Title: TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL
Abstract: Article1 September 1997free access TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL Henning Walczak Corresponding Author Henning Walczak Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Mariapia A. Degli-Esposti Mariapia A. Degli-Esposti Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Richard S. Johnson Richard S. Johnson Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Pam J. Smolak Pam J. Smolak Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Jennifer Y. Waugh Jennifer Y. Waugh Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Norman Boiani Norman Boiani Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Martin S. Timour Martin S. Timour Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Mary J. Gerhart Mary J. Gerhart Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Kenneth A. Schooley Kenneth A. Schooley Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Craig A. Smith Craig A. Smith Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Raymond G. Goodwin Raymond G. Goodwin Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Charles T. Rauch Charles T. Rauch Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Henning Walczak Corresponding Author Henning Walczak Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Mariapia A. Degli-Esposti Mariapia A. Degli-Esposti Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Richard S. Johnson Richard S. Johnson Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Pam J. Smolak Pam J. Smolak Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Jennifer Y. Waugh Jennifer Y. Waugh Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Norman Boiani Norman Boiani Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Martin S. Timour Martin S. Timour Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Mary J. Gerhart Mary J. Gerhart Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Kenneth A. Schooley Kenneth A. Schooley Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Craig A. Smith Craig A. Smith Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Raymond G. Goodwin Raymond G. Goodwin Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Charles T. Rauch Charles T. Rauch Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA Search for more papers by this author Author Information Henning Walczak 1, Mariapia A. Degli-Esposti1, Richard S. Johnson1, Pam J. Smolak1, Jennifer Y. Waugh1, Norman Boiani1, Martin S. Timour1, Mary J. Gerhart1, Kenneth A. Schooley1, Craig A. Smith1, Raymond G. Goodwin1 and Charles T. Rauch1 1Immunex Corporation, 51 University Street, Seattle, WA, 98101 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5386-5397https://doi.org/10.1093/emboj/16.17.5386 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2–Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family. Introduction Tumor necrosis factor (TNF) is the prototypic member of a family of cytokines that serve important functions in the immune system (reviewed by Cosman, 1994). The members of this family interact with a corresponding set of receptors that form the TNF receptor (TNFR) family (reviewed by Smith et al., 1994). Signals induced by these interactions serve such diverse functions as differentiation, proliferation, activation or induction of cell death by apoptosis (reviewed by Cosman, 1994). Apoptosis is the most common form of physiological cell death (Wyllie et al., 1980) and, together with cell proliferation, governs the homeostasis of tissues (reviewed by Thompson, 1995). A subgroup of the TNFR family has been shown to be critically involved in mediating apoptosis. The members of this subfamily, which includes TNFR-1, CD95 (APO-1/Fas), TRAMP (APO-3, DR3, WSL) (Chinnaiyan et al., 1996; Kitson et al., 1996; Marsters et al., 1996b; Bodmer et al., 1997) and CAR1 (Brojatsch et al., 