Title: The Involvement of Multiple Tumor Necrosis Factor Receptor (TNFR)-associated Factors in the Signaling Mechanisms of Receptor Activator of NF-κB, a Member of the TNFR Superfamily
Abstract: Receptor activator of NF-κB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-κB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-κB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-κB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events. Receptor activator of NF-κB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-κB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-κB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-κB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events. receptor activator of NF-κB tumor necrosis factor TNF receptor TNF receptor-associated factor RANK ligand electrophoretic mobility shift assay dendritic cell glutathione S-transferase TRAF binding site c-Jun NH2-terminal kinase polymerase chain reaction polyacrylamide gel electrophoresis interleukin 4-morpholinepropanesulfonic acid. RANK1and RANK ligand (RANKL) are a recently described cognate pair of the TNF receptor/ligand superfamilies (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar). The receptor (RANK) cDNA was originally isolated from a human DC cDNA library and shows the highest homology (40% identity within the extracellular domain) with CD40 among TNFR family members. Among antigen-presenting cells, RANK surface expression appears to be specific to DC and can be significantly up-regulated by a DC activator, CD40 ligand. However, RANK protein expression is not DC-specific as RANK is also expressed on human peripheral blood T cells treated with phytohemagglutinin and IL-4 or transforming growth factor-β (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar). In contrast to the relatively specific protein expression, RANK mRNA is broadly expressed in a variety of tissues including skeletal muscle, thymus, liver, colon, adrenal gland, and small intestine. The discrepancy between mRNA and surface protein expression suggests complex post-transcriptional regulatory mechanisms for RANK expression. Cells may therefore express RANK after discrete activation or differentiation conditions. The identification of the cognate ligand for RANK (RANKL) was performed by direct expression cloning from a mouse CD4+ thymoma cell line (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar). The same ligand has also been identified by screening a T cell hybridoma cell line (termed TRANCE) (2Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett III, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar) and as an osteoclast differentiation factor (3Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3571) Google Scholar) whose activity can be inhibited by a soluble TNF receptor family member, osteoprotegerin (4Lacey D.L. Timms E. Tan H.-L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4627) Google Scholar). RANKL mRNA appears to have a more restricted tissue expression pattern than RANK and has only been detected in mouse thymus, lymph node, spleen (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar, 2Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett III, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar), bone marrow stroma, and trabecular bone (3Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3571) Google Scholar). Specific lymphoid cells that express RANKL include both CD4+ and CD8+ T cells and B cell progenitors (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar, 2Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett III, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). TCR stimulation of T cell hybridomas leads to the rapid induction of RANKL/TRANCE mRNA (2Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett III, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). Studies of the biological function of the RANK/RANKL interaction demonstrate that RANKL promotes the survival of transforming growth factor-β-treated T cells and increases the clustering and allo-stimulatory capacity of human DC (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar). RANKL may promote DC survival by a BCL-XL-dependent mechanism (5Wong B.R. Josien R. Lee S.Y. Sauter B. Li H.-L. Steinman R.M. Choi Y. J. Exp. Med. 1997; 186: 2075-2080Crossref PubMed Scopus (752) Google Scholar). The recent characterization of RANKL as an essential factor for osteoclast differentiation and activation (3Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3571) Google Scholar, 4Lacey D.L. Timms E. Tan H.