Ligand-induced Formation of p55 and p75 Tumor Necrosis Factor Receptor Heterocomplexes on Intact Cells

The p55 and p75 tumor necrosis factor receptors are known to mediate their effects on cells through distinct signaling pathways. Under certain circumstances, the two classes of TNF receptors cooperate with each another to produce enhanced cellular responses. The only molecular mechanism proposed thus far to explain this effect is the process of “ligand passing,” whereby TNF is concentrated at cell surfaces by binding to p75 and then following dissociation from this receptor class binds with high efficiency to p55. Using thein vivo model of TNF-induced TNF receptor shedding we have uncovered a novel ligand-dependent interaction of the two TNF receptors that occurs upon exposure of cells to TNF. Using TNF receptor-specific monoclonal antibodies that bind TNF receptors in the presence or absence of ligand, we report that TNF induces the formation of heterocomplexes consisting of both p55 and p75 TNF receptors. Whereas immunoprecipitates from untreated or human TNF-treated cells formed with either p55 or p75 TNF receptor-specific monoclonal antibodies contained only the relevant TNF receptor class, anti-p55 or anti-p75 precipitated both receptor types from murine TNF-treated cells. Ligand-induced complex formation was transient, occurred at physiologically relevant concentrations of TNF, and occurred with receptors lacking intracellular domains or that contained irrelevant transmembrane domains. Formation of TNF receptor heterocomplexes may therefore 1) define a novel molecular mechanism of ligand passing and/or 2) contribute to cooperative TNF receptor signaling via the juxtaposition of the intracellular domains of the two receptor classes and the signaling proteins that they recruit. The p55 and p75 tumor necrosis factor receptors are known to mediate their effects on cells through distinct signaling pathways. Under certain circumstances, the two classes of TNF receptors cooperate with each another to produce enhanced cellular responses. The only molecular mechanism proposed thus far to explain this effect is the process of “ligand passing,” whereby TNF is concentrated at cell surfaces by binding to p75 and then following dissociation from this receptor class binds with high efficiency to p55. Using thein vivo model of TNF-induced TNF receptor shedding we have uncovered a novel ligand-dependent interaction of the two TNF receptors that occurs upon exposure of cells to TNF. Using TNF receptor-specific monoclonal antibodies that bind TNF receptors in the presence or absence of ligand, we report that TNF induces the formation of heterocomplexes consisting of both p55 and p75 TNF receptors. Whereas immunoprecipitates from untreated or human TNF-treated cells formed with either p55 or p75 TNF receptor-specific monoclonal antibodies contained only the relevant TNF receptor class, anti-p55 or anti-p75 precipitated both receptor types from murine TNF-treated cells. Ligand-induced complex formation was transient, occurred at physiologically relevant concentrations of TNF, and occurred with receptors lacking intracellular domains or that contained irrelevant transmembrane domains. Formation of TNF receptor heterocomplexes may therefore 1) define a novel molecular mechanism of ligand passing and/or 2) contribute to cooperative TNF receptor signaling via the juxtaposition of the intracellular domains of the two receptor classes and the signaling proteins that they recruit. TNF 1The abbreviations used are: TNF, tumor necrosis factor; HuTNF, human TNF; MuTNF, murine TNF; PBS, phosphate-buffered saline; NF-κB, nuclear factor-κB. interacts with two distinct receptors of M r 55,000 and 75,000, which are independently expressed on cell surfaces (1Kull Jr., F.C. Jacobs S. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5756-5760Crossref PubMed Scopus (256) Google Scholar, 2Ryffel B. Mihatsch M.J. Int. Rev. Exp. Pathol. 