1996), are characterized by the presence of an 80 amino acid cytoplasmic death domain (DD), which functions to initiate the intracellular apoptotic signaling cascade. Upon ligand-induced cross-linking of these receptors, a death-inducing signaling complex (DISC) is formed at the DD by recruitment of the cytoplasmic DD-containing molecule FADD/MORT1 (Kischkel et al., 1995). In the case of CD95, the interaction between FADD and the DD of the receptor is direct (Chinnaiyan et al., 1995; Kischkel et al., 1995), whereas in the case of TNFR-1 and TRAMP this recruitment is mediated via the DD adaptor molecule TRADD (Hsu et al., 1995; Chinnaiyan et al., 1996; Bodmer et al., 1997). FADD interacts with FLICE/MACH, the pro-form of caspase-8, through its death effector domain (DED) (Boldin et al., 1996; Muzio et al., 1996). The resulting activation of caspase-8 triggers a proteolytic cascade that ultimately leads to apoptosis (Boldin et al., 1996; Muzio et al., 1996). The two known apoptosis-inducing receptor–ligand systems, TNF and CD95, have been shown to play a major role in many physiological and pathophysiological situations. TNF is produced mainly by activated macrophages, and lymphotoxin α (LTα) by activated T lymphocytes. The role of TNF and LTα in septic shock, autoimmune disorders and graft-versus-host disease is well established (reviewed by Revel and Schattner, 1987; Cerami and Beutler, 1988; Cohen, 1988; Fiers, 1991). TNF has also been shown to be involved in activation-induced cell death (AICD) in peripheral T cells (Zheng et al., 1995; Sytwu et al., 1996), and TNF antagonists can interfere with up-regulated AICD in HIV-positive individuals (Badley et al., 1997). The CD95 receptor–ligand system is involved in several important physiological and pathophysiological processes (reviewed by Krammer et al., 1994; Nagata, 1997). Under physiological conditions, CD95 is present on many different tissues, while CD95L expression is restricted to activated T cells and sites of immune privilege. CD95L expression by activated T cells leads to AICD (Alderson et al., 1995; Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995), T-cell cytotoxicity (Kägi et al., 1994; Lowin et al., 1994), virus-induced hepatitis (Galle et al., 1995; Kondo et al., 1997) and auto-immune diabetes (Chervonsky et al., 1997). CD95L expressed at immune-privileged sites, like the anterior chamber of the eye (Griffith et al., 1995), the testis (Bellgrau et al., 1995) or tumors (French et al., 1996; Hahne et al., 1996; Strand et al., 1996), has been demonstrated to mediate immune evasion. Up-regulated CD95 and CD95L expression have also been shown to be causative for increased levels of AICD of T cells from HIV-infected individuals (Badley et al., 1996; Bäumler et al., 1996). The TNF-related apoptosis-inducing ligand (TRAIL) is the newest member of the TNF family of cytokines (Wiley et al., 1995). Among all family members, TRAIL is most closely related to CD95L (Wiley et al., 1995). Like CD95L, TRAIL induces apoptosis in a wide variety of transformed cell lines (Wiley et al., 1995; Pitti et al., 1996; Mariani et al., 1997) and previously activated T cells (Marsters et al., 1996a). It therefore belongs to a subgroup of apoptosis-inducing members of this family which, in addition to CD95L, also includes the tumor necrosis factors, TNF and LTα (reviewed by Smith et al., 1994). In contrast to the restricted expression patterns of CD95L and TNF, TRAIL mRNA is expressed in a wide variety of normal tissues (Wiley et al., 1995). In order to better understand the biological role of TRAIL, its apoptosis-inducing potential and the signals triggering the death pathway induced by this ligand, we set out to purify the receptor for TRAIL from cells that undergo TRAIL-induced apoptosis. This led to the identification of a receptor for TRAIL, TRAIL-R2. Recently, a