©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Self-association of the Death Domains of the p55 Tumor Necrosis Factor (TNF) Receptor and Fas/APO1 Prompts Signaling for TNF and Fas/APO1 Effects (*)

(Received for publication, October 4, 1994; and in revised form, November 4, 1994)

Mark P. Boldin (§) Igor L. Mett (§) Eugene E. Varfolomeev Irina Chumakov Yonat Shemer-Avni Jacques H. Camonis (1) David Wallach (¶)

From the Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel and Denis Diderot University, INSERM Unit 248, 75010 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H. J. and Mett, I.(1994) Cytokine 6, 556; Song, H. Y., Dunbar, J. D., and Bonner, D. B.(1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the ``death domain'', specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.


INTRODUCTION

Many cell surface receptors are triggered upon clustering. Unless restricted, this mode of triggering may result in their spontaneous signaling due to receptor chance encounters. The implications with regard to regulation of receptor function are underscored by the findings in the present study regarding the mechanisms of signaling by the p55 tumor necrosis factor (TNF) (^1)receptor (p55-R) and Fas/APO1. These two structurally related receptors provide signals that can cause the death of cells expressing them, via structurally related sequence motifs in their intracellular domains (the ``death domains''; Refs. 2, 5, and 6). Dominant negative effects of mutations in these domains (2) and mimetic effects of antibodies against the two receptors (1, 7, 8) indicate that their signaling is initiated as a consequence of their clustering and self-interaction. TNF, and quite likely also the closely similar Fas ligand(9) , occur as homotrimeric molecules (see, e.g., (10) and (11) ) and thus can induce clustering of receptors merely by binding to them. Data presented here (see also (33) ) and in another recent study (4) show, however, that the intracellular domains of p55-R and of Fas/APO1 can aggregate even in the absence of their ligands, prompted by the ability of their death domains to self-associate. Additionally, we show that an increase in expression of these receptors, or even just of their death domain, can result in the induction of TNF and Fas/APO1-like effects, suggesting that the self-association of the death domain suffices to trigger signaling. These findings emphasize the need to elucidate how spontaneous signaling as a consequence of chance encounters between receptors normally is prevented.


EXPERIMENTAL PROCEDURES

Two-hybrid Screen and Two-hybrid beta-Galactosidase Expression Test

cDNA inserts were cloned by polymerase chain reaction, either from the full-length cDNAs cloned previously in our laboratory, or from purchased cDNA libraries. beta-Galactosidase expression in yeasts (SFY526 reporter strain; (12) ) transformed with these cDNAs in the pGBT-9 and pGAD-GH vectors (DNA binding domain (DBD) and activation domain (AD) constructs, respectively) was assessed by a liquid test(13) , as well as by a filter assay, which yielded qualitatively the same results (not shown). Two-hybrid screening (14) of a Gal4 AD-tagged HeLa cell cDNA library (Clontech, Palo Alto, CA) for proteins that bind to the intracellular domain of the p55-R (p55-IC), was performed using the HF7c yeast reporter strain. Positivity of the isolated clones was assessed by (a) prototrophy of the transformed yeasts for histidine when grown in the presence of 5 mM 3-aminotriazole, (b) beta-galactosidase expression, and (c) specificity tests (interaction with SNF4 and lamin fused to Gal4 DBD).

