tnfrh1/mDcTrailr1 and
Tnfrh2/mDcTrailr2 genes are extremely homologous
with several stretches of genomic sequences that are nearly identical,
indicative of a very recent duplicating event and providing by far the
most convincing evidence supporting the gene duplication theory.
B activating capacity when overexpressed, are consistent with this hypothesis. Interestingly, there are considerable differences in the overall sequences between mDcTRAILR1 and -R2 and the previously known human decoy TRAIL receptors hTRAILR3 and -R4.
Whereas the human receptors have the N1-A1-B2-A1-B2 cysteine module
structure, both mDcTRAILR1 and mDcTRAILR2 have the A1-B2 domain instead
of the N1 module. In addition, mDcTRAILR1 and -R2 do not possess either
the membrane-proximal TAPE repeats that are characteristic of the
huTRAILR3 or the truncated death domain present in huTRAILR4. These
important differences suggest that, despite their apparent functional
equivalence, the murine and human TRAIL decoy receptors likely have
evolved independently.
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Fig. 5.
A, portion of the modeled
interface between mTRAIL (pink) and mDcTRAILR1
(green), mDcTRAILR2 (cyan), or mTNFRH-3
(blue). Intramolecular disulfide bonds are labeled
yellow and orange for the extra disulfide bond in
mTNFRH3. The hypothetic interface between mTRAIL and mTNFRH3 is likely
destabilized due to the clustering of positively charged residues
(Arg-86 and Arg-119 from mTNFRH3 and Arg-195 from mTRAIL).
B, comparative modeling of simulated ligand/receptor
interfaces between the CRD2 regions of mDcTRAILR1 or -R2 and human
versus murine TRAIL. Potential interactions (both
hydrophilic and hydrophobic) are marked in red. The strict
species selectivity of mDcTRAILR1 for murine but not human TRAIL is
likely due to an essential electrostatic interaction between Asp-89 of
mDcTRAILR1 and Lys-163 of muTRAIL that is not present between
mDcTRAILR1 and human TRAIL.
Whereas the overall modeled structure of mTNFRH3 with mTRAIL is similar to that of mTRAIL with mDcTRAILR1 or -R2, we have identified two distinct features in the mTNFRH3 structure that may be responsible for its inability to bind mTRAIL. Based on the modeling, cysteines 111 and 116 are positioned favorably to form a disulfide bond, resulting in the A1-B2-A2-B2 CRD module arrangement, rather than A1-B2-A1-B2 seen in mDcTRAILR1 or -R2. We also noticed that mTNFRH3 has two arginines in positions 86 and 119 that are not present in mDcTRAILR1 or -R2. When mapped on the hypothetical model of the mTRAIL-mTNFRH3 complex (Fig. 5A), these residues were found to be located proximally to Arg-195 of the mTRAIL, forming an unfavorable interaction between three positively charged residues. Both mDcTRAILR1 and -R2, however, can form a salt bridge with Arg-195 of mTRAIL via Asp-97 (mDcTRAILR1) or Asp-107 (mDcTRAILR2), stabilizing the interaction. The bulky nature of Arg-119 in mTNFRH3 also prevents its interaction with the side chain of Tyr-247 (mTRAIL) and instead promotes its reorientation toward Arg-86 (mTNFRH3) and Arg-195 (mTRAIL), resulting in the clustering of positive charges at the interface between mTNFRH3 and mTRAIL that makes the formation of a tight complex electrostatically unfavorable.