molecule termed DR4 was identified in a private expressed-sequence tag (EST) library based on sequence homology to the DD motif (Pan et al., 1997). Although DR4 protein has not been shown to be expressed by any cell, a soluble Fc fusion protein of its predicted extracellular domain is capable of binding to TRAIL and inhibits TRAIL-induced killing of Jurkat cells (Pan et al., 1997). These results imply that DR4 is a receptor for TRAIL (we therefore refer to it as TRAIL-R1). The results of our studies clearly demonstrate TRAIL-R2 to be distinct from TRAIL-R1 at both the biochemical and functional levels. Results Purification of TRAIL-R2 TRAIL-R2 protein was affinity purified from two different cell lines that undergo apoptosis upon incubation with TRAIL, the human T-cell line Jurkat and a spontaneous human B-cell line, termed PS-1. A doublet of biotinylated surface proteins with apparent molecular weights of 52 and 46 kDa, respectively, was affinity precipitated with an N-terminal Flag epitope-tagged version of TRAIL (Flag-TRAIL) (Wiley et al., 1995) from a lysate of surface-biotinylated PS-1 cells (Figure 1A, lane 2) or Jurkat cells (data not shown). Control affinity precipitations with either anti-Flag M2 antibody alone (Figure 1A, lane 1) or the CD95-precipitating monoclonal antibody M3 (Figure 1A, lane 3) (Alderson et al., 1995) did not yield these bands, thus demonstrating the specificity of the respective proteins for TRAIL binding. Biotinylated proteins of the same size were also TRAIL-affinity precipitated from the human B-cell lines Bjab and Ramos, and the human monocytic cell line U937 (data not shown). Figure 1.Purification of TRAIL-R2 from PS-1 cells. Identification of surface proteins binding specifically to TRAIL. PS-1 cells were surface biotinylated, lysed, and proteins were affinity precipitated with anti-Flag M2 (lane 1), Flag-TRAIL or anti-Flag M2 (lane 2 and B) or anti-CD95 (lane 3), and separated by SDS–PAGE (A) or subjected to 2D analysis (B) before a biotin-specific Western blot was carried out. (C) Preparative gel for TRAIL-R2 purification. A cell lysate was prepared from 2×010 PS-1 cells before affinity precipitating with Flag-TRAIL on anti-Flag M2–affigel. Bound proteins were 2D analyzed and silver stained as previously described (Shevchenko et al., 1996). Only the region of the first-dimensional IEF gel strip (pI 4–6.5) containing the spots corresponding to the two identified TRAIL-specific bands (see A, lane 2), as previously determined by 2D analysis (data not shown), was subjected to the second-dimensional SDS–PAGE (B and C). Boxes 1 and 2 in (C) indicate the gel regions cut out for in-gel trypsin digestion and subsequent separate mass spectrometric analysis. Download figure Download PowerPoint Two-dimensional (2D) analysis of the Flag-TRAIL affinity precipitate from biotinylated PS-1 cells (Figure 1B) or Jurkat cells (data not shown) resulted in the detection of the same two surface proteins, appearing as two rows of spots, with isoelectric points (pI) ranging from 4.5 to 5.5 (Figure 1B). Silver staining of 2D gels of preparative Flag-TRAIL affinity precipitates from lysates of 2×1010 Jurkat cells (data not shown) and PS-1 cells (Figure 1C) resulted in the detection of two rows of proteins matching the position of the specific biotinylated proteins (Figure 1C, Box 1 and Box 2). The two rows of spots were excised separately, trypsin digested and analyzed by nano electrospray tandem mass spectrometry (Nano-ES MS/MS). Comparison of the two mass spectra indicated that for PS-1 cells (Figure 2A) and Jurkat cells (data not shown) the same protein was contained within the two rows of spots. The data also demonstrate that the lower molecular weight form (Figure 1C, Box 2; Figure 2B) was missing one of the peptides (T1) and thus represents a shorter form of an otherwise identical