-L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4627) Google Scholar) demonstrates additional activities of RANKL on myeloid lineages. Although the role of RANK in bone resorption/osteoclast differentiation has not been elucidated, RANK and its ligand appear to be important in the regulation of T cell/DC interactions and may also function in other important cellular differentiation processes. Although the physiological functions of RANK and RANKL have been investigated, the mechanisms of RANK signal transduction have not been intensively studied. Multimerization of TNFR family members (as a result of ligand binding or receptor overexpression) is thought to lead to receptor activation and signal transduction, perhaps by revealing binding domains for enzymes or adaptor proteins. In recent years, substantial progress has been made to define the cytoplasmic proteins that function as adaptors (TRAFs 1–6, TRADD, FADD) or as serine/threonine kinases (RIP) and link TNF receptor stimulation with the induction of cell death, Jun kinase (JNK) or NF-κB activation pathways (reviewed in Ref. 6Darnay B.G. Aggarwal B.B. J. Leukocyte. Biol. 1997; 61: 559-566Crossref PubMed Scopus (168) Google Scholar). RANK stimulation leads to activation of the nuclear transcription complex NF-κB in RANK-expressing human T cells and transfected 293 cells (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar) and JNK (2Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett III, F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar) in mouse thymocytes. However, the RANK cytoplasmic determinants or the cytoplasmic effector/adaptor proteins necessary for downstream signaling have not been described. While the 383-amino acid cytoplasmic domain of RANK is the largest of any known TNFR, it does not contain sequences suggestive of catalytic activity or significant homology with any known protein. The amino acid sequences of the human and mouse RANK cytoplasmic domains include multiple sections that show striking homology (64% amino acid identity and 78% similarity) between species (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar), suggesting a conserved functional role for these structures. Since many members of the TNFR superfamily that do not contain a death domain (p80 TNFR, CD40, CD30, CD27, OX40, 4–1BB, LTβR, HVEM) interact with the cytoplasmic adaptor/effector TRAF proteins (7Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (978) Google Scholar, 8Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 9Hsu H. Solovyev I. Colombero A. Elliott R. Kelley M. Boyle W.J. J. Biol. Chem. 1997; 272: 13471-13474Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Marsters S.A. Ayres T.M. Skubatch M. Gray C.L. Rothe M. Ashkenazi A. J. Biol. Chem. 1997; 272: 14029-14032Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 11Gedrich R.W. Gilfillan M.C. Duckett C.S. Van Dongen J.L. Thompson C.B. J. Biol. Chem. 1996; 271: 12852-12858Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12Arch R.H. Thompson C.B. Mol. Cell. Biol. 1998; 18: 558-565Crossref PubMed Google Scholar, 13Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C.F. Malinin N.L. Wallach D. Yagita H. Okumura K. J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), we designed an approach to experimentally define TRAF binding to RANK. In this study we have identified the structural and functional features of RANK required for both NF-κB and JNK-mediated signaling. Our analysis delineates multiple distinct and independent domains capable of binding TRAF proteins and transmitting downstream signals. In addition, TRAF6 appears to mediate RANK function through unique determinants independent of other TRAF proteins and plays a selective role in RANKL-induced signaling. Deletion constructs of RANK were generated by PCR amplification, and point mutants were generated by PCR using primers containing the appropriate nucleotide substitutions. The appropriate inserts were subcloned into the pGEX-4T (Amersham Pharmacia Biotech) vector at the BamHI and EcoRI sites. The correct sequence of each of the PCR-generated inserts was confirmed by DNA sequencing. A GST-human FAS cytoplasmic domain construct was used as a specificity control. GST fusion proteins were expressed and purified from Escherichia coli by glutathione-agarose affinity media according to published procedures (14Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). Fusion protein concentrations of 0.5–2.5 mg/ml were typically obtained. Full-length TRAF cDNAs were subcloned into the pBluescript (Stratagene, La Jolla, CA) or pGEM-T (Promega Corp., Madison, WI) vectors. RNA was generated in vitro with either T7 or T3 polymerase using the Ambion (Austin, TX) mMessage Machine according to the manufacturer's protocol. RNA (1 μg) was translated in the presence of [35S]methionine/cysteine using 25 μl of rabbit reticulocyte lysate (TNT-coupled Reticulocyte