Our modeling further revealed possible structural basis for the strict species selectivity exhibited by mDcTRAILR1, which binds only mouse, but not human, TRAIL. Because mDcTRAILR2 is highly homologous to mDcTRAILR1 yet binds both human and mouse TRAIL, we closely examined the few residues involved in ligand binding that are different between mDcTRAILR1 and -R2, identifying a small cluster of such residues in the CRD2 regions of mDcTRAILR1 and -R2. As shown in Fig. 5B, it appears that the interface between mDcTRAILR1 and mTRAIL relies upon an important electrostatic interaction between Asp-89 of mDcTRAIL1 and the Lys-163 of mTRAIL. This salt bridge cannot occur with hTRAIL because it has the uncharged Ser-159 at this corresponding position. Although mDcTRAILR2 has Ala-99 at this position, which also precludes an electrostatic interaction with either Lys-163 in mTRAIL or Ser-159 in hTRAIL, the interface between mDcTRAILR2 and either human or mouse TRAIL is stabilized by two additional interactions that do not exist between mDcTRAILR1 and the TRAIL ligand. The aspartic acid residue Asp-98 of the mDcTRAILR2, but not the corresponding His-88 in mDcTRAILR1, can form a salt bridge with Arg-158 of the hTRAIL and the corresponding Arg-162 in mTRAIL. Similarly, a favorable hydrophobic interaction exists between the Tyr-225 of human TRAIL (Tyr-216 of mouse TRAIL) and Ile-101 of mDcTRAILR2 but not the charged corresponding residue Glu-98 in mDcTRAILR1.
The locus linkage of mDcTRAIL1 and -R2 to the murine syntenic region of the human BWS locus is quite intriguing. Beckwith-Wiedemann syndrome is a congenital overgrowth syndrome whose pathogenesis has been linked to the abnormal imprint regulation of several candidate genes located at chromosome 11p15.5, a major imprinting cluster in the human genome (34). Given the role of TRAIL in tumor surveillance, it is tempting to speculate that the dysregulated expression of decoy receptors for TRAIL as a result of loss of imprint could potentially contribute to the failure in tumor suppression that is often associated with BWS. Because the two known human TRAIL decoy receptors are not located in the BWS region, the potential presence in the BWS region of human homologs of mDcTRAILR1 and -R2 that might be involved in the molecular pathology of BWS needs to be investigated.
The TRAIL pathway has been a subject of intense research in the past
few years, largely due to the ability of TRAIL to preferentially kill
tumor cell lines in vitro and in vivo (35-37).
By using both TRAIL-deficient mice and a TRAIL-neutralizing antibody,
more recent studies (38-40) have established that the TRAIL-mediated
cytotoxicity pathway is critical for tumor surveillance by the immune
system in vivo. Given the presence of both signaling and
decoy receptors for TRAIL, it has been hypothesized that the ability of
TRAIL to selectively target certain tumor cells is determined by the relative expression levels of these antagonizing receptors (14, 41).
Repeated efforts, however, have failed to establish a clear link
between TRAIL responsiveness and the expression pattern of various
TRAIL receptors in many normal and tumor cells (39, 42). As a result,
the in vivo function of the decoy TRAIL receptors and their
relevance to the tumor-suppressing activity of TRAIL demonstrated in
TRAIL-deficient mice remains unclear. In this study, we report the
identification of putative decoy receptors for TRAIL in the mouse. Our
discovery thus reveals that a comparably complex set of interactions
between TRAIL ligand and its cognate as well as decoy receptors exists
in both human and mouse (Fig. 6). The
identification of these mDcTRAILR1 and -R2 will also make it possible
to generate mice deficient in these decoy receptors, thus allowing
definitive analysis of their possible in vivo contribution in modulating the sensitivity of different cell types to TRAIL.
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FOOTNOTES |
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* This work was supported in part by grants from the Swiss National Science Foundation (to P. S. and J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY165625, AY165626, AY165627, and AY165628.
To whom correspondence should be addressed: Dept. of
Exploratory Sciences, Biogen, Inc., 12 Cambridge Center, Cambridge, MA 02142. Tel.: 617-679-3348; Fax: 617-679-3208; E-mail:
timothy_zheng@biogen.com.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M210783200
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ABBREVIATIONS |
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The 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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Locksley, R. M., Killeen, N., and Lenardo, M. J. (2001) Cell 104, 487-501[CrossRef][Medline] [Order article via Infotrieve] |
2. | Koni, P. A., Sacca, R., Lawton, P., Browning, J. L., Ruddle, N. H., and Flavell, R. A. (1997) Immunity 6, 491-500[Medline] [Order article via Infotrieve] |
3. | Rennert, P. D., Browning, J. L., Mebius, R., Mackay, F., and Hochman, P. S. (1996) J. Exp. Med. 184, 1999-2006[Abstract] |
4. |
Schiemann, B.,
Gommerman, J. L.,
Vora, K.,
Cachero, T. G.,
Shulga-Morskaya, S.,
Dobles, M.,
Frew, E.,
and Scott, M. L.