In Vitro Self-association of Bacterially Produced p55-IC Fusion Proteins

Glutathione S-transferase (GST) and glutathione S-transferase-p55-IC fusion protein (GST-p55-IC) were produced as described elsewhere(15, 16) . Maltose-binding protein (MBP) fusion proteins were obtained using the pMalcRI vector (New England Biolabs) and purified on an amylose resin column. The interaction of the MBP and GST fusion proteins was investigated by incubating glutathione-agarose beads sequentially with the GST and MBP fusion proteins (5 µg of protein/20 µl of beads), first for 15 min and then for 2 h, at 4 °C. Incubation with MBP fusion proteins was carried out in a buffer solution containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 2 mM CaCl(2), 2 mM MgCl(2), 5 mM dithiothreitol, 0.2% Triton X-100, 0,5 mM phenylmethylsulfonyl fluoride, and 5% (v/v) glycerol or, when indicated, in that same buffer containing 0.4 M KCl or 5 mM EDTA instead of MgCl(2). Association of the MBP fusion proteins was assessed by SDS-polyacrylamide gel electrophoresis of the proteins associated with the glutathione-agarose beads, followed by Western blotting. The blots were probed with rabbit antiserum against MBP (produced in our laboratory) and with horseradish-peroxidase-linked goat anti-rabbit immunoglobulin.

Induced Expression in HeLa Cells of the p55-R, Fragments Thereof, and Fas-IC

HeLa cells expressing the tetracycline-controlled transactivator (the HtTA-1 clone; (17) ) were grown in Dulbecco's modified Eagle's medium, containing 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.5 mg/ml neomycin. cDNA inserts encoding the p55-R or parts thereof were introduced into a tetracycline-controlled expression vector (pUHD10-3, provided by H. Bujard). The cells were transfected with the expression construct (5 µg of DNA/6-cm plate) by the calcium phosphate precipitation method (16) . Effects of transient expression of the transfected proteins were assessed at the indicated times after transfection in the presence or absence of tetracycline (1 µg/ml). Clones of cells stably transfected with the human p55-IC cDNA in the pUHD10-3 vector were established by transfecting the cDNA to HtTA-1 cells in the presence of tetracycline together with a plasmid conferring resistance to hygromycin, followed by selection for clones resistant to hygromycin (200 µg/ml). Expression of the cDNA was obtained by removal of tetracycline, which was otherwise maintained constantly in the cell growth medium.

Assessment of TNF-like Effects Triggered by Induced Expression of the p55-R, Fragments Thereof, or Fas-IC

Effects of induced expression of the receptors and of TNF on cell viability were assessed by the neutral-red uptake method(18) . Induction of interleukin 8 (IL-8) gene expression was assessed by Northern analysis. RNA was isolated using TRI Reagent (Molecular Research Center, Inc.), denatured in formaldehyde/formamide buffer, electrophoresed through an agarose/formaldehyde gel, and blotted to a GeneScreen Plus membrane (DuPont) in 10 times SSPE buffer, using standard techniques. Filters were hybridized with an IL-8 cDNA probe ((19), nucleotides 1-392), radiolabeled by random primed DNA labeling kit (Boehringer, Mannheim, Germany), and washed stringently.

Assessment of TNF Receptor Expression

TNF receptor expression in samples of 1 times 10^6 cells was assessed by measuring the binding of TNF, labeled with I by the chloramine-T method, as described previously(20) . It was also assessed by ELISA, performed as described for the quantification of the soluble TNF receptors(21) , except for the use of radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) to lyse the cells (70 µl/10^6 cells) and to dilute the tested samples. The soluble form of the p55-R, purified from urine, served as the standard.


RESULTS

Self-association of the death domain in the p55-R was observed by happenstance, on screening a HeLa cell cDNA library by the two-hybrid system technique (14) for proteins that bind to the intracellular domain of this receptor. Among the cDNAs whose products bound specifically to the intracellular domain-GAL4 DBD fusion-protein, several clones encoded parts of the p55-R intracellular domain (p55-IC; marked with asterisks in Table 1).



The extent of specificity in the self-association of p55-IC and the particular region involved was evaluated by the two-hybrid test. Table 1shows the following. (a) The self-association of p55-IC is confined to a region within the death domain. Its N terminus is located between residues 328 and 344; its C terminus, close to residue 404, is somewhat upstream of the reported C terminus of this domain (residue 414). (b) Deletion of the membrane-proximal part of p55-IC upstream of the death domain enhanced self-association, suggesting that this region has an inhibitory effect. (c) Mouse p55-IC self-associates and also associates with the death domain of human p55-R. (d) Examination of the self-association of the intracellular domains of three other receptors of the TNF/NGF receptor family: Fas/APO1, CD40(22) , and the p75 TNF receptor(23) , showed that Fas-IC, which signals for cell death by a sequence motif related to the p55-R death domain, self-associates and associates to some extent with the p55-IC. However, CD40-IC, which provides growth stimulatory signals (even though also containing a sequence resembling the death domain), and p75-IC, which bears no structural resemblance to p55-IC, do not self-associate, nor do they bind p55-IC or Fas-IC.

