(Received for publication, October 4, 1994; and in revised form, November 4, 1994)
From the
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.
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) ()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.
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. ,
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 (,
)
and of p55-IC (
,
) 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.''
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. ()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.