(2001)
Science
293,
2111-2114 |
5. | Kong, 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., and Penninger, J. M. (1999) Nature 397, 315-323[CrossRef][Medline] [Order article via Infotrieve] |
6. | Headon, D. J., and Overbeek, P. A. (1999) Nat. Genet. 22, 370-374[CrossRef][Medline] [Order article via Infotrieve] |
7. | Taylor, P. C. (2001) Curr. Opin. Rheumatol. 13, 164-169[CrossRef][Medline] [Order article via Infotrieve] |
8. | Bodmer, J. L., Schneider, P., and Tschopp, J. (2002) Trends Biochem. Sci. 27, 19-26[CrossRef][Medline] [Order article via Infotrieve] |
9. | Naismith, J. H., and Sprang, S. R. (1998) Trends Biochem. Sci. 23, 74-79[CrossRef][Medline] [Order article via Infotrieve] |
10. | Inoue, J., Ishida, T., Tsukamoto, N., Kobayashi, N., Naito, A., Azuma, S., and Yamamoto, T. (2000) Exp. Cell Res. 254, 14-24[CrossRef][Medline] [Order article via Infotrieve] |
11. | Hofmann, K. (1999) Cell. Mol. Life Sci. 55, 1113-1128[CrossRef][Medline] [Order article via Infotrieve] |
12. | Marsters, S. A., Sheridan, J. P., Pitti, R. M., Huang, A., Skubatch, M., Baldwin, D., Yuan, J., Gurney, A., Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997) Curr. Biol. 7, 1003-1006[Medline] [Order article via Infotrieve] |
13. | Pitti, 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., and Ashkenazi, A. (1998) Nature 396, 699-703[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Sheridan, 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.,
and Ashkenazi, A.
(1997)
Science
277,
818-821 |
15. | Simonet, 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., and Boyle, W. J. (1997) Cell 89, 309-319[Medline] [Order article via Infotrieve] |
16. | Griffith, T. S., and Lynch, D. H. (1998) Curr. Opin. Immunol. 10, 559-563[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Emery, 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.,
and Young, P. R.
(1998)
J. Biol. Chem.
273,
14363-14367 |
18. |
Pan, G.,
O'Rourke, K.,
Chinnaiyan, A. M.,
Gentz, R.,
Ebner, R., Ni, J.,
and Dixit, V. M.
(1997)
Science
276,
111-113 |
19. |
Pan, G., Ni, J.,
Wei, Y. F., Yu, G.,
Gentz, R.,
and Dixit, V. M.
(1997)
Science
277,
815-818 |
20. |
Bucay, 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.,
and Simonet, W. S.
(1998)
Genes Dev.
12,
1260-1268 |
21. |
Wu, G. S.,
Burns, T. F.,
Zhan, Y.,
Alnemri, E. S.,
and El-Deiry, W. S.
(1999)
Cancer Res.
59,
2770-2775 |
22. | Schneider, P. (2000) Methods Enzymol. 322, 325-345[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Thompson, 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.,
and Ambrose, C.
(2001)
Science
293,
2108-2111 |
24. | Mongkolsapaya, J., Grimes, J. M., Chen, N., Xu, X. N., Stuart, D. I., Jones, E. Y., and Screaton, G. R. (1999) Nat. Struct. Biol. 6, 1048-1053[CrossRef][Medline] [Order article via Infotrieve] |
25. |
![]() |
26. | Koradi, R., Billeter, M., and Wuthrich, K. (1996) J. Mol. Graphics 14, 51-55[CrossRef][Medline] [Order article via Infotrieve], 29-32 |
27. |
Engemann, S.,
Strodicke, M.,
Paulsen, M.,
Franck, O.,
Reinhardt, R.,
Lane, N.,
Reik, W.,
and Walter, J.