An in vitro test of the interaction of a p55-IC-GST bacterial fusion protein with a p55-IC-MBP fusion protein confirmed that p55-R self-associates and ruled out involvement of yeast proteins (Fig. 1). The association was not affected by increased salt concentration or by EDTA (Fig. 1, lanes 3 and 4).


Figure 1: Self-association of the intracellular domain of p55-R in vitro: specific association of bacterially-produced fusion proteins containing the intracellular domain. Interaction between fusion of human p55-IC to MBP (MBP-p55-IC) and to GST (lane 2) and the effect of EDTA (lane 3) and increased salt concentration (0.4 M KCl, lane 4) on this interaction. Interaction of MBP-p55-IC with GST (lane 1) and of GST-p55-IC with the fusion product of MBP and an irrelevant peptide (residues 195-229 in the mouse p75 TNF-R, MBP-p75-EC, lane 5; position indicated by an arrow) were also tested. SDS-polyacrylamide gel electrophoresis (10% acrylamide) of the interacting proteins, followed by Western blotting, using anti-MBP antiserum, was performed as described under ``Experimental Procedures.''



To evaluate the functional implications of the self-association of the death domain, we examined the way in which induced expression of p55-R, or of parts of it, affects cells sensitive to TNF cytotoxicity. Using an expression vector that permits strictly controlled expression of transfected cDNAs by a tetracycline regulated transactivator(17) , we found that merely increasing p55-R expression in HeLa cells by expression of transiently transfected cDNA for the full-length receptor resulted in quite extensive cell death. Even greater cytotoxicity was observed when expressing just p55-IC ( Fig. 2and 3, A and B). Significant cytotoxicity was also observed when expressing just the death domain. In contrast, expression of parts of the p55-IC that lacked the death domain or contained only part of it (or expression of the luciferase gene, used as an irrelevant control) had no effect on cell viability. Expression of Fas-IC did not result in cytotoxicity, yet significantly enhanced the cytotoxicity of co-expressed p55-R (Fig. 2). The cytotoxicity of p55-IC was further confirmed using cells stably transfected with its cDNA; these cells continued to grow when p55-IC expression was not induced but died when p55-IC was expressed (Fig. 3C).


Figure 2: Ligand-independent triggering of a cytocidal effect in HeLa cells transfected with p55-R, or parts thereof, or with Fas-IC. TNF receptor expression (left and middle) and viability (right) in: A, HeLa cells expressing transiently the full-length p55-R (p55-R), p55-IC or parts thereof or, as a control, luciferase (LUC); and B, in cells expressing Fas-IC, alone or together with the p55-R, using a tetracycline-controlled expression vector. box, cells transfected in the presence of tetracycline (1 µg/ml), which inhibits expression; , cells transfected in the absence of tetracycline. TNF receptor expression was assessed 20 h after transfection, both by ELISA, using antibodies against the receptor's extracellular domain (left), and by determining the binding of radiolabeled TNF to the cells (middle). The cytocidal effect of the transfected proteins was assessed 48 h after transfection. Data shown are from one of three experiments with qualitatively similar results, in which each construct was tested in duplicate. ND, not determined.




