INTRODUCTION

Apoptosis is an intrinsic and fundamental biological process that plays a critical role in the normal development of multicellular organisms and in maintaining tissue homeostasis (1). Consequently, deregulation of apoptosis may contribute to diseases such as cancer and neurodegenerative disorders (2). Some of the well known regulators of apoptosis are cytokines of the tumor necrosis factor (TNF)¹ ligand family, such as Fas ligand (Fas L) and TNF, which induce apoptosis by activation of their corresponding receptors, Fas and TNFR-1 (3). These two receptors belong to a rapidly expanding family (collectively known as the TNF receptor family) containing at least 11 known members (3, 4). Members of this family contain an extracellular ligand-binding domain, of 2-6 cysteine-rich repeats, which is about 25% conserved between different family members. The cytoplasmic region is less conserved between various members except for a stretch of about 80 amino acids present in Fas, TNFR-1, DR3/Wsl-1/Apo-3/TRAMP, CAR-1, and DR4 (Refs. 3 and 4 and references therein). This intracellular region, which has been designated the cytoplasmic "death domain," is responsible for transducing the death signal.

Activation of Fas results in recruitment of the Fas-associated death domain-containing molecule FADD/MORT1 to the receptor complex (5-7). The resulting signaling complex then triggers activation of the caspase apoptotic pathway through interaction of the N-terminal death effector domain of FADD with the corresponding motifs in the prodomain of caspase-8 (Mch5/MACH/FLICE) and probably caspase-10 (Mch4) (8-11). In contrast to Fas, activation of TNFR-1 or DR3 results in recruitment of another death domain-containing adaptor molecule known as TRADD (12, 13). TRADD can associate with a number of signaling molecules, including FADD, TRAF2, and RIP, and as a result can transduce an apoptotic signal as well as activate NF-B (14, 15). Consequently, engagement of TNFR-1 or DR3 can signal an array of diverse biological activities.

Recently, a new member of the TNF family known as TRAIL or Apo-2 ligand was identified and shown to induce apoptosis in a variety of tumor cell lines (16-18). A human receptor for TRAIL was also recently identified and designated DR4 (4). DR4 contains a cytoplasmic death domain, and like Fas, it induces apoptosis but not NF-B activation. However, unlike Fas, TNFR-1, and DR3, DR4 does not recruit FADD to the receptor complex and thus signals death independently of FADD (4).

Here, we report the identification of two additional human receptors for the cytotoxic ligand TRAIL (designated DR5 and TRAIL-R3). These represent as yet undescribed members of the TNF receptor family. Interestingly, DR5 but not TRAIL-R3 contains a cytoplasmic death domain necessary for induction of apoptosis, and like DR-4, DR5 engages the apoptotic pathway independent of the adaptor molecule FADD/MORT1. TRAIL-R3 on the other hand, can bind TRAIL but does not induce apoptosis and thus may function as an antagonistic receptor.

MATERIALS AND METHODS

The full-length DR5 cDNA was cloned from a human Jurkat Uni-ZAP XR cDNA library (10) by PCR using specific primers derived from the nucleotide sequences of human GenBank^TM EST clones 650744 and 664665. Similarly, TRAIL-R3 was cloned by PCR using specific primers derived from the nucleotide sequences of human GenBank^TM EST clones 470799, 129137, and 504745.

T7-epitope tagging was done as described recently (19). To generate N-terminal Flag-tagged receptor and receptor mutants, PCR-generated cDNAs encoding Fas, DR4, DR4 (residues 86 to 351), DR5, and DR5 (residues 1 to 268) were inserted in a modified pcDNA-3 vector that allowed for in-frame fusion with a Flag epitope tag that is preceded by Fas signal peptide. To generate C-terminal Fc-tagged receptors, PCR generated cDNAs encoding Fas (residues 16 to 158), DR4 (residues 86 to 217, with N-terminal Fas signal peptide-Flag tag), DR5 (residues 51 to 133), and TRAIL-R3 (residues 63 to 217) extracellular domains were inserted into a modified pcDNA3 vector that allowed for in-frame fusion with the Fc portion of the mouse IgG. For apoptosis assays we used the mammalian double expression vector pRSC (20), which allows for expression of lacZ under the Rous sarcoma virus promoter, and the test cDNA (DR4, DR4, DR5, DR5, TRAIL-R3) under the CMV promoter. CrmA, FLAME-1, caspase-8-DN (C345A), or caspase-10-DN (C358A) (19) were expressed using pcDNA3 (Invitrogen).

