By
From the Department of Biochemistry and the Department of Molecular Biology, Immunex Corporation, Seattle, Washington 98101
TRAIL-R3, a new member of the TRAIL receptor family, has been cloned and characterized. TRAIL-R3 encodes a 299 amino acid protein with 58 and 54% overall identity to TRAIL-R1 and -R2, respectively. Transient expression and quantitative binding studies show TRAIL-R3 to be a plasma membrane-bound protein capable of high affinity interaction with the TRAIL ligand. The TRAIL-R3 gene maps to human chromosome 8p22-21, clustered with the genes encoding two other TRAIL receptors. In contrast to TRAIL-R1 and -R2, this receptor shows restricted expression, with transcripts detectable only in peripheral blood lymphocytes and spleen. The structure of TRAIL-R3 is unique when compared to the other TRAIL receptors in that it lacks a cytoplasmic domain and appears to be glycosyl-phosphatidylinositol-linked. Moreover, unlike TRAIL-R1 and -R2, in a transient overexpression system TRAIL-R3 does not induce apoptosis.
The TNF family of cytokines and receptors has been
shown to play a pivotal role in the maintenance of homeostasis in multiple biological networks, including the
immune system (1, 2). Particular interest has been generated by the finding that certain members of the TNF family
are capable of inducing programmed cell death or apoptosis. In addition to TNF and Fas ligand, TRAIL, a recently
identified member of the TNF family, has been shown to
induce apoptosis in a wide variety of transformed cell lines of diverse origin (3). Unlike Fas ligand, the expression of which is predominantly restricted to activated T cells (4) and sites of immune privilege (5), TRAIL message is
widely expressed (3). This suggests that either the receptor
for TRAIL is restricted in distribution, or that TRAIL is
capable of transducing different signals via one or multiple
receptors, as is the case for TNF. Two receptors for TRAIL,
TRAIL-R1 or DR4 (6) and TRAIL-R2 (7), have been recently characterized. Interestingly, both receptors show widespread distribution and are capable of mediating apoptosis.
In an attempt to identify novel proteins related to the
above-mentioned inducers of apoptosis, we searched for
sequences with homology to known members of the TNF
family of cytokines and receptors. Such molecules may
provide additional means to regulate the process of programmed cell death, and may lead not only to further understanding of immune regulation, but also to better intervention strategies in the battle against defects of the immune
system. Here we describe the identification and characterization of a new TRAIL receptor which, unlike the previously characterized TRAIL-R1 and -R2, does not signal
apoptosis and appears to be glycosyl-phosphatidylinositol (GPI)-linked to the plasma membrane.
Isolation of TRAIL-R3 cDNA.
A cDNA sequence (data available from EMBL/GenBank/DDBJ under accession number
T71406) with homology to TRAIL-R1 (6) and -R2 (7), was
identified using the full length TRAIL-R1 sequence to perform a
Blast search of the NCBI (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD) EST
(expressed sequence tag) database. A putative third TRAIL receptor was identified by sequencing the I.M.A.G.E. clone containing
this EST (I.M.A.G.E. Consortium Homo sapiens cDNA clone
110226; reference 8). Additional cDNAs encoding TRAIL-R3
were subsequently identified from a human foreskin fibroblast cDNA library using a 32P-dCTP random prime-labeled PCR
product encompassing the cysteine-rich extracellular domain of
the putative TRAIL-R3.
Plasmid Construction.
A full length TRAIL-R3 transcript, using Met 41 as the initiator methionine, was cloned into the
pDC409 mammalian expression vector (9) by PCR. The TRAIL-R3-Fc fusion chimera was constructed as described (9), by fusing
the extracellular domain, encoded between Met 41 and Ala 216, to the Fc portion of a mutein human IgG1 sequence.
Transient Transfection and Measurement of X-gal Expression.
CVI/EBNA cells (CRL 10478; American Type Culture Collection, Rockville, MD) (1.65 × 105 cells) were transfected with 1.5 µg (1.0 µg of test plasmid and 0.5 µg of pDC409-Escherichia coli
lacZ) of DNA by the DEAE-dextran method (10). After 48 h
cells were washed with PBS, fixed with glutaraldehyde, and
stained with X-gal (5-bromo-4-chloro-3-indoxyl- Scatchard Analysis.
