Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5.

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.

Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen.

The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).

Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.

TRAIL (also called Apo2L) belongs to the tumor necrosis factor family, activates rapid apoptosis in tumor cells, and binds to the death-signaling receptor DR4. Two additional TRAIL receptors were identified. The receptor designated death receptor 5 (DR5) contained a cytoplasmic death domain and induced apoptosis much like DR4. The receptor designated decoy receptor 1 (DcR1) displayed properties of a glycophospholipid-anchored cell surface protein. DcR1 acted as a decoy receptor that inhibited TRAIL signaling. Thus, a cell surface mechanism exists for the regulation of cellular responsiveness to pro-apoptotic stimuli.

Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA.

A new member of the tumor necrosis factor (TNF) cytokine family, designated Apo-2 ligand (Apo-21) [1] or TRAIL [2], has been shown recently to induce apoptosis in various tumor cell lines; however, its biological role is unknown. Here, we show that Apo-21, activated apoptosis in T-cell-enriched cultures of peripheral blood lymphocytes stimulated by interleukin-2 (IL-2), but not in unstimulated cells. This finding suggests that, like Fas/Apo-1 ligand and TNF [3-5], Apo-2L may play a role in regulating post-stimulation apoptosis of mature lymphocytes. Studies on the mechanism of Apo-2L action demonstrated marked membrane blebbing, a hallmark of apoptosis, within a few minutes of the addition of Apo-2L to tumor cells. Ectopic expression of a dominant negative mutant of FADD, a cytoplasmic protein that mediates death signalling by Fas/Apo-1 and by TNF receptor type 1 (TNFR1) [6-9], inhibited the induction of apoptosis by anti-Fas/Apo-1 antibody, but had little effect on Apo-2L function. In contrast, expression of CrmA, a cowpox virus-derived inhibitor of the Ced-2-like proteases ICE [10] and CPP32/Yama [11,12], blocked the induction of apoptosis by either Apo-2L or anti-Fas/Apo-1 antibody. These results suggest that Apo-2L activates a rapid, FADD-independent pathway to trigger a cell-death programme that requires the function of cysteine proteases such as ICE or CPP32/Yama.

Identification of a ligand for the death-domain-containing receptor Apo3.

The tumor necrosis factor (TNF) cytokine family regulates development and function of the immune system [1]. TNF is expressed primarily by activated lymphocytes and macrophages and induces gene transcription or apoptosis in target cells [2,3]. We have identified a novel relative of TNF that binds to the recently discovered, death-domain-containing receptor called Apo3 [4] (also known as DR3, WSL-1, TRAMP or LARD [5-9]). The Apo3 ligand (Apo3L) is a 249 amino-acid, type II transmembrane protein. The extracellular sequence of Apo3L shows highest identity to that of TNF. We detected Apo3L mRNA in many human tissues and mapped its encoding gene to chromosome 17p13, near the p53 tumor-suppressor gene. Soluble Apo3L induced apoptosis and nuclear factor kappaB (NF-kappaB) activation in human cell lines. Caspase inhibitors blocked apoptosis induction by Apo3L, as did a dominant-negative mutant of the cell death adaptor protein Fas-associated death domain protein (FADD/MORT1), which is critical for apoptosis induction by TNF [3]. Dominant-negative mutants of several factors that play a key role in NF-kappaB induction by TNF [10] inhibited NF-kappaB activation by Apo3L. Thus, Apo3L has overlapping signaling functions with TNF, but displays a much wider tissue distribution.

Interaction of the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and TACI.

BLyS (also called TALL-1, THANK, or BAFF) [1] [2] [3] [4] is a member of the tumor necrosis factor (TNF) gene family that stimulates proliferation and immunoglobulin production by B cells. BLyS interacts with the TNF receptor (TNFR) homologue TACI (transmembrane activator and CAML-interactor) [5], and treatment of mice with a TACI-Fc fusion protein abolishes germinal center formation after antigenic challenge [6]. Here we report a novel interaction between BLyS and another TNFR homologue, BCMA (B cell maturation antigen) [7] [8]. Further, the TNF homologue APRIL [9], a close relative of BLyS, also bound to BCMA and TACI. BLyS or APRIL activated nuclear factor-kappaB (NF-kappaB) through TACI and BCMA, and each ligand stimulated immunoglobulin M (IgM) production by peripheral blood B cells. These results define a dual ligand-receptor system that may play an important role in humoral immunity.