1993; 34: 149-155Crossref PubMed Scopus (35) Google Scholar). The p55 and p75 TNF receptors share 28% homology in their extracellular domains but show no homology in their intracellular domains (3Dembic Z. Loetscher H. Gubler U. Pan Y.E. Lahm H. Gentz R. Brockaus M. Lesslauer W. Cytokine. 1990; 2: 231-237Crossref PubMed Scopus (234) Google Scholar). The TNF receptors belong to a receptor family that displays extracellular domain homology largely through conservation of cysteine-rich repeating sequences and includes the low affinity nerve growth factor receptor, Fas, OX-40, CD30, CD40, and 4BB1 (4Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1850) Google Scholar). Recent work (5Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1767) Google Scholar, 6Darnay B.G. Singh S. Chaturvedi M.M. Aggarwal B.B. J. Biol. Chem. 1995; 270: 14867-14870Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 7Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (939) Google Scholar) has revealed that the two receptor classes interact with distinct families of cellular proteins through their intracellular domains. These observations have led to the suggestion that the two TNF receptors utilize different signaling mechanisms. The ability of each TNF receptor to signal biologic responses in cells has been extensively studied during the past few years. Engagement of p55 is now known to be both necessary and sufficient to induce a variety of proinflammatory TNF-mediated cellular responses including cytotoxicity (8Tartaglia L.A. Rothe M. Hu Y-F. Goeddel D.V. Cell. 1993; 73: 213-216Abstract Full Text PDF PubMed Scopus (307) Google Scholar), induction of inducible nitric-oxide synthase (9Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1173) Google Scholar) and manganous superoxide dismutase (10Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1008) Google Scholar), expression of intercellular adhesion molecule 1 (ICAM-1) (11Mackay F. Loetscher H. Stueber D. Gehr G. Lesslauer W. J. Exp. Med. 1993; 177: 1277-1286Crossref PubMed Scopus (367) Google Scholar, 12Trefzer U. Brockhaus M. Loetscher H. Parlow F. Kapp A. Schöpf E. Krutmann J. J. Invest. Dermatol. 1991; 97: 911-916Abstract Full Text PDF PubMed Google Scholar), and anti-viral activity (13Wong G.W.H. Tartaglia L.A. Lee M.S. Goeddel D.V. J. Immunol. 1992; 149: 3350-3353PubMed Google Scholar). In contrast, p75 appears to effect only a limited number of cellular responses, many of which are restricted to T cell populations and include effects on proliferation/cell viability (14Tartaglia L.A. Goeddel D.V. Reynolds C. Figari I.S. Weber R.F. Fendly B.M. Palladino Jr., M.A. J. Immunol. 1993; 151: 4637-4641PubMed Google Scholar, 15Zheng L. Fisher G. Miller R.E. Peschon J. Lynch D.H. Lenardo M.J. Nature. 1995; 377: 348-351Crossref PubMed Scopus (1057) Google Scholar) and cytokine production (16Vandenabeele P. Declercq W. Vercammen D. Van de Craen M. Grooten J. Loetscher H. Brockhaus M. Lesslauer W. Fiers W. J. Exp. Med. 1992; 176: 1015-1024Crossref PubMed Scopus (128) Google Scholar). In many biologic systems, p75 plays an accessory role in p55-mediated responses. In murine models, the engagement of both p55 and p75 by MuTNF leads to enhanced cellular responses compared with the selective engagement of p55 by HuTNF or p55 agonistic antibody. In addition, overexpression of p75 can result in the enhancement of p55-mediated signaling whereas p75-specific antagonist mAbs partially inhibit p55-induced responses (8Tartaglia L.A. Rothe M. Hu Y-F. Goeddel D.V. Cell. 1993; 73: 213-216Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 16Vandenabeele P. Declercq W. Vercammen D. Van de Craen M. Grooten J. Loetscher H. Brockhaus M. Lesslauer W. Fiers W. J. Exp. Med. 1992; 176: 1015-1024Crossref PubMed Scopus (128) Google Scholar, 17Tartaglia L.A. Pennica D. Goeddel D.V. J. Biol. Chem. 1993; 268: 18542-18548Abstract Full Text PDF PubMed Google Scholar, 18Sheehan K.C.F. Pinckard J.K. Arthur C.D. Dehner L.P. Goeddel D.V. Schreiber R.D. J. Exp. Med. 1995; 181: 607-617Crossref PubMed Scopus (152) Google Scholar). To some extent, this cooperativity has been explained by “ligand passing,” a process by which TNF is concentrated near the cell surface by selectively binding to p75 (because p75 binds ligand with a 10–20-fold higher affinity than p55 binds ligand), dissociates, and subsequently binds with increased efficiency to p55 (17Tartaglia L.A. Pennica D. Goeddel D.V. J. Biol. Chem. 1993; 268: 18542-18548Abstract Full Text PDF PubMed Google Scholar). Recent studies have shown that TNF can induce shedding of both classes of TNF receptors in mice (19Bemelmans M.H.A. Gouma D.J. Buurman W.A. J. Immunol. 