The EMBO Journal vol. 1 1 no.3 pp.943 - 950, 1992
Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor Cord Brakebusch, Yaron Nophar, Oliver Kemper, Hartmut Engelmann and David Wallach The Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot, Israel 76100 Communicated by S.Fuchs
The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hup55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4,(-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct. Key words: receptor mutants/shedding/soluble receptors/ structure-function relationship/TNF receptor hu p55
Introduction Tumour necrosis factor (TNF), a pro-inflammatory cytokine produced primarily by mononuclear phagocytes, contributes to the defence of the host against pathogens as well as to various detrimental manifestations of inflammation through a variety of different effects on cell function (Old, 1990; Beutler and Cerami, 1989). These effects are initiated by the binding of TNF to specific, high affinity receptors, which are expressed on the surface of most kinds of cells (Baglioni et al., 1985; Aggarwal et al., 1985; Beutler et al., 1985; Kull et al., 1985; Tsujimoto et al., 1985; Israel et al., 1986). The receptors provide the intracellular signals for cell response to TNF (Engelmann et al., 1990a). Two molecular species of the TNF receptors (TNF-Rs), expressed differentially in different types of cells, have been identified (Engelmann et al., 1990b; Brockhaus et al., 1990). Both exist also in soluble forms (Engelmann et al., 1989, 1990b; Olsson et al., 1989; Seckinger et al., 1989), which are derived proteolytically from the cell surface receptors (Nophar et al., 1990; Porteu and Nathan, 1990; Porteu et al., 1991). The soluble forms of the TNF receptors specifically bind TNF, and thus, by competing for its binding to the cell surface TNF-Rs, may function as inhibitors of ( Oxford University Press
TNF activity (Engelmann et al., 1989, 1990b; Olsson et al., 1989; Seckinger et al., 1989). Their formation in vitro is enhanced by certain stimulatory agents, including the tumour promoting phorbol diester 4,B-phorbol-12-myristate-13-acetate (PMA), and the chemotactic peptide formyl-methionine-leucine-phenylalanine (in granulocytes) (Porteu and Nathan, 1990; Lantz et al., 1990; Kohno et al., 1990). Their serum concentrations increase dramatically in various diseases (Aderka et al., 1991 and unpublished data), quite probably as a consequence of cell exposure in vivo to such stimulants. Little is known of the mechanisms of signalling, protein cleavage and membrane trafficking which take part in TNF-R function, or of the extent to which these different activities are interlinked. The recent cloning of the cDNAs for the two TNF receptors (Heller et al., 1990; Loetscher et al., 1990; Nophar et al., 1990; Schall et al., 1990; Smith et al., 1990) has provided access to structure -function analysis of the mechanisms of action and modulation of these receptors. In the present study, we explored the mechanistic relationship between the signalling activity and the inducible shedding of the p55 TNF-R by examining the effect of cytoplasmic truncation of the receptor on these activities.
Results Signalling for the cytocidal effect of TNF in rodent cells by transfected human p55 TNF receptors In order to identify the structural elements in the p55 TNF-R specifically involved in the mediation of distinct activities, mutated forms of this receptor were created and expressed in cultured cells. Since most cell lines express TNF receptors, it was necessary to find a way of distinguishing between the activities of the receptors encoded by the expression constructs and those endogenous to the cells. Cross-linking the p55 receptors by antibodies recognizing their extracellular domain triggers their signalling activity (Espevik et al., 1990; Engelmann et al., 1990a). We therefore expressed the human p55 receptor in rodent cells and used specific antibodies with the aim of triggering these receptors selectively, without affecting the receptors endogenous to the rodent cells. After deletion of large parts of the 3' and 5' non-coding sequences, the cDNA for the human p55 TNF receptor (hupS5-TNF-R) was introduced into the pMPSVEH expression vector, under the control of the myeloproliferative sarcoma virus promoter (Artelt et al., 1988) and co-transfected with an expression vector encoding the neomycin resistance gene into cells of the murine A9, L929 and NIH 3T3 lines and of the hamster BHK line. Cells constitutively expressing the transfected vectors were selected by growth in the presence of G418. As controls, cells transfected with the neomycin resistance vector alone were selected by the same procedure. A large proportion of the clones co-transfected with the vector containing the wild-type (or mutants, see below) 943
C.Brakebusch et al.
