ABSTRACT
Using the monoclonal antibody C1.7, which recognizes a signaling, membrane-bound molecule on human NK and a proportion of CD8(+) T cells, we cloned a novel molecule we refer to as NK cell activation-inducing ligand (NAIL). It is a 365-amino acid protein that belongs to the immunoglobulin-like superfamily with closest homology to murine 2B4, and human CD84 and CD48. Using a soluble NAIL-Fc fusion protein, we determined the counterstructure for NAIL, CD48, which it binds with high affinity. Stimulation of human B cells with recombinant NAIL in the presence of a suboptimal concentration of human CD40 ligand or IL-4 resulted in increased proliferation. Treatment of human dendritic cells with soluble NAIL-leucine zipper protein resulted in an increased release of IL-12 and TNF-alpha. Using recombinant CD48 protein, we demonstrated the ability of this molecule to increase NK cell cytotoxicity and induce IFN-gamma production. We also showed that 2B4 binds to mouse CD48, suggesting that interaction of these receptors may play a similar role in both species. Taken together these results indicate that the NAIL-CD48 interaction may be an important mechanism regulating a variety of immune responses.
Subject(s)
Antigens, CD/metabolism , Killer Cells, Natural/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , B-Lymphocytes/cytology , CD48 Antigen , Cell Division , Cloning, Molecular , Cytokines/biosynthesis , Cytotoxicity, Immunologic , Humans , Immunoglobulin Fc Fragments/metabolism , Interferon-gamma/biosynthesis , Jurkat Cells , K562 Cells , Ligands , Membrane Glycoproteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Molecular Sequence Data , Phosphorylation , RNA, Messenger , Receptors, Immunologic/metabolism , Recombinant Fusion Proteins/metabolism , Signaling Lymphocytic Activation Molecule Family , Tyrosine/metabolism , U937 Cells , Up-RegulationABSTRACT
mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.
Subject(s)
Antibodies, Monoclonal/immunology , Apoptosis , Receptors, Tumor Necrosis Factor/physiology , Animals , Apoptosis Regulatory Proteins , Caspase Inhibitors , GPI-Linked Proteins , Humans , Membrane Glycoproteins/antagonists & inhibitors , Mice , Mice, Inbred BALB C , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor, Member 10c , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor Decoy Receptors , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Membrane Glycoproteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Amino Acid Sequence , Apoptosis Regulatory Proteins , B-Lymphocytes/metabolism , Base Sequence , Carrier Proteins/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 8 , Cloning, Molecular , Cysteine Endopeptidases/metabolism , Fas-Associated Death Domain Protein , Humans , Molecular Sequence Data , RNA, Messenger/analysis , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/isolation & purification , Sequence Analysis , Sequence Homology, Amino Acid , Serine Proteinase Inhibitors/pharmacology , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand , Tissue DistributionABSTRACT
A fourth member of the emerging TRAIL receptor family, TRAIL-R4, has been cloned and characterized. TRAIL-R4 encodes a 386-amino acid protein with an extracellular domain showing 58%-70% identity to those of TRAIL-R1, TRAIL-R2, and TRAIL-R3. The signaling capacity of TRAIL-R4 is similar to that of TRAIL-R1 and TRAIL-R2 with respect to NF-kappaB activation, but differs in its inability to induce apoptosis. Yet TRAIL-R4 retains a C-terminal element containing one third of a consensus death domain motif. Transient overexpression of TRAIL-R4 in cells normally sensitive to TRAIL-mediated killing confers complete protection, suggesting that one function of TRAIL-R4 may be inhibition of TRAIL cytotoxicity. Like TRAIL-R1 and TRAIL-R2, this receptor shows widespread tissue expression. The human TRAIL-R4 gene has been mapped to chromosome 8p22-21, clustered with three other TRAIL receptors.