Lysate Systems, Promega). The integrity of the protein product was determined by SDS-PAGE, and relative protein amounts were normalized after image analysis using a Molecular Dynamics (Sunnyvale, CA) Storm 860 optical scanner. Proteins were diluted into binding buffer (50 mm HEPES (pH 7.4), 250 mm NaCl, 0.25% (v/v) Nonidet P-40, 10% glycerol, 2 mm EDTA) and precleared by overnight incubation with 100 μg of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). For each in vitro binding assay, translated protein was combined with 5 μg of the appropriate GST fusion protein complexed to glutathione-Sepharose beads and incubated with rotation for 2 h at 4 °C. The protein complexes were recovered by centrifugation, washed four times in binding buffer, and analyzed by SDS-PAGE. Gels were stained with Coomassie Blue to confirm equal loading of GST fusion proteins, treated with Amplify (Amersham Pharmacia Biotech), and subjected to fluorography for 6–24 h. A NF-κB-responsive reporter plasmid was constructed in pGL2-Basic (Promega) with the human IL-8 promoter containing a NF-κB binding site (15Bonnert T.P. Garka K.E. Parnet P. Sonoda G. Testa J.R. Sims J.E. FEBS Lett. 1997; 402: 81-84Crossref PubMed Scopus (112) Google Scholar) fused to a luciferase reporter. 293/EBNA cells (Invitrogen, San Diego, CA) were transiently transfected by the DEAE-dextran method with the reporter plasmid either alone or in combination with full-length human RANK cDNA in pDC304 (16Mosley B. Beckmann M.P. March C.J. Idzerda R.L. Gimpel S.D. VandenBos T. Friend D. Alpert A. Anderson D. Jackson J. Wignall J.M. Smith C. Gallis B. Sims J.E. Urdal D. Widmer M.B. Cosman D. Park L.S. Cell. 1989; 59: 335-348Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Full-length and deletion constructs of TRAF cDNAs (with an in-frame NH2-terminal FLAG tag) were generated by PCR amplification and cloned into pDC304. The TRAF2 NH2-terminal deletion (TRAF2-(87–501)) removed the first 86 amino acids (as described in Ref. 17Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar). The zinc-ring truncation of TRAF5-(234–558) was constructed similarly as described (18Nakano H. Oshima H. Chung W. Williams-Abbott L. Ware C.F. Yagita H. Okumura K. J. Biol. Chem. 1996; 271: 14661-14664Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The TRAF6 construct truncated the zinc ring and each of the zinc fingers (TRAF6-(289–522)) (19Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1122) Google Scholar). A β-galactosidase-expressing plasmid (pDC304/LACZ; 25 ng/well) was used as an internal control for transfection efficiency. Total DNA concentrations for each transfection were equalized by the addition of empty pDC304 vector. Transfections were performed in triplicate. Twenty-four hours after transfection, cells were treated with recombinant human RANKL for 16 h at 37 °C. Recombinant human RANKL is an NH2-terminal fusion of a leucine zipper trimerization domain (20Fanslow W.C. Srinivasan S. Paxton R. Gibson M.G. Spriggs M.K. Armitage R.J. Semin. Immunol. 1994; 6: 267-278Crossref PubMed Scopus (150) Google Scholar) with residues 138–317 of RANKL (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar). Luciferase activity in cell lysates was measured according to manufacturer's instructions (Promega) using a EG&G/Berthold luminometer. Relative luciferase activities were normalized to the β-galactosidase activity. Nuclear extracts were prepared from 293 cells transfected with full-length or cytoplasmic truncations of RANK or control vector 24 h after transfection as described (21Yao Z. Fanslow W.C. Seldin M.F. Rousseau A.-M. Painter S.L. Comeau M.R. Cohen J.I. Spriggs M.K. Immunity. 1995; 3: 811-821Abstract Full Text PDF PubMed Scopus (806) Google Scholar). Oligonucleotides containing an NF-κB binding site were annealed, radiolabeled with [γ-32P]ATP and combined with 10 μg of nuclear extracts for 20 min at room temperature. Specificity of the reaction was confirmed by competition with 50-fold molar excess of non-labeled wild-type oligonucleotides or oligonucleotides containing a mutated NF-κB binding site. The protein-DNA complexes were resolved by 6% PAGE in 0.25× TBE buffer and visualized by autoradiography. For JNK assays, whole cell extracts were prepared from 293 cells 24 h after transfection. Cells were lysed in a buffer containing 20 mm HEPES, pH 7.4, 2 mm EGTA, 50 mm β-glycerol phosphate, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 1% Triton X-100, 10% glycerol, and the protease inhibitors leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride. Clarified lysates were immunoprecipitated with 1 μg each of anti-JNK (FL) and anti-JNK (C17) (both from Santa Cruz Biotechnology, Inc. Santa Cruz, CA). The immune complexes