1993; 151: 5554-5562PubMed Google Scholar). The biologic relevance of this response has been ascribed to a protective effect of soluble TNF receptors which can block TNF binding to other cell surface receptors and to the loss of cell surface receptors rendering the cell TNF-insensitive. The molecular basis of the shedding response has not yet been determined. In the current study, we analyzed the receptor requirements for TNF-induced p75 shedding in vivo and observed that p75 shedding could be mediated by engagement of p55 alone but not by p75 alone. However, simultaneous engagement of both p55 and p75 led to an enhanced shedding response. No level of p55 ligation could induce the amount of p75 shedding effected when both receptors were engaged. These results suggest that the cooperativity between p55 and p75 in this experimental model may extend beyond ligand passing. One possible mechanism underlying this observation is that TNF may induce formation of a receptor heterocomplex containing both p55 and p75. However, previous studies by others have failed to demonstrate such a heterocomplex (17Tartaglia L.A. Pennica D. Goeddel D.V. J. Biol. Chem. 1993; 268: 18542-18548Abstract Full Text PDF PubMed Google Scholar, 20Higuchi M. Aggarwal B.B. J. Biol. Chem. 1992; 267: 20892-20899Abstract Full Text PDF PubMed Google Scholar, 21Moosmayer D. Dinkel A. Gerlach E. Hessabi B. Grell M. Pfizenmaier K. Scheurich P. Lymphokine Cytokine Res. 1994; 13: 295-301PubMed Google Scholar). In the current report we have used a unique set of monoclonal antibodies specific for murine p55 and p75 whose binding properties are not affected by the presence of ligand to demonstrate that murine TNF can indeed form heterocomplexes between p55 and p75 on the surface of intact cultured and primary cells. This result thereby suggests that 1) one mechanism of “ligand passing” may be a direct, contact-mediated hand-off of TNF from p75 to p55 in which a heterocomplex of p55 and p75 is an obligate intermediate and/or 2) formation of p55-p75 heterocomplexes and the subsequent juxtaposition of their intracellular domains could potentially lead to functional cooperativity between the signaling components that associate with the two types of TNF receptors. Monoclonal antibodies specific for murine TNFα (22Sheehan K.C.F. Ruddle N.H. Schreiber R.D. J. Immunol. 1989; 142: 3884-3893PubMed Google Scholar) and murine p55 and p75 (18Sheehan K.C.F. Pinckard J.K. Arthur C.D. Dehner L.P. Goeddel D.V. Schreiber R.D. J. Exp. Med. 1995; 181: 607-617Crossref PubMed Scopus (152) Google Scholar) were produced as described and biotinylated using the Enzotin reagent (ENZO Biochem, Inc., New York, NY) according to the manufacturer's protocol. Purified recombinant MuTNFα (1.2 × 107 units/mg) and rHuTNFα (5.6 × 107 units/mg) were generously supplied by Genentech, Inc., South San Francisco, CA. All reagents used in this study contained less than 0.5 pg/ml endotoxin as determined by the Limulus amebocyte lysate assay (BioWhittaker Inc., Walkerville, MD). Lipopolysaccharide (Escherichia colistrain O127:B8) was purchased from Difco Laboratories (Detroit, MI). Balb/c ByJ female mice (6–7 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice with genetic deficiency of the p55 TNF receptor were provided by Dr. Werner Lesslauer (Hoffman-La Roche, Basel, Switzerland). Mice deficient in p75 were provided by Dr. Mark Moore (Genentech, Inc.). Mice were injected intraperitoneally with 0.5-ml samples of monoclonal antibodies or pyrogen-free