A novel receptor for Apo2L/TRAIL contains a truncated death domain.

Apo2 ligand (Apo2L [1], also called TRAIL for tumor necrosis factor (TNF)-related apoptosis-inducing ligand [2]) belongs to the TNF family and activates apoptosis in tumor cells. Three closely related receptors bind Apo2L: DR4 and DR5, which contain cytoplasmic death domains and signal apoptosis, and DcR1, a decoy receptor that lacks a cytoplasmic tail and inhibits Apo2L function [3-5]. By cross-hybridization with DcR1, we have identified a fourth Apo2L receptor, which contains a cytoplasmic region with a truncated death domain. We subsequently named this protein decoy receptor 2 (DcR2). The DcR2 gene mapped to human chromosome 8p21, as did the genes encoding DR4, DR5 and DcR1. A single DcR2 mRNA transcript showed a unique expression pattern in human tissues and was particularly abundant in fetal liver and adult testis. Upon overexpression, DcR2 did not activate apoptosis or nuclear factor-kappaB; however, it substantially reduced cellular sensitivity to Apo2L-induced apoptosis. These results suggest that DcR2 functions as an inhibitory Apo2L receptor.

Apo-3, a new member of the tumor necrosis factor receptor family, contains a death domain and activates apoptosis and NF-kappa B.

BACKGROUND: Two receptors that contain the so-called "death domain' have been described to date: tumor necrosis factor receptor 1 (TNFR1) and Fas/Apo-1 (CD95); both belong to the TNFR gene family. The death domain of TNFR1 mediates the activation of programmed cell death (apoptosis) and of the transcription factor NF-kappa B, whereas the death domain of CD95 only appears to activate apoptosis. RESULTS: We have identified an additional member of the TNFR family, which we have named Apo-3. Apo-3 is a transmembrane protein of approximately 47 kDa that has similarity of members of the TNFR family in its extracellular, cysteine-rich domains. In addition, Apo-3 resembles TNFR1 and CD95 in that it contains a cytoplasmic death domain. The Apo-3 gene mapped to human chromosome 1p36.3, and Apo-3 mRNA was detected in several human tissues, including spleen, thymus, peripheral blood lymphocytes, small intestine and colon. Ectopic expression of Apo-3 in HEK293 or HeLa cells induced marked apoptosis. CrmA, a poxvirus inhibitor of Ced-3-like proteases which blocks death signaling by TNFR1 and CD95, inhibited Apo-3-induced apoptosis. Ectopic expression of Apo-3 also induced the activation of NF-kappa B. Apo-3 did not specifically bind to the Apo-2 ligand, suggesting the existence of a distinct ligand for Apo-3. CONCLUSIONS: These results identify Apo-3 as a third member of the TNFR family that activates apoptosis, and suggest that Apo-3, TNFR1 and CD95 engage a common apoptotic cell-death machinery. Apo-3 resembles TNFR1 because it can stimulate NF-kappa B activity and regulate apoptosis. Apo-3 mRNA is expressed in various tissues, consistent with the possibility that this receptor may regulate multiple signaling functions.

Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR.

The tumor necrosis factor (TNF) and TNF receptor (TNFR) gene superfamilies regulate diverse biological functions, including cell proliferation, differentiation, and survival [1] [2] [3]. We have identified a new TNF-related ligand, designated human GITR ligand (hGITRL), and its human receptor (hGITR), an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related (mGITR) protein [4]. The hGITRL gene mapped to chromosome 1q23, near the gene for the TNF homolog Fas/CD95 ligand [5]. The hGITR gene mapped to chromosome 1p36, near a cluster of five genes encoding TNFR homologs [1] [6]. We found hGITRL mRNA in several peripheral tissues, and detected hGITRL protein on cultured vascular endothelial cells. The levels of hGITR mRNA in tissues were generally low; in peripheral blood T cells, however, antigen-receptor stimulation led to a substantial induction of hGITR transcripts. Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB, via a pathway that appeared to involve TNFR-associated factor 2 (TRAF2) [7] and NF-kappaB-inducing kinase (NIK) [8]. Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death. Thus, hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues.