protein (Figure 2A and B). In addition to determining the mass of nine different peptides, the sequences of six of these peptides were determined either partially (Table I) or entirely (Figure 3A; Table I) by Nano-ES MS/MS. None of these peptide sequences, nor peptide sequence tags, matched any sequence in public databases, indicating they were derived from a heretofore unidentified protein. Figure 2.Mass spectrometric analysis of TRAIL-R2. (A and B) Mass spectra of peptides released after in-gel trypsin digestion (Shevchenko et al., 1996) of the proteins contained in the gel region indicated in Figure 1C by Box 1 (A) and Box 2 (B). Peptide T1 is present in (A) but not in (B), whereas all other specific peptides (peptides that are not trypsin autocatalysis products) are present in both (A) and (B). (C) Tandem mass spectrum of the peptide annotated as T6 in (A) and (B). The Biemann ion nomenclature is used to label the fragment ions that delineate the peptide sequence (Biemann, 1990). The ions labeled ‘+2’ indicate doubly-charged fragment ions, and the ion labeled ‘(M+2H)+2’ is the precursor ion. (D) Tandem mass spectrum of the methyl esterified derivative of peptide T6. Combined analysis of the two tandem mass spectra resulted in sequence determination of this peptide: (L/I)(L/I)VPANEGDPTET(L/I)R (see left column of Table I, for all other sequences determined by Nano-ES MS/MS). Download figure Download PowerPoint Figure 3.TRAIL-R2 is a novel member of the TNFR family. (A) DNA and amino acid sequence of TRAIL-R2. The N-terminal leader cleavage site is indicated by an arrowhead, the Nano-ES MS/MS-detected peptides by open boxes. The transmembrane domain is marked by a dotted line. The DD is boxed (shaded box). The positions and directions of the two peptide T6- and T8-derived oligonucleotides used for amplification of the TRAIL-R2-specific DNA molecule are indicated by long arrows above the boxed peptides T6 and T8, respectively. (B) TRAIL-R2 contains two extracellular cysteine-rich repeats. Extracellular cysteine-rich domain alignment of TRAIL-R2 and other apoptosis-inducing TNFR family members. Conserved cysteine residues are boxed. Three disulfide bonds of linked cysteines found in TNFR1 are indicated above the alignment as SS1, SS2 and SS3. (C) TRAIL-R2 has a cytoplasmic DD. DD alignment of TRAIL-R2 and other apoptosis-inducing TNFR family members. Conserved residues are boxed and a consensus sequence is shown. (D) Direct comparison of the amino acid sequences of TRAIL-R2 and TRAIL-R1. Identical residues are boxed. Download figure Download PowerPoint Table 1. TRAIL-R2-derived peptide sequences Nano-ES MS/MS Sequence in TRAIL-R2 Observed mass T1 (L/I)T(Q/K)(Q/K)D(L/I)AP(Q/K)(Q/K)R ITQQDLAPQQR 1297.2 T2 no sequence information YGQKYSTHWNDLLFCLR 2188.2 T3 [275]SGEVELS[560]R CDSGEVELSPCTTTR 1711.4 T4 [215]VC(Q/K)C[1996]R NTVCQCEEGTFREEDSPEMCR 2634.3 T5 no sequence information VGDCTPWSDIECVHK 1803.0 T6 (L/I)(L/I)VPANEGDPTET(L/I)R LLVPANEGDPTETLR 1624.6 T7 (L/I)G(L/I)M[358](L/I)K LGLMDNEIK 1032.2 T8 [216](L/I)Y[345](L/I)K DTLYTMLIK 1097.0 T9 no sequence information DASVHTLLDALETLGER 1839.9 Additional peptide sequence information was obtained from a subsequent preparation of this protein from 3×1010 PS-1 cells. After trypsin digestion, peptides were separated by reverse-phase high-pressure liquid chromatography (RP-HPLC). N-terminal sequencing of purified peptides and parallel analysis by Nano-ES MS/MS yielded two unambiguous peptide sequences: LLVPANEGDPTETLR and DTLYTMLIK. Molecular cloning of full-length TRAIL-R2 cDNA Polymerase chain reactions (PCRs), using combinations of degenerate oligonucleotides derived from these two peptide sequences (arrows in Figure 3A) and PS-1-derived cDNA as a template, yielded a PCR product of 177 bp in length. DNA sequencing of the PCR product and comparison with other