physiologic saline. At various time points thereafter lipopolysaccharide or purified recombinant murine or human TNF was diluted in 0.5 ml of pyrogen-free saline and was injected intraperitoneally into the mice. At specified time points mice were bled and the serum was immediately assayed for the presence of shed TNF receptor proteins by enzyme-linked immunosorbent assay. For detection of shed p75, the enzyme-linked immunosorbent assay consisted of plate-bound TR75-54 and biotinylated TR75-32 as the detection (18Sheehan K.C.F. Pinckard J.K. Arthur C.D. Dehner L.P. Goeddel D.V. Schreiber R.D. J. Exp. Med. 1995; 181: 607-617Crossref PubMed Scopus (152) Google Scholar, J. K. C. and R. D. in Scholar). MuTNF was with and according to the manufacturer's The number of TNF binding was by or of MuTNF to cells for 1 at The of or p75-specific binding were by the cells for 1 at with a of TR75-54 or which are monoclonal antibodies to p75 or of binding was in and of 2) of or cells were to TNF binding with and p75-specific The of p55 and p75 binding were and cells were in with and in were by with and with cells of either were with TNF for at and immediately on The cells were with and then in containing 1 1 1 1 and for at the was by at × for and the samples were with of a of Inc., of mAbs or were and the samples were for 1 at of was and for 1 at The was in in of and at for were then to on and for p55 or p75 as described (18Sheehan K.C.F. Pinckard J.K. Arthur C.D. Dehner L.P. Goeddel D.V. Schreiber R.D. J. Exp. Med. 1995; 181: 607-617Crossref PubMed Scopus (152) Google Scholar) using biotinylated of or were using the reagent Inc., The the murine p55 and p75 TNF receptors in the expression were from Dr. D. Goeddel Inc., South San Francisco, was to of p55 and p75. The used for the p55 were and used in the of the p75 were and These a to the and an to the that were subsequently used to into the expression The for of p55 and p75 receptor proteins that only or of their intracellular domains. The sequences of were on both by cells were with 1 and the cells were by One cells were in of ml and with different of recombinant MuTNFα or for at were as described A. A. U. P. A. T. J. Biol. Chem. 1994; Google Scholar) and using an assay that a from the of the D. W. Nature. PubMed Scopus Google Scholar). is now that TNF can effect shedding of the p75 TNF receptor in M.H.A. Gouma D.J. Buurman W.A. J. Immunol. 1993; 151: 5554-5562PubMed Google Scholar). K. K. C. F. and R. D. therefore to define the role of each TNF receptor class in the response. from untreated mice contained 1 of soluble p75 of MuTNFα effected shedding of soluble p75 which of by and to of 1 by mice were with a of MuTNFα the of p75 shedding was by but the of the response To p55 ligation was necessary for mediated p75 we murine TNF effected the shedding response p55 ligation could not MuTNFα an in the level of soluble p75 in mice for mice with of MuTNF or In contrast, no was observed in TNF-treated mice for or In with previous p75 shedding was not on TNF (19Bemelmans M.H.A. Gouma D.J. Buurman W.A. J. Immunol. 1993; 151: 5554-5562PubMed Google Scholar) and thus occurred to a in and deficient mice. results were using mice with In this effected a of TNF-induced p75 shedding such that of shedding was at the of used not These results thereby demonstrate that p55 ligation is for TNF-induced p75 To p55 engagement was sufficient to induce p75 shedding in the were using two which interact only with p55 and not human TNF M. Tartaglia L.A. Lee A. Wong G.H.W. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar) and the agonistic (18Sheehan K.C.F. Pinckard J.K. Arthur C.D. Dehner L.P. Goeddel D.V. Schreiber R.D. J. Exp. Med. 1995; 181: 607-617Crossref PubMed Scopus (152) Google Scholar). In both the p75 shedding with to effected by murine TNF 1 However, the of p75 shedding were only of by the murine when used at the To the p75 shedding by the p55 selective was either to an in affinity of TNF for cell ligand or a cooperativity between the two classes of TNF the in vivo shedding were using different TNF effected a in shed p75 of with a of 1 In contrast, both human TNF and the of p75 shedding which of only when the were at the high of and of mice with mAbs to p75 the of shedding of p75 to that observed when the were used or not These observations that p75 is in p75 receptor no amount of selective p55 ligation effected the level of p75 shedding upon engagement of both TNF the response could not be explained by the model of ligand passing. therefore the that TNF receptor cooperativity could via formation of a p55-p75 receptor described unique mAbs that murine p55 or p75 TNF receptors in a that was of receptor by therefore used mAbs to one TNF molecule can interact with both p55 and p75. were on the murine tumor cell which p55 and p75 receptor binding as by not were with or TNF and then and was with TNF receptor-specific were subsequently analyzed for the presence of each TNF receptor by TNF receptor immunoprecipitates of cells with either or HuTNF binds only to murine contained only the relevant TNF receptor class and and In contrast, p55 or p75 immunoprecipitates of cells to MuTNF binds both murine p55 and contained both TNF receptor anti-p75 immunoprecipitates of cells contained p55 and anti-p55 of cells contained p75 The of the and components were by that the by with the receptor that was from cell of cytokine The of the was by two p55 p75 contained the murine receptor thereby the that the TNF receptor contained irrelevant cell surface of the receptor not either p55 or p75 and murine TNF is of TNF receptor on the surface of this murine tumor cell To heterocomplex formation occurs on cells as as tumor we the using two different of primary murine cells. The results were to with cells. of p75 contained p55 only when were with MuTNF and only p55 from contained p75 1 and formation was observed in from mice not heterocomplex formation was not observed in from mice deficient for p55 or p75 not the of the ligand-dependent TNF receptor heterocomplex formation occurs in primary as as cultured cells. To the of TNF receptor cells were with MuTNF, and then in for various of time for p55 and p75 no heterocomplexes were observed in the absence of MuTNF but were immediately following cellular exposure to MuTNF 1 and The amount of heterocomplex observed 1 of in was to that in cells but thereafter revealed that the of the complex was at heterocomplexes a at the cell To the intracellular domains of the two TNF receptors to formation of TNF-induced we complex formation could with receptor For this we expressed of either p55 or p75 in thereby cell that contained both and receptors. Monoclonal antibodies specific for the extracellular domain of murine p55 both and of this receptor from cells cells were with either or HuTNF, p75 immunoprecipitates not either form of p55 However, following exposure of the cells to MuTNF, p75 immunoprecipitates contained both and of p55 for p75 from cells p75 revealed the presence of both and of p75 in cells of p55 from cells led to the of both and of p75 the of to p75 receptor in the was to the of to p75 receptor expressed on the cell that the receptor is not into the This result thus the that heterocomplexes contained an of the and receptors. the receptor in the cell was not to heterocomplex To the transmembrane domains of p55 or p75 to heterocomplex the were using cells that a that consisted of the murine p75 extracellular the murine receptor transmembrane domain and the intracellular domain of p55 from cells in the of both and of p75 not These results demonstrate that the formation of p55-p75 heterocomplexes on cell surfaces not between the intracellular or transmembrane domains of the receptors. appears that the of the extracellular domains by MuTNF is sufficient for heterocomplex to a heterocomplex with soluble of p55 and p75 have not This result that heterocomplex formation a specific or of the receptor extracellular domains. The previous were on cells to high concentrations of MuTNF To ligand-dependent receptor heterocomplex formation occurs at physiologic of MuTNF, the were on cells to different concentrations of the murine formation was by p55 at a MuTNF of 1 and at ligand concentrations of These results were by of the in cells to the of concentrations of MuTNF used in the was at MuTNF concentrations of and at a TNF of heterocomplex formation can at a of TNF concentrations that a TNF-induced that heterocomplexes are not to an of high ligand previous in vivo studies using HuTNF that engagement of p55 was sufficient for the TNF-induced shedding of p75 (19Bemelmans M.H.A. Gouma D.J. Buurman W.A. J. Immunol. 