Characterization of Structural Domains of Human Osteoclastogenesis Inhibitory Factor

Osteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a ∼60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1–4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs. Osteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a ∼60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1–4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs. In the vertebrate, homeostasis and remodeling of bone are by strictly controlled by mostly unrevealed mechanisms. Much effort has been made to clarify the mechanisms, and several protein factors were found to participate in bone homeostasis (1Chambers T.J. Hall T.J. Vitam. Horm. 1991; 46: 41-86Google Scholar, 2Suda T. Takahashi N. Martin J. Endocr. Rev. 1992; 13: 66-80Google Scholar, 3Suda T. Udagawa N. Nakamura I. Miyaura C. Takahashi N. Bone. 1995; 17: 87S-91SGoogle Scholar). Recently, we have isolated one such factor termed osteoclastogenesis inhibitory factor (OCIF) 1The abbreviations used are: OCIF, osteoclastogenesis inhibitory factor; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; DDH, death domain homologous region; TRAP, tartaric-resistant acid phosphatase; CHAPS, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; IMDM, Iscove's modified Dulbecco's medium; FPLC, fast protein liquid chromatography; X-gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; bp, base pair(s); kb, kilobase pair(s). from the conditioned medium of human embryonic lung fibroblasts, IMR-90 (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). Both a ∼60-kDa monomer and a disulfide-linked homodimer are present in the conditioned medium, and the two forms have similar specific activity in inhibition of osteoclast formation in vitro (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). However, the mechanism by which OCIF inhibits osteoclastogenesis is not yet known. Based on the partial amino acid sequence, cDNA for human OCIF was molecularly cloned. The amino acid sequence deduced from the nucleotide sequence of OCIF cDNA predicted that it consists of 401 amino acid residues, including a putative 21-amino acid residue signal sequence (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar). The nucleotide sequence analysis has revealed that OCIF is identical to osteoprotegerin (6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). OCIF has seven major domains (domains 1–7) and has overall similarity to proteins of the tumor necrosis factor receptor (TNFR) family, although OCIF lacks an apparent transmembrane region (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). Domains 1–4 are cysteine-rich structures with a characteristic of extracellular domains of the TNFR family proteins. Domains 5 and 6 share structural features with “death domains” of TNFR 1, Fas, DR 3 (also designated as Apo 3, Wsl 1, and TRAMP), the TRAIL receptor, and the several recently identified cytoplasmic proteins mediating apoptosis (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 7Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Google Scholar, 8Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Google Scholar, 9Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Google Scholar, 10Cleveland J.L. Ihle J.M. Cell. 1995; 81: 479-482Google Scholar, 11Chinnaiyan A.M. O'Rourke K. Yu G.-L. Lyons R.H. Garg M. Duan D.R. Xing L. Gentz R. Ni J. Dixit V.M. Science. 1996; 274: 990-992Google Scholar, 12Marsters S.A. Sheridan J.P. Donahue C.J. Pitti R.M. Gray C.L. Goddard A.D. Bauer K.D. Ashkenazi A. Curr. Biol. 1996; 6: 1669-1676Google Scholar, 13Kitson J. Raven T. Jiang Y.-P. Goeddel D.V. Giles K.M. Pun K.-T. Grinham C.J. Brown R. Farrow S.N. Nature. 1996; 384: 372-375Google Scholar, 14Bodmer J.-L. Burns K. Schneider P. Hofmann K. Steiner V. Thome M. Bornard T. Hahne M. Schroter M. Becker K. Wilson A. French L.E. Browning J.L. Macdonald H.R. Tschopp J. Immunity. 1997; 6: 79-88Google Scholar, 15Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit M. Science. 1997; 276: 111-113Google Scholar, 16Nagata S. Cell. 1997; 88: 355-365Google Scholar, 17Duan H. Dixit V.M. Nature. 1997; 385: 86-89Google Scholar). However, unlike previously characterized death domains, two death domain homologous regions (DDHs), domains 5 and 6 of OCIF, exist in extracellular environments, because OCIF is secreted into conditioned medium. Domain 7, which does not resemble any protein motifs characterized thus far, consists of 50 amino acid residues and has a relatively high net positive charge; it contains eight basic amino acid residues (Lys and Arg) and only one acidic residue (Glu). To determine which residue(s) or domain(s) is/are involved in the in vitro biological activity, binding to heparin, and dimer formation, we generated and characterized various mutants of OCIF. We also examined the potential of domains 5 and 6 for mediating cell death by overexpressing chimeric proteins in which portions containing the transmembrane domain derived from Fas were inserted into OCIF. Escherichia coli DH5α (Life Technologies, Inc.) was used to propagate and amplify plasmids. 