Figure 3: Ligand-independent triggering of a cytocidal effect in HeLa cells transfected with p55-R or its intracellular domain: kinetic study of transient expression of the receptor and its expression in a stable transfectant. A, TNF receptor expression (assessed by ELISA); B, cell viability, in transient transfection of the full-length receptor (, box) and of p55-IC (circle, bullet) in the presence or absence of tetracycline (empty and solid notes, respectively), assessed at various times after incubation with the transfected DNA; C, effect of p55-IC expression on the viability of cells transfected stably with this cDNA, assessed at various times after replacement of the cell growth medium with fresh medium either with or without tetracycline. Photographs were taken 36 h after tetracycline removal.



We examined also the effects of increased expression of p55-R and expression of just the intracellular domain of the receptor on the transcription of IL-8, known to be activated by TNF(19) . As shown in Fig. 4, transfection of HeLa cells with a tetracycline-controlled construct encoding the p55-R cDNA induced IL-8 transcription. An even stronger induction was observed in cells transfected with the cDNA for p55-IC. In both cases, the induction occurred only when tetracycline was excluded from the cell growth medium, indicating that it occurs as a consequence of expression of the transfected p55-R or p55-IC. Transfection with luciferase cDNA, as a control, had no effect on IL-8 transcription.


Figure 4: Ligand-independent induction of IL-8 gene expression in HeLa cells transfected with p55-R or its intracellular domain. A, Northern analysis of RNA (7 µg/lane), extracted from HeLa (HTta-1) cells, untreated or treated with TNF (500 units/ml for 4 h; autoradiography performed for 6 h), or the HTta-1 cells 24 h after their transfection (in the presence or absence of tetracycline) with p55-IC, the p55-R or luciferase cDNA (autoradiography for 18 h). B, methylene blue staining of 18 S rRNA. For other details, see ``Experimental Procedures.''




DISCUSSION

Studies employing the two-hybrid technique suggested that the intracellular domain of the p55-R self-associates and located this self-association to a part of a region found to be critical for signaling by this receptor ( (4) and the present report; see also (33) ). Further tests confirmed that this association is not artifactual, as may well occur in the yeast genetic test(24) , and indicated that it has functional consequences. The self-association could be shown to occur also in vitro, using GST and MBP p55-IC fusion proteins, thus ruling out involvement of yeast proteins or of the Gal4 DBD or AD in this association. Moreover, the expected functional consequence of this association could be demonstrated, namely occurrence of spontaneous signaling under conditions that permit receptor aggregation. A mere increase in p55-R expression, or even expression just of the intracellular domain of the receptor or of its death domain, was found to be sufficient to trigger signaling for cytotoxicity as well for expression of the TNF-inducible IL-8 gene within cells.

Normally, cells expressing the p55-R do not exhibit TNF effects unless exposed to this cytokine. Presumably, cells possess some mechanisms that reduce the self-association of the receptor and impose on it ligand dependence. Probably self-association of the receptors is in part restricted by mechanisms that maintain their self-surface expression at a low level. It may also be restricted by constraints imposed on the death domain in the receptor by other regions in the p55-R molecule. To some extent, self-association of the death domain seems to be inhibited by the membrane-proximal part of the intracellular domain (Table 1). Crystallographic studies of the extracellular domain of the receptor suggest that also this domain mediates an inhibitory effect; they indicate that, in the absence of TNF, the extracellular domains of neighboring p55-R molecules are capable of interacting in a way that obviates association of their intracellular domains. (^2)Such interaction may well prevent spontaneous signaling by the receptors and allow their intracellular domains to self-associate only after TNF binding.

The intracellular domain of Fas/APO1, which bears marked structural similarity to that of the p55-R and that likewise signals for cell death, was found also to self-associate and thus trigger signaling, suggesting that this receptor, too, plays an active role in its aggregation and is subject to control mechanisms that antagonize its propensity to self-associate. This may well be the case also for a number of other receptors, for example several tyrosine-kinase receptors, including Neu/HER-2 and the epidermal growth factor receptor, that are found, just like the p55-R, to signal spontaneously when expressed at high levels as well as after deletion of their extracellular domain (see, e.g., (25, 26, 27) , and references therein).