Antiserum
Monoclonal Antibodies
120 100 0
UX
Serum Dilution
Monoclonal Antibodies [jgg / ml]
Fig. 1. Cytocidal effects of rabbit antiserum against the hu-p55-TNF-R (left panel), and murine monoclonal antibodies against the receptor (right panel) in HeLa cells (0), A9 cells (0) and A9 cells expressing the wild type hu-p55-TNF-R (-). The antiserum, or the monoclonal antibodies (numbers 18 and 20, Engelmann et al., 1990b), were applied simultaneously with CHI (50 itg/ml in the A9 cells and 25 gg/ml in the HeLa cells). The two monoclonal antibodies were applied in equal amounts, to the concentration specified in the figure.
Effect of Antibodies against
the hu-p55-TNF-R
Effect of TNF
120
._
m
*5co ._
0
0
Monoclonal Antibodies [jgg / ml] Fig. 2. Response of the L929, NIH 3T3 and BHK cells (0) and of those same cells cytocidal effect of monoclonal antibodies against the hu-p55-TNF-R (left panels) and Figure 1.
944
TNF [pM] when expressing the wild type hu-p55-TNF-R (a), to the of TNF (right panels). The assay was performed as in
_B,
50% of the 25-50 clones picked up from each transfected culture) showed a significant increase in TNF binding, up to 10-fold that observed in the non-transfected cells, indicating effective expression of the cDNAs. However, TNF binding by cells transfected only with the neomycin resistance vector was indistinguishable from the binding observed in the non-transfected cells. Cells showing highest TNF binding were chosen for further analysis, testing at least three and in most cases more than five clones for each DNA construct. Effective cross linking of the p55 TNF receptors in order to trigger their signalling activity can be achieved with polyclonal antisera or by simultaneous application of two different monoclonal antibodies each recognizing different epitopes in the extracellular domain of the receptor (Engelmann et al., 1990a). While in the human HeLa cells, antibodies against the TNF receptor induced a pronounced TNF-like cytocidal effect (Figure 1), in non-transfected murine A9 cells, which are equally sensitive to the cytocidal effect of TNF, rabbit antiserum against the hu-p55-TNF-R had ony a slight effect (Figure 1, left panel), and mouse monoclonal antibodies had no effect at all (Figure 1, right panel). The antibodies had no effect either in controltransfected cells expressing only the neomycin resistance gene (not shown). In contrast, both the polyclonal and monoclonal antibodies had a strong cytocidal effect on A9 cells in which the hu-p55-TNF-R was expressed, comparable to their effect on the HeLa cells (Figure 1). Similarly, the monoclonal antibodies against the hu-p55-TNF-R were markedly cytocidal to L929, NIH 3T3 and BHK cells expressing the hu-p55-TNF-R, though no such effect could be observed in the non-transfected cells, nor in cells expressing only the neomycin resistance gene (Figure 2, left panels, and data not shown). Comparison of the response to the antibodies in different clones of the transfected cells (3-20 clones of each transfected line) revealed a rough proportionality between the extent of response and the extent of expression of the transfected receptors, as reflected in TNF binding to the cells. Thus, in a given experiment, applying the rabbit antiserum at a dilution of 1:2500 to cells of seven different transfected A9 clones, exhibiting increased TNF binding of 0.5-fold, 1.3-fold, 3.3-fold, 5.6-fold, 7-fold and 12-fold (above the binding of 510 CPM measured in the control cell cultures, expressing only the neomycin resistance gene), resulted in death of 7, 25, 43, 52, 87 and -
.
Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor Cord Brakebusch, Yaron Nophar, Oliver Kemper, Hartmut Engelmann and David Wallach The Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot, Israel 76100 Communicated by S.Fuchs
The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hup55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4,(-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct. Key words: receptor mutants/shedding/soluble receptors/ structure-function relationship/TNF receptor hu p55
Introduction Tumour necrosis factor (TNF), a pro-inflammatory cytokine produced primarily by mononuclear phagocytes, contributes to the defence of the host against pathogens as well as to various detrimental manifestations of inflammation through a variety of different effects on cell function (Old, 1990; Beutler and Cerami, 1989). These effects are initiated by the binding of TNF to specific, high affinity receptors, which are expressed on the surface of most kinds of cells (Baglioni et al., 1985; Aggarwal et al., 1985; Beutler et al., 1985; Kull et al., 1985; Tsujimoto et al., 1985; Israel et al., 1986). The receptors provide the intracellular signals for cell response to TNF (Engelmann et al., 1990a). Two molecular species of the TNF receptors (TNF-Rs), expressed differentially in different types of cells, have been identified (Engelmann et al., 1990b; Brockhaus et al., 1990). Both exist also in soluble forms (Engelmann et al., 1989, 1990b; Olsson et al., 1989; Seckinger et al., 1989), which are derived proteolytically from the cell surface receptors (Nophar et al., 1990; Porteu and Nathan, 1990; Porteu et al., 1991). The soluble forms of the TNF receptors specifically bind TNF, and thus, by competing for its binding to the cell surface TNF-Rs, may function as inhibitors of ( Oxford University Press
TNF activity (Engelmann et al., 1989, 1990b; Olsson et al., 1989; Seckinger et al., 1989). Their formation in vitro is enhanced by certain stimulatory agents, including the tumour promoting phorbol diester 4,B-phorbol-12-myristate-13-acetate (PMA), and the chemotactic peptide formyl-methionine-leucine-phenylalanine (in granulocytes) (Porteu and Nathan, 1990; Lantz et al., 1990; Kohno et al., 1990). Their serum concentrations increase dramatically in various diseases (Aderka et al., 1991 and unpublished data), quite probably as a consequence of cell exposure in vivo to such stimulants. Little is known of the mechanisms of signalling, protein cleavage and membrane trafficking which take part in TNF-R function, or of the extent to which these different activities are interlinked. The recent cloning of the cDNAs for the two TNF receptors (Heller et al., 1990; Loetscher et al., 1990; Nophar et al., 1990; Schall et al., 1990; Smith et al., 1990) has provided access to structure -function analysis of the mechanisms of action and modulation of these receptors. In the present study, we explored the mechanistic relationship between the signalling activity and the inducible shedding of the p55 TNF-R by examining the effect of cytoplasmic truncation of the receptor on these activities.
Results Signalling for the cytocidal effect of TNF in rodent cells by transfected human p55 TNF receptors In order to identify the structural elements in the p55 TNF-R specifically involved in the mediation of distinct activities, mutated forms of this receptor were created and expressed in cultured cells. Since most cell lines express TNF receptors, it was necessary to find a way of distinguishing between the activities of the receptors encoded by the expression constructs and those endogenous to the cells. Cross-linking the p55 receptors by antibodies recognizing their extracellular domain triggers their signalling activity (Espevik et al., 1990; Engelmann et al., 1990a). We therefore expressed the human p55 receptor in rodent cells and used specific antibodies with the aim of triggering these receptors selectively, without affecting the receptors endogenous to the rodent cells. After deletion of large parts of the 3' and 5' non-coding sequences, the cDNA for the human p55 TNF receptor (hupS5-TNF-R) was introduced into the pMPSVEH expression vector, under the control of the myeloproliferative sarcoma virus promoter (Artelt et al., 1988) and co-transfected with an expression vector encoding the neomycin resistance gene into cells of the murine A9, L929 and NIH 3T3 lines and of the hamster BHK line. Cells constitutively expressing the transfected vectors were selected by growth in the presence of G418. As controls, cells transfected with the neomycin resistance vector alone were selected by the same procedure. A large proportion of the clones co-transfected with the vector containing the wild-type (or mutants, see below) 943
C.Brakebusch et al.