293-EBNA (CLONTECH), a human fetal kidney cell line, was grown in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum (FBS) and 250 μg/ml geneticin (Sigma). A mouse bone marrow-derived stromal cell line, ST2 (Riken Cell Bank RCB0224, Japan) was grown in minimum essential medium-α containing 10% FBS. Mammalian expression plasmid pCEP4 (CLONTECH) was used for expression of OCIF mutants, Fas, and OCIF-Fas chimeric proteins. Full-length OCIF cDNA was subcloned into the XhoI and BamHI sites of pCEP4 to in which the cDNA is expressed the of the Fas cDNA N. S. A. M. S.-I. M. A. Y. Nagata S. Cell. 1991; was by polymerase and and A human cell cDNA (CLONTECH) was used as a for the and were to a polymerase chain R. Scholar). To the expression for OCIF mutants, by were with for and and and and and and and were for the region of OCIF cDNA in The thus were designated and To a expressing for domains 1–4 of OCIF and and that for the transmembrane domain the region of Fas and were as previously R. Scholar). The was with and and the was for the of OCIF cDNA in to To a the transmembrane region of Fas was by with and and the human cell cDNA as a the was with and the was with the of OCIF the was with and and a was a from the and an from were To a sequence domains 5 to 7 was from by R. Scholar). were by the of and 293-EBNA cells were on a cell of and the cells in were with of the expression on the (Life Technologies, the was and the cells were in medium for with were in containing 10% and μg/ml to was used to OCIF mutants in the conditioned medium. The was as OCIF in 293-EBNA was to as previously (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). were with the OCIF. was from serum of the protein was a Osteoclastogenesis inhibitory activity was by the of cell cell formation was by acid activity in of mouse cells and ST2 cells for in the of and with various of OCIF mutants as previously (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). activity is expressed in as previously (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). was from conditioned medium of a 293-EBNA The conditioned medium was to the a of the with of 50 containing and and with of containing CHAPS, proteins were from the with acid containing and a of containing were by an and to a with containing the with the proteins to the were with a from to a of containing were and The affinity of OCIF and OCIF mutants for heparin was by on heparin medium containing OCIF was to the with 50 containing The was with a of to in a of and were The of the in was by were on or were used as were a OCIF or OCIF mutants were horseradish and the was to was isolated from cells (Life Technologies, was with (Life Technologies, Inc.) of the for the were and The were to amplify a to the putative signal and domains of OCIF. of the was by 293-EBNA cells were into in a a cell of The cells in were on the with 250 of or cells were with for the cells with of was to the and the was for the cells with of PBS, of and in was to the and the was for 3 The was by 50 of 6 to and the was 293-EBNA cells were into in a a cell of The cells in were on the with of or with of an expression plasmid for the was and containing 10% was to of cells were by with for 5 with of PBS, and with Cell was examined a and the of cells was the of activity in the medium, of conditioned medium were the activity was a was as previously M. R. M. W. Res. 1994; cells for A of OCIF and mutants of OCIF used in is in We a of expression and into 293-EBNA cells to mutants, and We also expression for C-terminal mutants, and to a residue responsible for dimer formation of OCIF, a of to mutants, and in which residue in domains 6, and 7 was with and a which lacks two C-terminal amino acid residues, Cys-400 and were two mutants and were in the conditioned medium by analysis OCIF mutants were by and were not secreted by or The of of the mutants in conditioned from to of inhibitory activity of was to A the osteoclastogenesis inhibitory activity of the mutants on the inhibition of cell formation in the OCIF the cell formation in a of 5 to inhibitory of and to inhibit the cell formation and In the