(2000)
Hum. Mol. Genet.
9,
2691-2706 |
28. | Udenfriend, S., and Kodukula, K. (1995) Methods Enzymol. 250, 571-582[Medline] [Order article via Infotrieve] |
29. | Hymowitz, S. G., Christinger, H. W., Fuh, G., Ultsch, M., O'Connell, M., Kelley, R. F., Ashkenazi, A., and de Vos, A. M. (1999) Mol. Cell 4, 563-571[Medline] [Order article via Infotrieve] |
30. |
Cha, S. S.,
Sung, B. J.,
Kim, Y. A.,
Song, Y. L.,
Kim, H. J.,
Kim, S.,
Lee, M. S.,
and Oh, B. H.
(2000)
J. Biol. Chem.
275,
31171-31177 |
31. | Taguchi, R., Suzuki, K., Nakabayashi, T., and Ikezawa, H. (1984) J. Biochem. (Tokyo) 96, 437-446[Abstract] |
32. |
Chan, F. K.,
Chun, H. J.,
Zheng, L.,
Siegel, R. M.,
Bui, K. L.,
and Lenardo, M. J.
(2000)
Science
288,
2351-2354 |
33. |
Truneh, A.,
Sharma, S.,
Silverman, C.,
Khandekar, S.,
Reddy, M. P.,
Deen, K. C.,
McLaughlin, M. M.,
Srinivasula, S. M.,
Livi, G. P.,
Marshall, L. A.,
Alnemri, E. S.,
Williams, W. V.,
and Doyle, M. L.
(2000)
J. Biol. Chem.
275,
23319-23325 |
34. |
Maher, E. R.,
and Reik, W.
(2000)
J. Clin. Invest.
105,
247-252 |
35. |
Ashkenazi, A.,
Pai, R. C.,
Fong, S.,
Leung, S.,
Lawrence, D. A.,
Marsters, S. A.,
Blackie, C.,
Chang, L.,
McMurtrey, A. E.,
Hebert, A.,
DeForge, L.,
Koumenis, I. L.,
Lewis, D.,
Harris, L.,
Bussiere, J.,
Koeppen, H.,
Shahrokh, Z.,
and Schwall, R. H.
(1999)
J. Clin. Invest.
104,
155-162 |
36. |
Chinnaiyan, A. M.,
Prasad, U.,
Shankar, S.,
Hamstra, D. A.,
Shanaiah, M.,
Chenevert, T. L.,
Ross, B. D.,
and Rehemtulla, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1754-1759 |
37. | Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R. G., Rauch, C. T., Schuh, J. C., and Lynch, D. H. (1999) Nat. Med. 5, 157-163[CrossRef][Medline] [Order article via Infotrieve] |
38. | Takeda, K., Hayakawa, Y., Smyth, M. J., Kayagaki, N., Yamaguchi, N., Kakuta, S., Iwakura, Y., Yagita, H., and Okumura, K. (2001) Nat. Med. 7, 94-100[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Griffith, T. S.,
Wiley, S. R.,
Kubin, M. Z.,
Sedger, L. M.,
Maliszewski, C. R.,
and Fanger, N. A.
(1999)
J. Exp. Med.
189,
1343-1354 |
40. |
Cretney, E.,
Takeda, K.,
Yagita, H.,
Glaccum, M.,
Peschon, J. J.,
and Smyth, M. J.
(2002)
J. Immunol.
168,
1356-1361 |
41. | Degli-Esposti, M. A., Dougall, W. C., Smolak, P. J., Waugh, J. Y., Smith, C. A., and Goodwin, R. G. (1997) Immunity 7, 813-820[Medline] [Order article via Infotrieve] |
42. |
Griffith, T. S.,
Rauch, C. T.,
Smolak, P. J.,
Waugh, J. Y.,
Boiani, N.,
Lynch, D. H.,
Smith, C. A.,
Goodwin, R. G.,
and Kubin, M. Z.
(1999)
J. Immunol.
162,
2597-2605 |