Interestingly, the p75 TNF receptor, even though it has, like p55-R and Fas/APO, the ability to signal for cell death(28) , does not display self-association, nor does a high level of expression of this receptor result in spontaneous signaling(29) . Apparently, the mode of signaling for cell death by this receptor differs from that of the p55-R(29) .

Most likely, the self-associations of p55-R and Fas/APO1 serve to fortify the aggregated state imposed on them by their ligands. Such a mechanism has certain functional advantages. It may augment signaling and also provide ways for modulation of signaling by mechanisms that act within the cell. An intriguing possibility for such modulation is indicated by the slight association between p55-IC and Fas-IC, which may allow cross-talk between the two cell death-inducing receptors(7) .

The propensity of these receptors to self-associate may permit also a kind of derangement of regulation that would not be expected if their aggregation occurred in a passive manner. It can lead to spontaneous signaling, independent of the ligand, in situations in which the mechanisms restricting the self-association of the receptors fail to function properly. Such ligand-independent function is, in the case of growth factor receptors, a well known cause for the uncontrolled growth of malignant cells. In receptors that signal for cytotoxicity, it may contribute to uncalled-for death of cells, as observed, for example, in response to cytopathic viruses and various other pathogens.


FOOTNOTES

*
This work was supported in part by grants from Inter-Lab Ltd. (Ness-Ziona, Israel), from Ares Trading S.A. (Switzerland), and from the Israeli Ministry of Arts and Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Equivalent contributions were made by these two authors.

To whom correspondence should be addressed. Tel: 972-8-343941. Fax: 972-8-343165.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; AD, activation domain; DBD, DNA binding domain; GST, glutathione S-transferase; IC, intracellular domain; IL-8, interleukin 8; MBP, maltose-binding protein; p55-IC, intracellular domain of the p55-R; p55-R, p55 tumor necrosis factor receptor; ELISA, enzyme-linked immunosorbent assay.

(^2)
J. Naismith and S. Sprang, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Drs. Herman Bujard and Sabina Freundlieb for providing us with the reagents for tetracycline-controlled expression and to Ada Dibeman for careful handling of the cultured cells.