Protection against endotoxic shock by bactericidal/permeability-increasing protein in rats.

Bactericidal/permeability-increasing protein (BPI) is a neutrophil primary granule protein that inhibits effects of LPS in vitro. The current study examined the effects of BPI on hemodynamics, mortality, and circulating endotoxin and cytokines in conscious rats with endotoxic shock. Catheters were implanted into the right femoral artery and vein. 1 d later, human recombinant BPI (10 mg/kg) or vehicle was intravenously injected immediately, 30 min, or 2 h after intravenous injection of LPS (7.5 mg/kg). Mean arterial pressure (MAP) and heart rate were monitored and blood was collected before and after injection. BPI given immediately or 30 min after LPS prevented the LPS-induced reduction in MAP at 4-8 h and markedly reduced mortality. BPI given 2 h after LPS injection had no protective effect. BPI treated immediately after LPS reduced the circulating levels of endotoxin and IL-6 but increased the circulating levels of TNF. We propose that BPI exerts its protective effect through a TNF-independent mechanism, by inhibiting endotoxin-stimulated production of IL-6.

Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Ligand-induced assembly and activation of the gamma interferon receptor in intact cells.

Functionally active gamma interferon (IFN-gamma) receptors consist of an alpha subunit required for ligand binding and signal transduction and a beta subunit required primarily for signaling. Although the receptor alpha chain has been well characterized, little is known about the specific role of the receptor beta chain in IFN-gamma signaling. Expression of the wild-type human IFN-gamma receptor beta chain in murine L cells that stably express the human IFN-gamma receptor alpha chain (L.hgR) produced a murine cell line (L.hgR.myc beta) that responded to human IFN-gamma. Mutagenesis of the receptor beta-chain intracellular domain revealed that only two closely spaced, membrane-proximal sequences (P263PSIP267 and I270EEYL274) are required for function. Coprecipitation studies showed that these sequences are necessary for the specific and constitutive association of the receptor beta chain with the JAK-2 tyrosine kinase. These experiments also revealed that the IFN-gamma receptor alpha and beta chains are not preassociated on the surface of unstimulated cells but rather are induced to associate in an IFN-gamma-dependent fashion. A chimeric protein in which the intracellular domain of the beta chain was replaced by JAK-2 complemented human IFN-gamma signaling and biologic responsiveness in L.hgR. In contrast, a c-src-containing beta-chain chimera did not. These results indicate that the sole obligate role of the IFN-gamma receptor beta chain in signaling is to recruit JAK-2 into the ligand-assembled receptor complex.

Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage.

The extracellular portion of the interleukin-1 receptor (IL-1R) is sufficient for high-affinity binding to IL-1; however, the structural basis for binding of the receptor to IL-1 is not known. To produce individual domains of IL-1 receptor for structural studies, we constructed a molecular fusion of IL-1 receptor with immunoglobulin G heavy chain that contains a protease specific sequence joining the two portions of the molecule (IL-1R-G-IgG). We introduced the hexapeptide sequence AAHY:TL (where ":" denotes the scissile bond) at the junction of the IL-1R and IgG regions, for specific cleavage by an H64A variant of subtilisin BPN' (Genenase I), an endoprotease that cleaves selectively at this sequence (Carter et al., (1989) Proteins 6, 240-248). Plasmid DNA encoding the fusion protein was used to transfect human embryonic kidney 293 cells transiently, and secreted IL-1R-G-IgG was purified from cell supernatants by protein A chromatography. The IL-1 receptor's extracellular region was then generated by enzymatic cleavage with Genenase I which was immobilized on controlled-pore glass. Incubation of IL-1R-G-IgG with immobilized Genenase I resulted in specific cleavage at the target site, as confirmed by SDS-PAGE, immunoblotting and direct sequencing of the newly generated termini. The resulting soluble IL-1R was separated from the immunoglobulin Fc cleavage product by re-chromatography on protein A. The purified, soluble IL-1R retained quantitatively the ability to bind to its ligand, IL-1 beta. This approach offers a generic means by which the extracellular region of a given type I transmembrane receptor can be expressed as an immunoadhesin, released enzymatically and then easily purified for crystallographic or ligand binding studies.

