75 kDa Tumour Necrosis Factor (TNF) receptors transduces TNF signals: necessity for the 75 kDa-TNF receptor transmembrane domain

DIMERIZATION OF CHIMERIC ERYTHROPOIETIN/75 kDa TUMOUR NECROSIS FACTOR (TNF) RECEPTORS TRANSDUCES TNF SIGNALS: NECESSITY FOR THE 75 kDa-TNF RECEPTOR TRANSMEMBRANE DOMAIN Wim Declercq, Peter Vandenabeele,

Walter Fiers

We developed a transfection-based assay for evaluating human (h) Tumour Necrosis Factor receptor (TNF-R) activities in a rat/mouse T-cell hybridoma, viz. PC60. Here we report on the role of TNF-R75 cross-linking in induction of GM-CSF secretion and apoptosis. The effect of TNF-R75 dimerization, in contrast to trimerization, was analysed by replacing the extracellular domain of this receptor with the equivalent domain of the murine erythropoietin receptor (EPO-R), which dimerizes upon ligand interaction. To determine the role of the transmembrane region in signal transduction, chimeric EPO-R/TNF-R75 were constructed in which the respective transmembrane domains were interchanged. The hybrid receptors were introduced into PC60hTNFR55 cells, which already expressed functional, transfected hTNF-R55. By this approach we demonstrated that dimerized chimeric EPOR/TNF-R75 receptors act synergistically with hTNF-R55-induced cytokine production and apoptosis as does trimerized wild-type hTNF-R75. Dimeric triggering of these hybrid receptors with EPO alone was less efficient than trimerization of hTNF-R75. Furthermore, EPO-R/TNFR75 only responded to EPO when the matching transmembrane region of TNF-R75 was present. Our results also prove that the hTNF-R75 extracellular part per se is not required for signalling. Finally, our data indicate that the expression of chimeric EPOR/TNF-R75 in PC60hTNF-R55 cells, regardless of the presence of the TNF-R75 transmembrane region, facilitates TNF-R55-dependent signal transduction leading to apoptosis. This means that introduction of the intracellular domain of hTNF-R75, even without triggering, is sufficient to promote hTNF-R55-dependent activities in PC60 cells. © 1995 Academic Press Limited

Tumour Necrosis Factor (TNF) is a homotrimeric monokine originally described for its antitumour activity. Now it is known as one of the most pleiotropic cytokines mediating a broad spectrum of biological activities, such as cell proliferation, cytotoxicity, antiviral responses, and activation of transcription factors and cellular genes. TNF is considered a key regulatory molecule in immune and inflammatory reactions, and in a number of pathological conditions.1–3 Two distinct, high-affinity TNF receptors of 55 kDa (TNF-R55) and 75 kDa (TNF-R75) have been identified and cloned.4–7 Based on the characteristic, repeating, cysteine-rich motifs in the extracellular domain, both TNF-R55 and TNF-R75 belong to the nerve growth factor/TNF-R

From the Laboratory of Molecular Biology, Gent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium Correspondence to: Walter Fiers Received 26 January 1995; accepted for publication 24 April 1995 © 1995 Academic Press Limited 1043-4666/95/07070119 $12.00/0 KEY WORDS: apoptosis/EPO/TNF/TNF-R/transmembrane CYTOKINE, Vol. 7, No. 7 (October), 1995: pp 701–709

family.8 The intracellular domains of the two TNF-Rs show no sequence homology, suggesting distinct biological functions. Although the signalling mechanism of the TNF-Rs is still incompletely understood, oligomerization of receptors is believed to be a first essential step. This is supported by the fact that TNF activities can be mimicked with specific anti-TNF-R antibodies.9–12 In particular pentameric IgM anti-TNF-R antibodies or cross-linked, monoclonal anti-TNF-R IgG antibodies are effective in eliciting biological activities in several cell lines. The trimeric TNF molecule contains three receptor-binding sites, located in the clefts between the subunits.13 Indeed, the stoichiometry of binding in solution for the soluble, extracellular domains of TNF-R55 or TNF-R75 to the homotrimeric TNF molecule ranged between 2 and 3.14–16 Lymphotoxin is closely related to TNF and binds to the same receptors. The X-ray structure of the soluble human (h) TNF-R55/human lymphotoxin complex confirmed the previous data and showed three receptor molecules bound to one lymphotoxin trimer.17 701

702 / Declercq et al.