members of the TNFR family indicated that it encoded a polypeptide with striking homology to the DDs of TNFR-1, CD95, CAR1 and TRAMP. Screening of a human foreskin fibroblast (HFF) cDNA library with a single-stranded probe of the obtained PCR product resulted in the cloning of the entire coding sequence of human TRAIL-R2 (Figure 3A). TRAIL-R2 is a novel member of the TNFR family After isolation of the cDNA for TRAIL-R2, the deduced amino acid sequence was analyzed and compared with other members of the TNFR family. A computer-predicted signal peptide of 55 amino acids is present at the N-terminus of the protein. All non-trypsin-derived peptides (T1–T9; Table I; Figure 3A) detected and sequenced by Nano-ES MS/MS are present in the predicted TRAIL-R2 protein (open boxes in Figure 3A), indicating that the 2D preparations of TRAIL-R2 were pure. Peptide T1 (Table I; Figure 3A), defined and sequenced by Nano-ES MS/MS, is not preceded by a tryptic cleavage site, but instead is located four amino acids C-terminal of the highest scoring computer-predicted signal peptide cleavage site. Thus, the N-terminus of mature TRAIL-R2 is at Ile56. Two cysteine-rich repeats are present in the extracellular domain spanning amino acids 56–210, demonstrating that TRAIL-R2 is a new member of the TNFR family (Figure 3B). No consensus sites for potential N-linked glycosylation are present in the extracellular domain of TRAIL-R2. The cytoplasmic domain (amino acids 232–440) following the transmembrane stretch (211–231) contains a region (345–420) homologous to the DDs of the other apoptosis-inducing members of the TNFR family (Figure 3C). The DD of TRAIL-R2 is 34, 30, 30 and 18% identical to the DDs of CAR1, TRAMP, TNFR-1 and CD95, respectively. Comparison of this new receptor for TRAIL with the recently identified TRAIL-R1 (Pan et al., 1997) revealed that the two proteins are homologous but not identical (58% identity) (Figure 3D). The two receptors for TRAIL are similar in their ligand-binding extracellular domains and also in the portions of their cytoplasmic domains that represent the DD (Figure 3D). In addition, TRAIL-R2 contains sequences adjacent to the transmembrane region that are not present in TRAIL-R1 (Figure 3D). Thus, the data demonstrate that we have identified and cloned a receptor for TRAIL (TRAIL-R2) distinct from TRAIL-R1. TRAIL binds to recombinant cell surface-expressed TRAIL-R2 To determine whether the cloned cDNA for TRAIL-R2 encodes a functional receptor for TRAIL, we tested whether cells transfected with TRAIL-R2 bind purified, soluble TRAIL. Upon transfection of CV-1/EBNA cells with expression plasmids for either TRAIL-R2 and CrmA or CrmA alone, the TRAIL-R2 co-transfected cells showed binding of TRAIL (Figure 4A). Thus, TRAIL-R2 encodes a functional receptor for TRAIL. Figure 4.TRAIL-R2 is a cellular receptor for TRAIL. (A) TRAIL binds to cells transfected with TRAIL-R2. CV-1/EBNA cells were transfected with pDC409-CrmA together with pDC409-TRAIL-R2 (thick-lined histogram) or with pDC409-CrmA alone (fine-lined histogram). After 48 h, cells were detached non-enzymatically and stained with LZ-TRAIL, biotinylated anti-LZ antibody M15 and phycoerythrin-conjugated streptavidin. The cells were then analyzed cytometrically. (B) Inhibition of TRAIL-induced apoptosis by TRAIL-R2–Fc and a monoclonal antibody to TRAIL. Jurkat cells were treated with serial dilutions of LZ-TRAIL (-□-) starting at 1 μg/ml or were left untreated (-▪-). Additionally, LZ-TRAIL was used at a constant concentration of 500 ng/ml (-•-, -○-, -▵-) in the presence of serially diluted TRAIL-R2–Fc (-•-), TNFR2–Fc (-○-) or M180 (-▵-), starting at 5 μg/ml, and LZ-CD95L was used at 500 ng/ml in the presence of serially diluted TRAIL-R2–Fc (-▴-). Cell death was quantitated as previously described using an MTT assay (Mosmann, 1983). Download figure