Molecular and biological properties of an interleukin-1 receptor immunoadhesin.

Overproduction of the cytokine interleukin 1 (IL-1) is an important factor in the pathogenesis of several autoimmune and inflammatory disease. To develop a recombinant inhibitor of IL-1 with an extended pharmacologic half-life, we constructed an IL-1 receptor immunoadhesin (IL-1R-IgG), by fusing the extracellular domain of the type IL-1 receptor with the hinge and Fc regions of human IgG1 heavy chain. Transfected human 293 cells express IL-1R-IgG as a secreted, disulfide-bonded homodimer. The secreted protein contains an intact antibody Fc region, as indicated by immunoblotting, and a functional IL-1 receptor region, as indicated by ligand-blotting. Saturation binding analysis indicates an equilibrium dissociation constant (KD) of 350 pM for the binding of IL-1R-IgG to its ligand, IL-1 beta. Kinetic analysis of the binding reveals an off rate of 0.1 min-1 and an on rate of 1.5 x 10(8) min-1 M-1, yielding a calculated KD of 770 pM. These binding properties are similar to those of cell-surface type I IL-1 receptor. IL-1R-IgG is capable of inhibiting the biological activity of IL-1 beta in vitro, as evidenced in a thymocyte proliferation assay. Pharmacokinetic analysis in mice indicates that IL-1R-IgG has a terminal half-life of 91 hr in the blood circulation. This half-life is markedly longer than the values reported for other recombinant inhibitors of IL-1 such as the IL-1 receptor antagonist or soluble IL-1 receptor. Thus, IL-1R-IgG may be useful for investigating the interaction of IL-1 with its receptor and the role of IL-1 in disease, as well as for potential intervention in pathological situations involving overproduction of IL-1.

TRAF2 is a biologically important necroptosis suppressor.

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)-driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)-previously implicated in apoptosis suppression-also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα-driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.

Identification of cysteine-rich domains of the type 1 tumor necrosis factor receptor involved in ligand binding.

The extracellular portion of the type 1 (p55) and type 2 (p75) tumor necrosis factor (TNF) receptors contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs). This pattern is found also in several other cell surface proteins, including the p75 nerve growth factor receptor and the CD40, 4-1BB, OX40, Fas, and CD27 antigens. To investigate whether CRDs play a role in TNF binding, we have constructed soluble variants of the extracellular portion of human type 1 TNF receptor (sTNFR1), in which CRD1 (N-terminal) or CRD4 (C-terminal) was deleted by mutagenesis. These variants or a wild type sTNFR1 were linked in their C terminus to the hinge and Fc portion of IgG1 heavy chain to create sTNFR1-IgG chimeras (immunoadhesins). Deletion of either CRD1 or -4 did not cause any major perturbations in the structure of the sTNFR1 variants, as evidenced by their efficient expression and secretion from transfected cells, and by their binding to conformation-dependent monoclonal antibodies that recognize diverse epitopes on sTNFR1. The wild type sTNFR1 immunoadhesin exhibited high affinity binding to TNF alpha (Kd = 65 pM) and TNF beta (Kd = 640 pM). Deletion of CRD4 resulted in about a 10-fold reduction in affinity for TNF alpha (Kd = 660 pM) and for TNF beta (Kd = 5.7 nM). In contrast, deletion of CRD1 resulted in a complete loss of binding to TNF alpha and to TNF beta. These results indicate that CRD4 is important but not necessary for TNF binding, while CRD1 is required. In addition, the results suggest some overlap between the TNFR1 binding sites for TNF alpha and TNF beta, despite low amino acid sequence homology between these cytokines.

Inhibition of murine erythroid colony formation in vitro by interferon gamma and correction by interferon receptor immunoadhesin.