Equilibrium-binding isotherms between
the TRAIL ligand and three TRAIL receptors were determined
by Scatchard analysis using either purified Fc proteins (TRAIL-R1-Fc and -R2-Fc) bound to plates previously coated with goat
anti-human Fc, or transfected CVI/EBNA cells transiently expressing TRAIL-R3. Transfected cells were replated after 24 h in
24 well plates at a density of 50,000 cells/well and left to recover
overnight. The cells were then incubated (30 min at room temperature) in binding medium (3% BSA, 20 mM Hepes, pH 7.4, 0.15 M NaCl, 0.04% NaN3) with serial dilutions (2×) of soluble
Leucine-Zipper (LZ) human TRAIL (7) starting at a concentration of 5 µg/ml. The cells were washed with binding medium to
remove unbound LZ-TRAIL, and incubated (30 min at room temperature) in binding medium with 125I-labeled M15 anti-LZ
mAb (125 ng/ml). The cells were then washed, removed from
the plates with trypsin, and the radioactivity in the cell suspension
was measured. A similar procedure was used for plates carrying
bound Fc proteins, except that specifically bound ligand was released with 0.1 M glycine HCl, pH 3. In both experiments, nonspecific binding was determined by inclusion of a 500-fold molar
excess of unlabeled M15 anti-LZ antibody in two duplicate reaction mixtures containing the first two dilutions of LZ-TRAIL.
Specific binding values were calculated by subtracting linearly extrapolated nonspecific binding from each data point. The data
were plotted in a Scatchard coordinate system with a nonlinear
least squared fit using RS1 software (BBN Software Products Corporation, Cambridge, MA).
Jurkat Killing Assay.
Concentrated supernatants (25×) containing equivalent amounts (75-125 µg/ml) of TRAIL-R1-Fc,
-R2-Fc, -R3-Fc, and CD30-Fc were added to Jurkat cells (104
cells/well) simultaneously with LZ-TRAIL (150 ng/ml). Percentage viability was measured after 16-20 h by MTT dye conversion (12). The highest MTT reading, obtained in the absence
of LZ-TRAIL, was used as the maximum viability value.
GPI Linkage Analysis.
CVI/EBNA cells (105) transfected
with (a) TRAIL-R1, (b) -R2, (c) -R3, (d) LERK-3 (13), or (e)
pDC409 vector were harvested 48 h after transfection with 50 mM EDTA (10 min at 37°C), and incubated with or without 1 U/ml recombinant phosphatidylinositol-specific phospholipase C
(PI-PLC; Oxford Glyco Sciences, Bedford, MA) (1 h, 37°C).
The cells were then washed and incubated (30 min at room temperature) with either LZ-TRAIL (5 µg/ml) followed by 125I-labeled
M15 anti-LZ antibody (125 ng/ml) or Hek-Fc, the LERK-3 cognate (5 µg/ml) followed by 125I-labeled goat anti-human Fc
(125 ng/ml). Free and bound probes were separated by microfuging duplicate 60-µl aliquots of the suspension through a pthalate
oil mixture (14). The counts in the free (supernatant) and bound
(cell pellet) probes were measured. Each experiment was repeated
four times.
Northern Analysis.
A human multiple tissue Northern blot
(Clontech, Palo Alto, CA) was probed with a 32P-dCTP random
prime-labeled PCR product encompassing the extracellular domain of TRAIL-R3 from Met 41 to Thr 201. Hybridization was
performed at 63°C in Stark's buffer overnight (15). The blot was
then washed three times in 2× SSC, 0.1% SDS at 68°C and exposed to film (X-OMAT; Eastman Kodak Co., Rochester, NY)
16-72 h at Chromosomal Mapping.
Chromosomal location was determined
using two independent radiation hybrid panels, the Stanford G3
Radiation Hybrid Panel and the Genebridge 4 Radiation Hybrid
Panel (Research Genetics, Huntsville, AL). The panels were screened
with two oligonucleotide primers (5 The NCBI EST database was screened with the TRAIL-R1 sequence to determine the potential existence of additional TRAIL receptors. Three EST sequences (data available from EMBL/GenBank/
DDBJ under accession numbers T71406, AA150849, and
AA150541) were identified showing partial identity to the
nucleotide sequence of TRAIL-R1 (6) and -R2 (7). An
897-nucleotide open reading frame (ORF) was obtained
by direct sequencing of T71406 (I.M.A.G.E. Consortium
Clone 110226) (8) (Fig. 1 A). This ORF, referred to as
TRAIL-R3, has 54 and 52% nucleotide and 58 and 54%
amino acid (aa) identity to TRAIL-R1 and -R2, respectively. The authenticity of this cDNA was confirmed by analysis of a second full length cDNA clone isolated by screening the human foreskin fibroblast library with a probe
encompassing the cysteine-rich extracellular domain of the
putative TRAIL-R3.