Download PowerPoint Inhibition of TRAIL-induced apoptosis by TRAIL-R2–Fc and the TRAIL-specific monoclonal antibody M180 Fusion proteins of other TNFR family members have been used successfully in blocking the activities of their respective ligands in vitro (Alderson et al., 1995; Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995) and in vivo (Mohler et al., 1993). The molecular cloning of TRAIL-R2 allowed for the production of a protein containing the extracellular domain of TRAIL-R2 fused to the Fc region of human IgG (TRAIL-R2–Fc). In order to test whether the extracellular domain of TRAIL-R2 is sufficient to block TRAIL-induced apoptosis of the human T-cell line Jurkat, we incubated cells with TRAIL either in the absence or presence of TRAIL-R2–Fc, TNFR2–Fc or the mouse monoclonal antibody anti-TRAIL M180, specific for human TRAIL (Figure 4B). Like anti-TRAIL M180, TRAIL-R2–Fc, but not TNFR2–Fc, blocked TRAIL-induced apoptosis in a dose-dependent fashion (Figure 4B). In addition, TRAIL-R2–Fc did not inhibit CD95L-induced apoptosis (Figure 4B). Thus, the extracellular domain of TRAIL-R2 is capable of binding specifically to TRAIL, thereby inhibiting its function. With the binding of TRAIL to native TRAIL-R2 on Jurkat and PS-1 cells (Figure 1), and to recombinant full-length TRAIL-R2 expressed on transfected cells (Figure 4A), these results demonstrate that TRAIL-R2 is a cellular receptor for TRAIL. TRAIL-R2 mRNA is widely expressed To assess in which tissues TRAIL-R2-mediated apoptosis might be of importance, we examined the tissue distribution of TRAIL-R2 mRNA by Northern blot analysis. A single transcript of 4.4 kb was present in all tissues examined, including spleen, thymus, peripheral blood lymphocytes (PBLs), prostate, testis, ovary, uterus and multiple tissues along the gastro-intestinal tract (Figure 5). The cells and tissues with the highest levels of TRAIL-R2 mRNA are PBLs, spleen and ovary, as shown by comparison with control hybridizations with a GAPDH-specific probe. Figure 5.TRAIL-R2 mRNA is widely expressed. Northern blot analysis of poly(A)+ RNA of various human tissues. A probe specific for TRAIL-R2 was used under high-stringency conditions. A glyceraldehyde-phosphate dehydrogenase (GAPDH)-specific probe was used to standardize for RNA loadings. Download figure Download PowerPoint Chromosomal localization of the human TRAIL-R2 gene to 8p22-21 In order to find out whether any known genetic defect could potentially be caused by an alteration in the gene that encodes TRAIL-R2, we determined the localization of the human TRAIL-R2 gene. PCR screening of two independent radiation hybrid panels mapped the TRAIL-R2 gene to human chromosome 8p22-21, 49 cM from the telomere (data not shown). Sequencing of the PCR product determined that the amplified DNA was specific for TRAIL-R2. None of the other known members of the TNFR family has been mapped to this region of the human genome, although the recently identified osteoprotegerin gene (Simonet et al., 1997) also maps to human chromosome 8, but to a distant position (Simonet et al., 1997). Therefore, these two genes do not form a cluster, as has been shown for other members of the TNFR family. Interestingly, two tumor suppressor genes also map to 8p (Takle and Knowles, 1996; Tanaka et al., 1996). TRAIL-R2 overexpression induces cell death by apoptosis Other DD-containing members of the TNFR family induce apoptosis upon overexpression in a ligand-independent fashion (Boldin et al., 1996; Chinnaiyan et al., 1996; Marsters et al., 1996b; Muzio et al., 1996; Bodmer et al., 1997). Overexpression of TRAIL-R2 or CD95 resulted in membrane blebbing and nuclear condensation of the transfected CV-1/EBNA cells (Figure 6A). In addition, TRAIL-R2 overexpression also induced DNA fragmentation (Figure 6B), as determined by measurement of nuclear DNA content