1993; 151: 5554-5562PubMed Google Scholar), we have shown that engagement of both p55 and p75 is for p75 In addition, we demonstrate that p75 engagement alone is not sufficient to induce shedding but in with p55 in a cooperative not this effect to a ligand passing the of p75 shedding by and p55 a that is only of that by ligation of both receptors by ligand passing were the mechanism underlying p75 in the shedding responses to HuTNF MuTNF be by the of the human TNF a response was not therefore that some other mechanism of receptor is for Using a unique set of mAbs to the two murine TNF we have shown in cultured and primary murine cells that MuTNF is of with p55 and p75, and thereby can form a Formation of p55-p75 heterocomplexes occurs at physiologically relevant concentrations of TNF and is in ligand-dependent of p55 and p75 not the of the intracellular or transmembrane domains of the receptors. The of a role for the intracellular and transmembrane domains of the TNF receptors in heterocomplex formation appears to with the to demonstrate heterocomplexes with soluble receptors in However, could for this the interaction may that the receptors be in the between in the are to be than potentially between soluble the receptors may be in to one thereby heterocomplex the receptors may a different when they are not in a which could to form the specific p55 and p75 transmembrane domains are not in the cell domain appears to be necessary to the formation of heterocomplexes between the two receptor whereas with anti-p55 of the p75 with anti-p75 only a of the p55 This can partially be to the that the cells used in as many p55 than p75 receptors. In addition, is possible that the binding of the p75-specific to the TNF-induced p55-p75 receptor heterocomplex results in a dissociation of p55. cooperativity between p55 and p75 TNF receptors is the only molecular for this has been from the model of ligand passing proposed by Tartaglia (17Tartaglia L.A. Pennica D. Goeddel D.V. J. Biol. Chem. 1993; 268: 18542-18548Abstract Full Text PDF PubMed Google Scholar). In this model p75, which has a high affinity but for TNF, to low of TNF near the cell TNF from p75, ligand then binds to which displays a ligand binding affinity (17Tartaglia L.A. Pennica D. Goeddel D.V. J. Biol. Chem. 1993; 268: 18542-18548Abstract Full Text PDF PubMed Google Scholar). This model was proposed a heterocomplex could not be with the reagents at that study the formation of a receptor heterocomplex and thereby a intermediate in the ligand passing ligand passing may be the result of a direct, contact-mediated of ligand from p75 to p55. In addition, the interaction between p55 and p75 may have for TNF p75 receptor shedding results that p55 and p75 may be signaling in a cooperative which could be for by the formation of a In of this is the observation that and which bind to p55 and p75 intracellular are of with one another H. H. Pan M. Goeddel D.V. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). interaction has been observed at the p55 intracellular domain the of p75 H. M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, the of to the complex may be when can be by p75 during heterocomplex formation with p55. The formation of p55-p75 heterocomplexes and the juxtaposition of their intracellular domains may thus be a mechanism by which interaction between their signaling proteins is be to in functional to this at this time no in model of TNF-induced receptor shedding in a murine cell are to