were washed three times in lysis buffer, two times with wash buffer (500 mm LiCl, 100 mm Tris, pH 7.5, 0.1% Triton X-100, 1 mm dithiothreitol) and three times in assay buffer (20 mm MOPS, pH 7.0, 2 mm EGTA, 10 mm MgCl2, 1 mm dithiothreitol, 0.1% Triton X-100). JNK activity was determined by an immune-complex assay using 1 μg of GST-c-Jun-(1–169) (Upstate Biotechnology Inc., Lake Placid, NY) and 5 μCi of [32P]ATP as substrate in 40 μl of assay buffer at 30 °C for 20 min. Reaction products were resolved on 4–20% SDS-PAGE and visualized by autoradiography. In order to define whether TRAFs may bind to RANK, each of the known TRAFs was transcribed and translated in vitro in the presence of [35S]methionine/cysteine and coprecipitation assays were performed to determine the interaction with a GST fusion expressing the full-length RANK cytoplasmic domain. This domain (RANK amino acids 206–616) was able to interact with TRAFs 1, 2, 3, 5, and 6 in a specific manner (Fig.1). We observed strong binding of RANK with TRAFs 1, 2, 3, and 6; weak interaction with TRAF5; and no binding with TRAF4. In contrast, none of the TRAFs interacted with a control GST protein (Fig. 1) or a GST-FAS cytoplasmic domain fusion protein (data not shown). To confirm that the in vitro interaction of TRAFs with RANK also occurred in cells, co-immunoprecipitation experiments were performed in 293 cells cotransfected with RANK and epitope-tagged TRAFs and revealed that full-length RANK associated with the same repertoire of TRAFs (TRAFs 1, 2, 3, 5, and 6) but not TRAF4 (data not shown). To identify the binding sites in the RANK cytoplasmic domain, four individual COOH-terminal deletions were constructed and the in vitro binding assays were repeated. In contrast to the full-length RANK cytoplasmic domain, a deletion construct that lacks the COOH-terminal 72 amino acids (RANK Δ544) was unable to interact with TRAFs 1, 2, 3, and 5 (Fig. 1), and TRAF6 binding was reduced by approximately 50%. The deletion of an additional 68 amino acids (RANK Δ476) or 123 amino acids (RANK Δ421) showed no difference in TRAF6 binding activity relative to RANK 206–544. However, when the COOH-terminal 277 amino acids were deleted (RANK Δ339), the remaining interaction with TRAF6 was lost. These data demonstrate that two separate regions of the RANK cytoplasmic domain are each capable of binding TRAF proteins. Examination of the protein sequence within the COOH-terminal 72-amino acid RANK domain capable of TRAF binding revealed multiple regions with limited homology with experimentally defined TRAF binding sites (TBS) in other TNFR family members. We defined two potential RANK TBS in this region (residues 569–574 (P-V-Q-E-E-T) and residues 607–611 (P-V-Q-E-Q)) due to their similarity to the P-X-Q-X-T TBS found in CD40 and CD30 (22Boucher L.-M. Marengère L.E. Lu Y. Thukral S. Mak T.W. Biochem. Biophys. Res. Commun. 1997; 233: 592-600Crossref PubMed Scopus (89) Google Scholar) as well as the HVEM TBS (V-E-E-T) (9Hsu H. Solovyev I. Colombero A. Elliott R. Kelley M. Boyle W.J. J. Biol. Chem. 1997; 272: 13471-13474Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and the OX40 TBS (P-I-Q-E-E) (12Arch R.H. Thompson C.B. Mol. Cell. Biol. 1998; 18: 558-565Crossref PubMed Google Scholar). To determine if these residues in RANK were important for TRAF binding, we substituted certain amino acids within these putative TBS with alanine and assayed TRAF binding. Mutagenesis of amino acids 609–610 (Q-E) to alanine within the full-length RANK cytoplasmic domain significantly abolished TRAF1, TRAF2, and TRAF5 binding (Fig.2), similar to that seen with the GST-RANK Δ544 deletion. Binding of TRAF3 and TRAF6 were unaffected. Substitution of residues 571–573 (Q-E-E) with alanine resulted in a loss of TRAF3 binding without reducing the binding of any other TRAF. These results identify two separate regions critical for binding TRAFs 1, 2, 3, and 5. By combining mutations of these two sites (571–573 and 609–610), loss of TRAFs 1, 2, and 3 binding was more pronounced (Fig.2), suggesting that these two sites cooperate in binding. None of the mutations examined, either alone or in combination, reduced TRAF6 binding, indicating that TRAF6 recognizes distinct TBS in RANK. We have previously reported that activation of RANK either by receptor overexpression or by RANKL treatment leads to the activation of NF-κB complexes (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. Dubose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar). In order to study the role of RANK cytoplasmic sequences and other cellular protein effectors in RANK signaling, we established a transient transfection/NF-κB-responsive reporter system. We first examined reporter activity resulting