and quantification of nuclei with subdiploid DNA content (Nicoletti et al., 1991). Thus, TRAIL-R2 overexpression results in cell death by apoptosis. Figure 6.TRAIL-R2-induced apoptosis is blocked by caspase inhibitors and FADD-DN. (A) CV-1/EBNA cells were transfected with expression plasmids for TRAIL-R2 (pDC-409-TRAIL-R2) or CD95 (pDC409-CD95) together with a 3-fold excess of empty expression vector (pDC409) in the presence or absence of z-VAD-fmk (10 μM) or together with a 3-fold excess of pDC409-CrmA, pDC409-p35 or pDC409-FADD-DN. The cells were assayed 48 h after transfection by confocal microscopy as described (Wiley et al., 1995). (B) CV-1/EBNA cells transfected with pDC409-TRAIL-R2 or pDC409 were harvested 72 h after transfection. Apoptotic cell death was quantified as described (Nicoletti et al., 1991). (C) Four hundred ng/slide of an expression vector for the E.coli lacZ gene was co-transfected together with all DNA mixes as in (A) and the cells assayed 48 h after transfection as described (Mosmann, 1983). The values plotted represent the mean and standard deviation of at least three separate experiments. Download figure Download PowerPoint TRAIL-R2-induced apoptosis involves caspases and FADD/MORT1 CD95-mediated apoptosis has been shown to involve caspases and the intracellular adaptor molecule FADD/MORT1 (Kischkel et al., 1995; Muzio et al., 1996). We therefore tested whether these molecules might also be involved in TRAIL-R2-mediated apoptosis. Co-transfection of the viral caspase inhibitors poxvirus CrmA or baculovirus P35, or the presence of the caspase-inhibitory peptide zVAD-fmk, was found to inhibit TRAIL-R2-induced apoptosis (Figure 6A and C). This demonstrates that, as with TRAIL-R1, the death pathway engaged by TRAIL-R2 involves caspases. However, in contrast to TRAIL-R1-induced apoptosis (Pan et al., 1997), TRAIL-R2-induced apoptosis was blocked by co-expression of a dominant-negative form of FADD/MORT1 (FADD-DN) (Figure 6A and C), the intracellular adaptor molecule recruited directly by CD95 and indirectly by TNFR-1 and TRAMP. Discussion We have identified a cellular receptor for TRAIL, TRAIL-R2, that is distinct from the recently described TRAIL-R1 (Pan et al., 1997). By virtue of the biochemical cloning approach pursued, we have shown that native TRAIL-R2 is expressed on the surface of both Jurkat cells and PS-1 cells. Recombinant TRAIL-R2 has likewise been shown to be expressed on the surface of transiently transfected CV-1/EBNA cells by binding of TRAIL as determined by FACS staining. Thus, TRAIL-R2 is the first receptor for TRAIL that has been shown to be expressed on the surface of cells in both native and recombinant forms. TRAIL-R2 and TRAIL-R1 are proteins with similar structures. The N-termini of the two mature TRAIL receptors are at homologous positions since the computer-predicted cleavage site for TRAIL-R1 is after amino acid 51 (amino acid 109 according to Pan et al., 1997), exactly matching the biochemically determined N-terminus of TRAIL-R2 (Figure 3A and D). In this regard, the DNA sequence preceding the codon for Met1 in TRAIL-R1 resembles a well-conserved Kozak consensus sequence, whereas the sequence preceding the previously reported putative start codon for Met58 (Pan et al., 1997) does not (reference: GenBank accession Nos AA102745 and AA100865). The two TRAIL receptors also exhibit homology in their extracellular cysteine-rich repeats, which are important for TRAIL binding, and in their cytoplasmic domains in the region resembling the DD. In contrast to TRAIL-R1, TRAIL-R2 does not contain any potential N-linked glycosylation sites. However, when expressed as soluble Fc fusion proteins, both TRAIL receptors are capable of blocking the apoptosis-inducing activity of TRAIL on Jurkat cells (Figure 4B and Pan et al., 1997). This suggests that the extracellular dom