These were done as described recently (19).

Recombinant soluble TRAIL with N-terminal T7 and His₆ tags was obtained by nickel affinity purification from bacteria transformed with a pET28c-TRAIL (residues 95 to 281) vector. Receptor-Fc chimeras were obtained by harvesting conditioned media of 293 cells transfected with constructs encoding Fas-, DR4-, DR5-, or TRAIL-R3-Fc fusion proteins as described (4). Binding of TRAIL to the receptor-Fc chimeras was performed as described (4).

RESULTS AND DISCUSSION

To identify additional members of the TNF receptor family, we searched the GenBank^TM EST data base for sequences that are homologous to the TRAIL receptor-1, DR4. Several EST clones were identified, and their 3 and 5 sequences were compiled. Based on the compiled sequences, PCR primers were generated and used to clone two cDNAs that encode two new DR4-related sequences (Fig. 1, A and B). The first cDNA encodes a protein of 411 amino acids with an overall ~59% identity to DR4 (Fig. 1A). Its predicted domain structure is highly related to DR4 and the other members of the TNF receptor family. It contains a putative N-terminal signal peptide (amino acids 51 to 1) followed by an extracellular domain containing two cysteine-rich pseudorepeats. Following the extracellular domain is a transmembrane domain (amino acids 132 to 152) and a cytoplasmic domain. Within the cytoplasmic domain there is a stretch of 67 amino acids (amino acids 273 to 339) comprising a death domain homology region (Fig. 1C). Based on these criteria and its apoptotic activity (see below) the new protein was designated death receptor-5 (DR5).

The second cDNA encodes a protein of 299 amino acids with overall ~40 and 36% identity to DR4 and DR5, respectively (Fig. 1B). This protein contains a putative N-terminal signal peptide (amino acids 63 to 1) followed by an extracellular domain containing two cysteine-rich pseudorepeats and five nearly identical PAAEETMN(T)TSPGTPA repeats. Following the extracellular domain is a C-terminal transmembrane domain (amino acids 217 to 236). Unlike DR4 and DR5, this molecule does not contain a cytoplasmic domain. Based on these criteria and its ability to bind TRAIL (see below), this protein was designated TRAIL-R3.

Northern blot analysis of equivalent amounts of mRNA samples from normal human tissues and tumor cell lines with a DR5 riboprobe detected a ~4-kilobase transcript in all the samples (Fig. 1D, upper panels). Interestingly, the amount of DR5 transcript was at least 100-fold more in most tumor cell lines than in normal tissues. Autoradiography for less than 2 h was sufficient to detect the DR5 message in tumor cell lines, compared with 48 h in the case of the normal tissues. Other normal tissues such as testes, ovary, colon, small intestine, and lymphoid tissues had detectable but low expression of DR5 transcript (not shown), similar to that observed in the normal tissues shown in Fig. 1D. The TRAIL-R3 riboprobe detected ~a 5-kilobase message in both normal human tissues and tumor cell lines (Fig. 1D, lower panels). A significantly elevated expression of TRAIL-R3 mRNA in normal compared with tumor cells was observed. Given the activities of these two receptors (see below), this could explain the high sensitivity of tumor cell lines to TRAIL compared with normal cells (16-18).