Antiserum
Monoclonal Antibodies
120 100 0
UX
Serum Dilution
Monoclonal Antibodies [jgg / ml]
Fig. 1. Cytocidal effects of rabbit antiserum against the hu-p55-TNF-R (left panel), and murine monoclonal antibodies against the receptor (right panel) in HeLa cells (0), A9 cells (0) and A9 cells expressing the wild type hu-p55-TNF-R (-). The antiserum, or the monoclonal antibodies (numbers 18 and 20, Engelmann et al., 1990b), were applied simultaneously with CHI (50 itg/ml in the A9 cells and 25 gg/ml in the HeLa cells). The two monoclonal antibodies were applied in equal amounts, to the concentration specified in the figure.
Effect of Antibodies against
the hu-p55-TNF-R
Effect of TNF
120
._
m
*5co ._
0
0
Monoclonal Antibodies [jgg / ml] Fig. 2. Response of the L929, NIH 3T3 and BHK cells (0) and of those same cells cytocidal effect of monoclonal antibodies against the hu-p55-TNF-R (left panels) and Figure 1.
944
TNF [pM] when expressing the wild type hu-p55-TNF-R (a), to the of TNF (right panels). The assay was performed as in
_B,
50% of the 25-50 clones picked up from each transfected culture) showed a significant increase in TNF binding, up to 10-fold that observed in the non-transfected cells, indicating effective expression of the cDNAs. However, TNF binding by cells transfected only with the neomycin resistance vector was indistinguishable from the binding observed in the non-transfected cells. Cells showing highest TNF binding were chosen for further analysis, testing at least three and in most cases more than five clones for each DNA construct. Effective cross linking of the p55 TNF receptors in order to trigger their signalling activity can be achieved with polyclonal antisera or by simultaneous application of two different monoclonal antibodies each recognizing different epitopes in the extracellular domain of the receptor (Engelmann et al., 1990a). While in the human HeLa cells, antibodies against the TNF receptor induced a pronounced TNF-like cytocidal effect (Figure 1), in non-transfected murine A9 cells, which are equally sensitive to the cytocidal effect of TNF, rabbit antiserum against the hu-p55-TNF-R had ony a slight effect (Figure 1, left panel), and mouse monoclonal antibodies had no effect at all (Figure 1, right panel). The antibodies had no effect either in controltransfected cells expressing only the neomycin resistance gene (not shown). In contrast, both the polyclonal and monoclonal antibodies had a strong cytocidal effect on A9 cells in which the hu-p55-TNF-R was expressed, comparable to their effect on the HeLa cells (Figure 1). Similarly, the monoclonal antibodies against the hu-p55-TNF-R were markedly cytocidal to L929, NIH 3T3 and BHK cells expressing the hu-p55-TNF-R, though no such effect could be observed in the non-transfected cells, nor in cells expressing only the neomycin resistance gene (Figure 2, left panels, and data not shown). Comparison of the response to the antibodies in different clones of the transfected cells (3-20 clones of each transfected line) revealed a rough proportionality between the extent of response and the extent of expression of the transfected receptors, as reflected in TNF binding to the cells. Thus, in a given experiment, applying the rabbit antiserum at a dilution of 1:2500 to cells of seven different transfected A9 clones, exhibiting increased TNF binding of 0.5-fold, 1.3-fold, 3.3-fold, 5.6-fold, 7-fold and 12-fold (above the binding of 510 CPM measured in the control cell cultures, expressing only the neomycin resistance gene), resulted in death of 7, 25, 43, 52, 87 and -
.