The TRAIL-R3 transcript encodes a 299-aa protein
with a predicted signal sequence cleavage site after aa 69, a
121-aa extracellular cysteine-rich domain, an 88-aa extracellular linker sequence, and a 21-aa hydrophobic COOH-terminal sequence. Like TRAIL-R1, this transcript carries
two methionines within the first 60 aa of the ORF. Analysis of signal peptide cleavage sites predicts the mature protein to start at Arg 70, suggesting that Met 41 is probably
the start codon. Unlike the previously characterized TRAIL
receptors, the predicted TRAIL-R3 protein does not contain a cytoplasmic domain (Fig. 1 A). Like TRAIL-R1 and
-R2, the extracellular domain of TRAIL-R3 contains only
two of the four cysteine-rich pseudo-repeats characteristic
of the extracellular domain of most members of the TNF
receptor family (Fig. 1 B). The TRAIL-R3 cysteine-rich extracellular domain is separated from a COOH-terminal
hydrophobic region by an 88-aa linker sequence. This
linker contains five copies of a 15-aa pseudo-repeat (Fig. 1
A); a single copy of this repeat is found in the 31-aa linker
region of TRAIL-R2, but is absent in TRAIL-R1.
Given the observed aa identity
in the extracellular ligand binding domains of TRAIL-R3,
-R2, and -R1, we predicted that TRAIL-R3 would bind
TRAIL. To test this hypothesis, TRAIL-R3 was transiently expressed in CVI/EBNA cells. Transfected cells
were then tested for their ability to bind TRAIL using the
very sensitive autoradiographic analysis previously described (16). Binding of TRAIL was observed to CVI/
EBNA cells transfected with TRAIL-R3, but not to vector transfected cells (data not shown), demonstrating that this
receptor is expressed on the cell surface and that it is a cognate for the TRAIL ligand.
The equilibrium-binding isotherm between soluble TRAIL
ligand and TRAIL-R3 was determined using a recombinant receptor transiently expressed on CVI/EBNA cells.
For comparison, the isotherms of TRAIL-R1 and -R2 were
determined using purified Fc proteins as the substrate for
the binding studies (see Materials and Methods). Binding of
the receptors to TRAIL was achieved using LZ-TRAIL and 125I-labeled M15 anti-LZ antibody. All three receptors
bound LZ-TRAIL in a specific, saturable fashion, and Scatchard analysis using nonlinear least squared regression revealed binding sites with comparable affinities (Kd(HIGH) = 0.04-0.36 nM; Kd(LOW) = 0.38-9.0 nM), indicating that
TRAIL binds equally well to all three receptors (Fig. 2).
Soluble fusion proteins comprising the ligand
binding domain of receptors fused to the Fc domain of human IgG1 have proven to be potent inhibitors of ligand-mediated activity (17, 18). Thus, a soluble TRAIL-R3-Fc
was constructed to determine its ability to impede TRAIL-mediated activities. The Jurkat T-cell line, previously shown
to die in response to TRAIL, was cultured with soluble LZ-TRAIL in the presence of concentrated supernatants from
CVI/EBNA cells transiently expressing soluble (a) TRAIL-R3-Fc, (b) -R2-Fc, (c) -R1-Fc, or (d) CD30-Fc. Specific
and complete inhibition of LZ-TRAIL-mediated apoptosis
was obtained with TRAIL-R3-Fc, -R1-Fc, and -R2-Fc,
but not with CD30-Fc (Fig. 3).
Fas and TNF-R1 are prototypic triggers of apoptosis. In
addition, four new members of the TNFR family are able
to induce apoptosis. A common feature of these receptors,
which include DR-3 (19), CAR-1 (20), and the two receptors for TRAIL (6, 7), is that they share an ~80-aa region of homology, referred to as the "death domain,"
within their cytoplasmic domains. This region appears to
be critical for the induction of apoptosis by Fas, TNFR-1
and DR-3 (19, 21, 22).
As for Fas, TNFR-1, and DR3, overexpression of TRAIL
receptors 1 and 2 in a transient transfection system results in
ligand-independent apoptosis (6, 7). Unlike TRAIL-R1
and -R2, TRAIL-R3 lacks a cytoplasmic domain, the region that normally encodes the death domain. Therefore,
we expected TRAIL-R3 to be unable to transduce an apoptotic signal. Indeed, transient overexpression of TRAIL-R3 did not lead to cell death (data not shown).
The sequence of TRAIL-R3 is unusual in that the COOH-terminal hydrophobic
region is not followed by a cytoplasmic domain. However,
despite the lack of a typical type I transmembrane protein structure, we have shown that a recombinant form of
TRAIL-R3 is expressed on the cell surface of transfected
cells and is capable of binding TRAIL with high affinity.