inhibitory activity with an of a specific activity to that of OCIF. A C-terminal which lacks domains 6 and 7, the osteoclastogenesis inhibitory activity, although in the inhibitory activity was that of OCIF of the of the inhibitory activity of which lacks we it an affinity The osteoclastogenesis that of domains of the C-terminal does not the biological However, the of was 10% of that of OCIF as from results that the N-terminal portion containing the domains is sufficient for the osteoclastogenesis inhibitory To the of the binding of OCIF to heparin in the inhibition of osteoclastogenesis, we the affinity of and for heparin by on heparin medium of the cells expressing OCIF was on the and was from the with OCIF was of and which to which the monomer and the dimer form of OCIF are was as a an of only on the binding of OCIF to heparin proteins were as because are present as results that domain 7, which the C-terminal 50 amino acid residues, contains a heparin-binding The that of domain 7 not the inhibition of osteoclastogenesis the binding of OCIF to heparin that binding of OCIF to heparin does not correlate with osteoclastogenesis inhibitory activity in OCIF from medium is present as two a monomer with an of and a disulfide-linked dimer with an of (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). To domain(s) responsible for the dimer formation, the of the domain mutants to form was as in A. A protein with a of was for and as a major In is present as a is present as a and are present in two a monomer and a dimer a in the conditioned medium, that domain 7 is involved in the dimer and both domain 7, as as in and is to a of are potential sites in domain 3, is such site in domain (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). is only one residue in domain 7 (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google of the residue in the was To two mutants, one with of a residue for Cys-400 and the with a of the two C-terminal amino acid residues Cys-400 and were a a of to mutants, and in which residue in domains 5 and 6 was with a were The mutants were expressed in 293-EBNA and the of the mutants was by as in The results that both and exist as a monomer with a of OCIF with a of was in the conditioned medium of or mutants the dimer form OCIF with a of The derived from results that Cys-400 is responsible for the dimer formation of OCIF. we that the protein in the conditioned medium of cells is a homodimer of two by an two Cys-400 and which are present as a monomer in the conditioned medium, were as as OCIF in the inhibition of the in vitro osteoclast formation results that formation of the dimer is not essential for the in vitro osteoclastogenesis inhibitory and which are present as as in the biological activity not We domains 5 and 6 have a potential for mediating cytotoxic we various OCIF/Fas fusion proteins with an expression plasmid for in 293-EBNA The of OCIF, Fas, and fusion proteins used in is in 6 A. The of derived from chimeric in the cells was by were to amplify a to the portion of OCIF in 6 from the cells with or generated the not with pCEP4 or to the cells with or not with the or 6 results that chimeric cDNA was and the fusion were to the of the cells were with to the and the of the cells expression of the cells with or revealed that of the cells were and of cell death 7 In when with the or of the cells the and 7 activity in the conditioned medium of the cells with or was that with the or 7 that of OCIF-Fas or cell The was the in signal by OCIF-Fas was that by Fas for a To the cell death was by we the of in the cells with the or with or of a of as with 7 results that OCIF is of apoptosis when the Fas transmembrane region is inserted cysteine-rich regions and and that domains have a potential to the death domain of Fas in the of of cytotoxic signal by of on 293-EBNA of 293-EBNA cells with expression on a were with the expression the cells were with X-gal, and a in the medium. were as in and 50 of the conditioned medium were to the for are expressed as of in 293-EBNA of a were with the expression was isolated and the was on a as in M. R. M. W. Res. 1994; Scholar). 