REFERENCES

  1. Engelmann, H., Holtmann, H., Brakebusch, C., Avni, Y. S., Sarov, I., Nophar, Y., Hadas, E., Leitner, O., and Wallach, D. (1990) J. Biol. Chem. 265, 14497-14504 [Abstract/Free Full Text]
  2. Brakebusch, C., Nophar, Y., Kemper, O., Engelmann, H., and Wallach, D. (1992) EMBO J. 11, 943-950 [Abstract]
  3. Dhein, J., Daniel, P. T., Trauth, B. C., Oehm, A., Moller, P., and Krammer, P. H. (1992) J. Immunol. 149, 3166-3173 [Abstract/Free Full Text]
  4. Song, H. Y., Dunbar, J. D., and Bonner, D. B. (1994) J. Biol. Chem. 269, 22492-22495 [Abstract/Free Full Text]
  5. Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goeddel, D. V. (1993) Cell 74, 845-853 [Medline] [Order article via Infotrieve]
  6. Itoh, N., and Nagata, S. (1993) J. Biol. Chem. 268, 10932-10937 [Abstract/Free Full Text]
  7. Yonehara, S., Ishii, A., and Yonehara, M. (1989) J. Exp. Med. 169, 1747-1756 [Abstract]
  8. Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Moller, P., Falk, W., Debatin, K. M., and Krammer, P. H. (1989) Science 245, 301-305 [Medline] [Order article via Infotrieve]
  9. Suda, T., Takahashi, T., Goldstein, P., and Nagata, S. (1993) Cell 75, 1169-1178 [Medline] [Order article via Infotrieve]
  10. Jones, E. Y., Stuart, D. I., and Walker, N. P. C. (1989) Nature 338, 225-228 [CrossRef][Medline] [Order article via Infotrieve]
  11. Eck, M. J., and Sprang, S. R. (1989) J. Biol. Chem. 264, 17595-17605 [Abstract/Free Full Text]
  12. Bartel, P. L., Chien, C. T., Sternglanz, R., and Fields, S. (1993) BioTechniques 14, 920-924 [Medline] [Order article via Infotrieve]
  13. Guarente, L. (1983) Methods Enzymol. 101, 181-191 [Medline] [Order article via Infotrieve]
  14. Fields, S., and Song, O. (1989) Nature 340, 245-246 [CrossRef][Medline] [Order article via Infotrieve]
  15. Frangioni, J. V., and Neel, B. G. (1993) Anal. Biochem. 210, 179-187 [CrossRef][Medline] [Order article via Infotrieve]
  16. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., Albright, L. M., Coen, D. M., and Varki, A. (eds) (1994) Current Protocols in Molecular Biology , pp. 8.0.1-8.1.6, 9.1.1-9.1.2, and 16.7-16.7.8, Greene Publishing Associates/Wiley & Sons, Inc., New York
  17. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551 [Abstract]
  18. Wallach, D. (1984) J. Immunol. 132, 2464-2469 [Abstract/Free Full Text]
  19. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, H. F., Leonard, E. J., and Oppenheim, J. J. (1988) J. Exp. Med. 167, 1883-1893 [Abstract]
  20. Holtmann, H., and Wallach, D. (1987) J. Immunol. 139, 1161-1167 [Abstract/Free Full Text]
  21. Aderka, D., Engelmann, H., Hornik, V., Skornick, Y., Levo, Y., Wallach, D., and Kushtai, G. (1991) Cancer Res. 51, 5602-5607 [Abstract]
  22. Stamenkovic, I., Clark, E. A., and Seed, B. (1989) EMBO J. 8, 1403-1410 [Abstract]
  23. Smith, C. A., Davis, T., Anderson, D., Solam, L., Beckmann, M. P., Jerzy, R., Dower, S. K., Cosman, D., and Goodwin, R. G. (1990) Science 248, 1019-1023 [Medline] [Order article via Infotrieve]
  24. Fields, S., and Sternglanz, R. (1994) Trends Genet. 10, 286-292 [CrossRef][Medline] [Order article via Infotrieve]
  25. Lonardo, F., Di Marco, E., King, C. R., Pierce, J. H., Segatto, O., Aaronson, S. A., and Di Fiori, P. P. (1990) New Biol. 2, 992-1003 [Medline] [Order article via Infotrieve]
  26. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A., Schlessinger, J., and Waterfield, M. D. (1984) Nature 307, 521-527 [Medline] [Order article via Infotrieve]
  27. Haley, J. D., Hsuan, J. J., and Waterfield, M. D. (1989) Oncogene 4, 273-283 [Medline] [Order article via Infotrieve]
  28. Heller, R. A., Song, K., Fan, N., and Chang, D. J. (1992) Cell 70, 47-56 [Medline] [Order article via Infotrieve]
  29. Bigda, J., Beletsky, I., Brakebusch, C., Varfolomeev, Y., Engelmann, H., Bigda, J., Holtmann, H., and Wallach, D. (1994) J. Exp. Med. 180, 445-460 [Abstract]
  30. Loetscher, H., Pan, Y.-C. E., Lahm, H.-W., Gentz, R., Brockhaus, M., Tabuchi, H., and Lesslauer, W. (1990) Cell 61, 351-359 [Medline] [Order article via Infotrieve]
  31. Goodwin, R. G., Anderson, D., Jerzy, R., David, T., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Smith, C. A. (1991) Mol. Cell. Biol. 11, 3020-3026 [Medline] [Order article via Infotrieve]
  32. Watanabe-Fukanaga, R., Brannan, C. I., Itoh, N., Yonehara, S., Copeland, N. G., Jenkins, N. A., and Nagata, S. (1992) J. Immunol. 148, 1274-1279 [Abstract/Free Full Text]
  33. Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H. J., and Mett, I. (1994) Cytokine 6, 556

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.