Several proteins can stably associate with the external
surface of the cell membrane by covalent linkage to glycolipids. These include several members of the immunoglobulin superfamily (23), as well as some leucocyte surface
proteins such as Ly-6 (24). The distinguishing features of
such proteins include (a) the presence of a hydrophobic
NH2-terminal signal peptide, which typically directs the
protein to the endoplasmic reticulum, (b) a second hydrophobic region at the COOH terminus, and (c) the lack of a cytoplasmic domain. Since the structure of TRAIL-R3 fulfills all of the above requirements, we tested the possibility
that this receptor is membrane bound through a GPI anchor. Given that most, but not all, GPI-anchored proteins
can be released from cell surfaces by PI-PLC, we treated
CVI/EBNA cells transfected with TRAIL-R3 with this
enzyme. TRAIL-R1 and -R2 were used as negative controls and the GPI-linked LERK-3 protein (13) as a positive
control. Analysis of TRAIL binding to TRAIL-R3-expressing cells after treatment with PI-PLC indicates that ~30%
of the membrane-bound TRAIL-R3 protein is displaced
from the cell surface by PI-PLC (Fig. 4). Approximately
80% of the GPI-linked LERK-3 was displaced by PI-PLC treatment (Fig. 4). As expected, PI-PLC treatment did not
affect the TRAIL-R1 and -R2 transmembrane proteins.
The poor sensitivity of TRAIL-R3 to PI-PLC-mediated
release suggests that anchoring of TRAIL-R3 to the cell
membrane is mediated by phospholipid bonds only partially hydrolyzed by phospholipase C (25). The functional significance of GPI-anchoring TRAIL-R3 to the cell surface, as indeed is the case for GPI-linked proteins in general, remains to be determined. It has been suggested that
GPI anchors allow differential protein release. Therefore, it
is plausible that the structure of TRAIL-R3 developed as a
mechanism for expeditious release of this receptor, thus
providing a soluble inhibitor of TRAIL-mediated activities. Alternatively, rapid downregulation of TRAIL-R3
may be required for TRAIL to signal through TRAIL-R1 and -R2. In addition, it is possible that, like other GPI-anchored molecules, TRAIL-R3 may be capable of direct
signaling (26).
The tissue distribution of TRAIL-R3 mRNA was determined by
Northern blot analysis (Fig. 5). TRAIL-R3 message was
clearly detected only in PBLs. A weak signal (not visible in
Fig. 5) was observed, after prolonged exposure, in spleen.
Five transcripts of ~1.3, 2.5, 3.0, 4.0, and 7.0 kb were detected. The restricted distribution of TRAIL-R3 markedly
contrasts with the wide-spread distribution of -R1 (6) and
-R2 (7).
The chromosomal location of TRAIL-R3 was determined by PCR analysis of two independent radiation hybrid panels. The TRAIL-R3 gene has been mapped to human chromosome 8p22-21, ~49 cM from the telomere, in
close proximity to a cluster which also encodes the genes
for TRAIL-R1 and -R2. This cluster is reminiscent of the
TNFR gene clusters on chromosome 1p (TNFR-2,
CD30, OX40) and on chromosome 12p (CD27, LT In conclusion, we have cloned and characterized TRAIL-R3, a new member of the TRAIL receptor
family. We have clearly demonstrated that TRAIL-R3 exists as a cell surface molecule capable of binding TRAIL.
However, the structure of this protein is unusual. Unlike TRAIL-R1 and -R2, -R3 appears to be GPI-linked and
lacks a cytoplasmic region, including the death domain.
Thus, as expected, TRAIL-R3 is unable to induce apoptosis. Of further interest is the restricted distribution of the
TRAIL-R3 message. Unlike the other two TRAIL receptors, which are widely expressed, TRAIL-R3 transcripts
are only present in PBLs and spleen. The significance of
this finding remains to be determined; it is tantalizing to
speculate that in these tissues TRAIL-R3 acts as an inhibitor of TRAIL-mediated apoptosis by competing with
TRAIL-R1 and -R2 for binding to the ligand.
The identification of TRAIL-R3 adds new complexity
to the emerging TRAIL receptor subfamily and is reminiscent of the intricacy surrounding the dual receptors for
TNF. By analogy to the latter system, it is possible that
novel TRAIL ligands are yet to be characterized. Continued evaluation of the biological activities of TRAIL-R3
and detailed characterization of this receptor family will
provide important insights into the in vivo functions of these proteins.