1, from from 3, from OCIF belongs to the TNFR family, containing cysteine-rich domains and two by a domain with a high net positive (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). In OCIF has that not of the TNFR family proteins (i) it is a secretory protein with apparent transmembrane region; (ii) it is present in two a monomer and a and (iii) it with In the present we examined which structural domains are involved in the inhibition of osteoclastogenesis, the binding to heparin, and the formation of the We also examined the two have potential for mediating the cytotoxic signal when a chimeric protein in which the Fas transmembrane region is inserted in front of is in The results are in the osteoclastogenesis inhibitory activity of and C-terminal mutants, we found that the N-terminal portion containing domains 1–4 is sufficient to inhibit Domains 1–4 to the extracellular cysteine-rich regions of the TNFR family proteins. the TNFR family proteins, regions the cells and are involved in the with for the form the cysteine-rich regions have the to with A secreted form of Fas the transmembrane domain is present in with that the as an Fas J. Y. C. J.P. Science. 1994; Scholar). A form of TNFR of the cysteine-rich which is generated by a inhibits the activity of both and Y. C. H. R. D. H. D. J. Scholar, C. L. T. M. S. C. K. Goodwin R.G. 1991; Scholar, A. S.A. D. Goeddel D.V. S. A. 1991; 88: Scholar, K. D. J. 1991; Scholar). of the of the TNFR family proteins as the the that OCIF with a receptor of osteoclast formation, by binding to a with a structural similarity to (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). Recently, (6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google have the of a cDNA for a protein identical to OCIF. that the domains inhibitory activity in vitro by the biological activity of C-terminal mutants (6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). However, the biological activity of the mutants was not in We found that conditioned medium of cells was of osteoclastogenesis in a not the was to to determine the the in vitro biological activity of the we that the N-terminal portion of OCIF is sufficient to inhibit osteoclastogenesis, although the is one of that of OCIF of heparin binding of the mutants revealed that domain 7 is involved in heparin to heparin or is to for such factors as basic factor to in vitro and in A. M. J. P. Cell. 1991; Scholar). The affinity of OCIF for heparin, not correlate with the in vitro biological proteins, in heparin-binding of and cell in a in that of affinity for heparin results in a in the specific vitro J. P. K. J. Biol. Chem. 1994; Scholar). it is the that affinity of OCIF for heparin of in of heparin binding sites of factors of a of basic amino acid residues A.D. Scholar). apparent of amino acid residues is present in domain 7, of to residues an in which and exist on one of the and residues such as or on the not basic residues to the binding of OCIF to was by the results that the mutants with for and in OCIF a in affinity for Yamaguchi and M. Domain 7 is also responsible for the of OCIF. was derived from analysis of the mutants identified Cys-400 as the residue essential for the dimer formation or of Cys-400 not the activity of OCIF the that both the monomer and the dimer form OCIF have similar specific activity in inhibition of in vitro osteoclastogenesis (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). The by is for receptor to inhibitory a TNFR is secreted as both a monomer and a and to with a similar the dimer is a M. K. G. J. Biol. Chem. 1996; Scholar). The of dimer formation of OCIF for the Domains 5 and 6 have a to death domains that are involved in in Domains 5 and 6 are not essential for the inhibitory activity A and heparin binding or the although of both domains in a in in vitro biological activity we examined the two domains have a potential for mediating when expressed in the of 293-EBNA has been that expression of death receptor proteins including TNFR 1, Fas, (also as or and the TRAIL receptor to cell death apoptosis A.M. O'Rourke K. Yu G.-L. Lyons R.H. Garg M. Duan D.R. Xing L. Gentz R. Ni J. Dixit V.M. Science. 1996; 274: 990-992Google Scholar, 12Marsters S.A. Sheridan J.P. Donahue C.J. Pitti R.M. Gray C.L. Goddard A.D. Bauer K.D. Ashkenazi A. Curr. Biol. 1996; 6: 1669-1676Google Scholar, 13Kitson J. Raven T. Jiang Y.-P. Goeddel D.V. Giles K.M. Pun K.-T. Grinham C.J. Brown R. Farrow S.N. Nature. 1996; 384: 372-375Google Scholar, 14Bodmer J.-L. Burns K. Schneider P. Hofmann K. Steiner V. Thome M. Bornard T. Hahne M. Schroter M. Becker K. Wilson A. French L.E. Browning J.L. Macdonald H.R. Tschopp J. Immunity. 1997; 6: 79-88Google Scholar). of OCIF does not cell a receptor OCIF which contains the transmembrane region of Fas domains and 5 of OCIF, an to apoptosis in 293-EBNA cells 7, of the the cell that domains 5 6 have a high potential for mediating the of the OCIF to any a potential that OCIF only in N. H. E. and K. it is that OCIF apoptosis in a similar to TNFR or OCIF apoptosis in an We N. T. and A. for and an affinity We also A. N. and H. for