It has been previously reported that inhibition of human erythroid colony-forming units (CFU-E) in vitro by interleukin-1 (IL-1) is an indirect effect, occurring through the production of interferon gamma (IFN gamma). IFN gamma, in turn, inhibits CFU-E colony formation directly, and its inhibitory effect can be overcome by exposure to high concentrations of erythropoietin (EPO). To develop an in vitro animal model for investigating inhibition of erythropoiesis by IFN gamma, the effects of recombinant murine (rm) IFN gamma on highly purified CFU-E from the spleens of mice infected with the anemia strain of the Friend virus (FVA) were studied. rmIFN gamma inhibited CFU-E colony formation in a dose-dependent manner. This inhibition occurred with large (> or = 8 cell) colonies only; smaller colonies were not affected. The inhibitory effect was corrected to 72% of control by high EPO concentrations of 64 U/mL. Murine CFU-E were then cultured with rmIFN gamma in the presence of a soluble murine IFN gamma receptor fused to the hinge and Fc domains of the human IgG1 heavy chain (mIFN gamma R-IgG). Inhibition of CFU-E colony formation by rmIFN gamma (100 U/mL) was corrected by mIFN gamma R-IgG in a dose-dependent manner, with an approximate IC50 of 0.05 nmol/L, and complete or near complete correction at 0.5 nmol/L. Similarly, a human IFN gamma R-IgG greatly reduced the inhibitory effect of recombinant human IFN gamma on human CFU-E. These experiments provide an in vitro animal model for studying the inhibitory effects of IFN gamma on erythropoiesis and indicate that IFN gamma R-IgG may be a useful agent for reducing the toxicity of IFN gamma in vivo.

Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.

Signaling by interferon gamma (IFN-gamma) requires two structurally related cell surface proteins: a ligand-binding polypeptide, known as the IFN-gamma receptor (IFN-gamma R), and an accessory factor. However, it is not known whether IFN-gamma forms a ternary complex with the IFN-gamma R and accessory factor to initiate signaling. Here we demonstrate complex formation between IFN-gamma and the two proteins, both in solution and at the cell surface. We observe complexes containing ligand, two molecules of IFN-gamma R (designated the IFN-gamma R alpha chain), and one or two molecules of accessory factor (designated the IFN-gamma R beta chain). Transfected cells expressing both IFN-gamma R chains bind IFN-gamma with higher affinity than do cells expressing alpha chain alone. Anti-beta-chain antibodies prevent the beta chain from participating in the ligand-receptor complex, reduce the affinity for IFN-gamma, and block signaling. Soluble alpha- or beta-chain extracellular domains also inhibit function. These results demonstrate that IFN-gamma signals via a high-affinity multisubunit complex that contains two types of receptor chain and suggest a potential approach to inhibiting specific actions of IFN-gamma by blocking the association of receptor subunits.

Control of apoptosis signaling by Apo2 ligand.

Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. The closest homolog of Apo2L is CD95 (Fas/Apo1) ligand, to which it has 24% amino acid sequence identity. Similar to CD95L, Apo2L activates rapid apoptosis in many types of cancer cells; however, whereas CD95L mRNA expression is restricted mainly to activated T cells, natural killer cells, and immune-privileged sites, Apo2L mRNA occurs in a wide variety of tissues. Most normal cells appear to be resistant to Apo2L's cytotoxic action, suggesting the existence of mechanisms that can protect against apoptosis induction by Apo2L. The first receptor described for Apo2L, called death receptor 4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits the apoptosis signal carried by Apo2L. We have identified three additional receptors that bind to Apo2L. One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines. The second receptor, designated decoy receptor 1 (DcR1), is a phospholipid-anchored cell-surface protein that lacks a cytoplasmic tail. The third receptor, called DcR2, is structurally similar to DR4 and DR5 but has a truncated cytoplasmic death domain and does not transmit a death signal. The mRNAs for DcR1 and DcR2 are expressed in multiple normal tissues but in few tumor cell lines. Transfection experiments indicate that DcR1 and DcR2 act as decoys that prevent Apo2L from inducing apoptosis through DR4 and DR5. These decoy receptors thus represent a novel mechanism for regulating sensitivity to a pro-apoptotic cytokine directly at the cell's surface. The preferential expression of these inhibitory receptors in normal tissues suggests that Apo2L may be useful as an anticancer agent that induces apoptosis in cancer cells while sparing normal cells.