-D-galactopyranoside) for
-galactosidase activity. A reduction in cells
stained indicates loss of
-galactosidase expression and correlates
with death of cells that express the protein(s) cotransfected with
the lacZ gene (11). Each experiment was repeated three times.
70°C.
CTTCCTTACCTGAAAGGTTCAGGTAGG3
and 5
CTCTTGGACTTGGCTGGGAGATGTG3
) capable of reliably and specifically amplifying
human TRAIL-R3 from genomic DNA. The results were electronically submitted to the appropriate servers for linkage analysis.
Isolation of TRAIL-R3 cDNA.
Fig. 1.
TRAIL-R3 is a novel member of the TRAIL receptor family. (A) The nucleotide and aa sequence of TRAIL-R3 is shown. The aa
sequence is started at the first Met; the two potential initiator codons are
boxed. The predicted NH2-terminal leader cleavage site is indicated by
the triangle. The COOH-terminal hydrophobic domain is marked by a
dashed line. Five pseudo-repeats in the linker region, separating the extracellular domain from the hydrophobic COOH terminus, are shown in
boxes. (B) Alignment of the extracellular domains of TRAIL-R3, -R2,
and -R1 shows conservation of two of the cysteine-rich pseudo-repeats
characteristic of the TNF receptor family. Conserved cysteine residues are
boxed. Predicted disulfide bonds are shown. These sequence data are
available from EMBL/GenBank/DDBJ under accession number AF014794.
[View Larger Versions of these Images (51 + 16K GIF file)]
Fig. 2.
Equilibrium binding isotherms of TRAIL-R3, -R2, and -R1. Full length TRAIL-R3 transiently expressed on CVI/EBNA cells and purified
TRAIL-R1 and -R2 Fc proteins were used in equilibrium binding assays with LZ-TRAIL + 125I-labeled M15 anti-LZ antibody, as described above (see
Materials and Methods). The binding data plotted in the Scatchard coordinate system is shown. The membrane-bound TRAIL-R3 protein binds TRAIL with similar affinity as TRAIL-R1 and -R2 soluble Fc proteins.
[View Larger Version of this Image (13K GIF file)]
Fig. 3.
TRAIL-R3-Fc inhibits TRAIL-mediated killing. Jurkat cells
treated with LZ-TRAIL (150 ng/ml) were cultured for 16 h with concentrated supernatants containing soluble TRAIL-R3-Fc, -R2-Fc, -R1-
Fc or CD30-Fc proteins. All three TRAIL-R-Fc proteins block TRAIL-mediated apoptosis of Jurkat cells as measured by MTT conversion (12). The maximum viability value corresponds to the MTT reading in the absence of LZ-TRAIL (MEDIUM ONLY). The minimum viability value corresponds to the MTT reading in the presence of LZ-TRAIL (LZ-TRAIL ONLY).
[View Larger Version of this Image (12K GIF file)]
Fig. 4.
TRAIL-R3 can be
partially displaced from the cell
membrane by phospholipase C. The effect of PI-PLC treatment
of CVI/EBNA cells transiently
transfected with TRAIL-R1, -R2,
-R3, LERK-3, or empty
pDC409 vector is shown. After
treatment or no treatment with
PI-PLC, the cells were incubated
with either LZ-TRAIL followed by 125I-labeled M15 anti-LZ antibody or Hek-Fc followed by
125I-labeled goat anti-human-Fc.
The radioactivity in the free and bound probes, separated by microfuging through a pthalate oil mixture,
was counted and the results plotted. PI-PLC treatment resulting in lowered bound probe counts (cpm) indicates displacement of cell surface
proteins that bind the labeled probe.
[View Larger Version of this Image (12K GIF file)]
Fig. 5.
TRAIL-R3 shows restricted
tissue expression. Northern analysis showing the distribution of TRAIL-R3
mRNA in whole human tissues. The
source of the mRNA is shown above
each lane. The position of RNA size
markers is shown on the left. Multiple
transcripts are detected in PBLs.
[View Larger Version of this Image (84K GIF file)]
R,
TNFR-1). However, the high degree of nucleotide identity shared by the three TRAIL receptors, combined with
their close chromosomal proximity, suggests that these loci have arisen recently as duplications of a precursor sequence.
Address correspondence to Mariapia A. Degli-Esposti, Immunex Corporation, 51 University St., Seattle, WA 98101. Phone: 206-587-0430, extension 4687; FAX: 206-233-9733; E-mail: mdegliesposti{at}immunex.com
Received for publication 16 July 1997 and in revised form 31 July 1997.
The authors are grateful to C. Rauch for purification of LZ-TRAIL; K. Maggiora and A. Learned for transfections; and M. Petersen for purification of soluble Fc proteins.
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