from increased RANK expression in the 293 cells. NF-κB-dependent reporter activity increased in a RANK dose-dependent manner until optimal (30-fold) induction was achieved with 6.4 ng of RANK DNA transfected (Fig. 3 A). These data illustrate that ectopic overexpression of RANK can lead to ligand-independent NF-κB signaling, similar to that seen with other TNFR family members, p80 TNFR and CD40 (7Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (978) Google Scholar, 23Cheng G. Baltimore D. Genes Dev. 1996; 10: 963-973Crossref PubMed Scopus (265) Google Scholar). RANKL-dependent signaling was next examined by the transfection/reporter system using a suboptimal RANK DNA concentration (0.4 ng/transfection). Co-transfection of RANK and the transmembrane form of RANKL stimulated the NF-κB-reporter more than that seen after transfection with RANK only (Fig. 3 B). We also demonstrated that soluble RANKL protein enhanced reporter activity (Fig.3 B). Similar effects of RANK overexpression or RANKL treatment were also seen with a minimal NF-κB promoter (data not shown), confirming that these responses are due to NF-κB activation. The interaction of TRAFs with multiple RANK cytoplasmic domains and the ability of some TRAFs to mediate NF-κB activation (7Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (978) Google Scholar, 24Song H.Y. Régnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (507) Google Scholar) suggests that the deletion of TBS may abrogate NF-κB-dependent signaling. To address this possibility, COOH-terminal cytoplasmic deletions of RANK were expressed in 293 cells and reporter activity was measured. Equivalent expression levels of each RANK construct were confirmed by immunoprecipitation of metabolically labeled proteins and by flow cytometry (data not shown). Transfection of 293 cells with the RANK deletion construct lacking the COOH-terminal 72 amino acids (RANK Δ544) resulted in reduced NF-κB reporter activity in the absence of RANKL activation (Fig.4 A). However, RANKL treatment of RANK Δ544-expressing cells induced NF-κB activation to levels similar to that seen with full-length RANK. Further deletion of COOH-terminal sequences had minimal effects on the constitutive and RANKL-mediated reporter activity until the removal of amino acids 339–422 (construct RANK Δ339), which completely abrogated both constitutive signaling and responsiveness to RANKL. The deletion of RANK cytoplasmic determinants had similar effects on NF-κB activation as determined by direct EMSA assays (Fig. 4 B). Taken together, these data suggest that RANK contains two domains (amino acids 339–422 and 544–616) within its cytoplasmic tail important for NF-κB signaling. These two RANK functional regions correspond with the two domains that affect binding of TRAFs 1, 2, 3, 5, and 6 (amino acids 544–616) and TRAF6 (amino acids 339–422). Moreover, the deletional analysis of RANK domains important for NF-κB signaling expose differences between RANK signal transduction as a result of receptor overexpression and RANKL treatment. Constitutive signaling resulting from RANK overexpression was significantly affected by the loss of the COOH-terminal 72 amino acids (RANK Δ544). However, RANKL-mediated signaling was only affected by the deletion of amino acids 339–422 correlating to the loss of direct TRAF6 binding in vitro. In the absence of receptor expression, TRAFs 2, 5, and 6 activated NF-κB (data not shown) as has been reported previously (7Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (978) Google Scholar, 24Song H.Y. Régnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (507) Google Scholar). To examine the functional involvement of these TRAFs in RANK-mediated NF-κB activation, 293 cells were co-transfected with RANK and expression vectors encoding NH2-terminal truncations of TRAF2, TRAF5, and TRAF6, each of which has been demonstrated to suppress NF-κB signaling in a dominant negative manner (7Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (978) Google Scholar, 18Nakano H. Oshima H. Chung W. Williams-Abbott L. Ware C.F. Yagita H. Okumura K. J. Biol. Chem. 1996; 271: 14661-14664Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 24Song H.Y. Régnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (507) Google Scholar). Co-expression of the zinc ring-deleted forms of TRAFs 2, 5, or 6 each inhibited the RANKL-inducible reporter activity in a concentration-dependent fashion (Fig.5). However, only NH2-truncated TRAF2 and TRAF5 suppressed constitutive signaling from the full-length RANK. The NH2-truncated TRAF6 selectively inhibited RANKL-induced signaling, but not constitutive signaling, revealing a specific role for TRAF6 in RANKL-induced signaling. To determine whether human RANK r