Because of the high degree of sequence homology between the extracellular domains of DR4, DR5, and TRAIL-R3, we decided to test whether DR5 and TRAIL-R3 are capable of binding TRAIL. The extracellular ligand-binding domains of Fas, DR4, DR5, and TRAIL-R3 were expressed as fusion proteins with the Fc region of mouse IgG (Fig. 2A, lower panel). As shown in Fig. 2A (upper panel), DR4-Fc, DR5-Fc, and TRAIL-R3-Fc were all capable of binding TRAIL to the same extent (lanes DR5-Fc, DR4-Fc, and TR3-Fc). As expected Fas-Fc was unable to bind TRAIL (lane 4). Furthermore, DR4-Fc, DR5-Fc, and TRAIL-R3-Fc but not Fas-Fc were capable of blocking TRAIL-induced apoptosis in MCF7 cells (Fig. 2B). These data suggest that, like DR4, DR5 and TRAIL-R3 are receptors for TRAIL.

Ectopic expression of death domain-containing members of the TNF receptor family induces apoptosis in a ligand-independent manner. Consistent with this observation, we found that transient expression of DR5 in MCF7 or 293 cells triggers apoptosis (Fig. 3, A and B). The level of apoptosis induction was similar to that observed with DR4. (Fig. 3A). Induction of apoptosis was dependent on the presence of the cytoplasmic death domain, because deletion of this domain abolished the ability of DR4 and DR5 to induce apoptosis (Fig. 3, A and B). Accordingly, TRAIL-R3, which does not naturally contain a death domain, was also incapable of inducing apoptosis (Fig. 3, A and B). Interestingly, transient expression of TRAIL-R3 in MCF7 cells significantly blocked TRAIL-induced apoptosis (Fig. 3C), suggesting that it may function as an antagonistic decoy receptor.

Like DR4 and the other TNF receptor family members, DR5-induced apoptosis was efficiently blocked by the caspase inhibitors z-VAD-fmk and CrmA (Fig. 3D). DR4- and DR5-induced apoptosis was also significantly inhibited by the dominant negative inhibitors FLAME-1 (also known as Casper, FLIP, I-FLICE, and CASH) (Refs. 19 and 21 and references therein), caspase-8-DN, and caspase-10-DN (Fig. 3E). Among these, caspase-10-DN was the most effective in blocking DR4- and DR5-induced apoptosis. Inhibition of DR4- and DR5-induced apoptosis by FLAME-1 is consistent with recent observations that TRAIL-induced apoptosis is blocked by expression of FLIP (FLAME-1) (22). These data also suggest that the upstream caspases-8 and -10 are involved in both the DR4 and DR5 death signaling pathways.

Death domain containing adaptor molecules such as FADD/MORT1, CRADD/RAIDD, TRADD, and RIP are recruited by some members of the TNF receptor family to engage the upstream caspases (3, 19). Using co-immunoprecipitation experiments, we tested if DR5 could interact with these molecules to transmit the apoptotic signal. Unlike Fas, DR5 did not interact with FADD or CRADD (Fig. 4A) nor with RIP or TRADD (data not shown). A similar observation was reported with DR4 (4). Interestingly, full-length Fas, DR4, and DR5, but not death domain-deleted mutants, were all capable of forming complexes with caspase-8, caspase-10, and FLAME-1 (Fig. 4B). Because these proteins do not interact directly, this suggests that formation of these complexes would require an adaptor molecule distinct from FADD.

Taken together, we report the identification of DR5 and TRAIL-R3 as two new receptors for the cytotoxic ligand TRAIL. Consistent with the observation that TRAIL-induced apoptosis is independent of FADD (18), we show that DR5, like DR4 (4), does not bind FADD. However, both receptors recruit the upstream caspases-8 and -10 and the anti-apoptotic protein FLAME-1, suggesting that an as yet unidentified adaptor molecule is involved in the mechanism of apoptosis signaling by these receptors. Because TRAIL-R3 does not contain a cytoplasmic death domain and is capable of attenuating the cytotoxicity of TRAIL, it may function physiologically as an antagonist to DR4 and DR5. A correlation may exist between the high sensitivity of tumor cells to TRAIL and the elevated levels of DR5 in these cells. Further analysis of the regulation of DR4, DR5, and TRAIL-R3 expression in normal and tumor cells should lead to a better understanding of their normal physiological function.