Studies with agonistic antibodies and receptorspecific TNF muteins demonstrate that TNF-R55 is responsible for the majority of TNF activities, including cytotoxicity, manganese superoxide dismutase induction,11 antiviral activity,18 fibroblast proliferation,10 induction of ICAM-1, E-selectin and VCAM-1 expression on endothelium19,20 as well as interleukin 6 and GM-CSF secretion by endothelial cells.21 TNF-R75 signalling involves mainly effects on lymphoid cells, such as T-cell proliferation22,23 and induction of GMCSF secretion by the T-cell hybridoma PC60.12 In the mouse, hTNF can be considered a murine (m) TNF-R55-specific agonist because it does not bind on mTNF-R75.24 Since mTNF, but not hTNF, showed substantial biological activity on the rat/mouse T-cell hybridoma PC60,25 we developed a transfection-based assay for evaluating hTNF-R75 activities in these cells.12 PC60hTNF-R75 cells showed significant GMCSF production upon selective triggering of this human receptor. Recently, we found that selective hTNF-R75 triggering was also capable of inducing apoptosis in PC60 cells, but, surprisingly, only in cells which were also transfected with hTNF-R55.26 Transfection of PC60 cells with TNF-R genes or TNF-R gene-derived constructs provides us with a simple test system to evaluate receptor function. Here, we report on the role of TNF-R75 cross-linking in induction of GM-CSF secretion and apoptosis in PC60 cells. The effect of TNF-R75 dimerization was analysed by replacing the extracellular domain of this receptor with the equivalent domain of the murine erythropoietin receptor (mEPO-R). It is well-documented that the extracellular domain of the latter plays an essential role in ligand-induced receptor dimerization.27,28 To determine the role of the transmembrane region in signal transduction, chimeric EPO-R/TNF-R75 genes were constructed in which the respective transmembrane domains were interchanged. By this approach we demonstrated that dimerization of EPO-R/TNF-R75 hybrids is capable of inducing GM-CSF production and acts synergistically with TNF-R55-mediated apoptosis in PC60 cells. Our data also suggest that the TNF-R75 transmembrane region contributes to the process leading to signalling. Furthermore, introduction of a chimeric receptor containing the intracellular domain of hTNF-R75 in PC60hTNF-R55 cells proved to be sufficient to render these cells susceptible to TNF-R55mediated apoptosis.

RESULTS Expression of chimeric EPO-R/TNF-R in PC60 cells We constructed two different hybrid EPO-R/TNFR75: one carrying the extracellular and transmembrane

CYTOKINE, Vol. 7, No. 7 (October 1995: 701–709)

domains of mEPO-R linked to the cytoplasmic region of hTNF-R75 (EPO-R/TNF-R75 Hybrid 1, H1), and a second containing the extracellular domain of mEPOR fused to the transmembrane and intracellular regions of hTNF-R75 (Hybrid 2, H2) (Fig. 1). The plasmids coding for these receptors were introduced into PC60hTNF-R55 cl 8 cells, which already expressed functional, transfected hTNF-R55. This allowed us to evaluate the cooperation between hTNF-R55 and hybrid EPO-R/TNF-R75. This is important since we previously observed a functional cooperation between the two transfected, wild-type hTNF-Rs in the induction of apoptosis in PC60 cells.26 G418-resistant colonies were isolated by limiting dilution and hybrid receptor expression was examined by staining the cells with an antiserum against an NH2-terminal peptide of EPO-R and analysis by flow cytometry. EPO-R/TNF-R75 H1 and H2 were expressed at similar levels on all clones tested, while mock-transfected cells showed no binding of anti-EPO-R antibody (data not shown). The number of cell surface H1-R and H2-R was estimated at 125 000/cell and 103 000/cell, respectively, by Scatchard analysis (Fig. 2). The affinity of the hybrid EPO-RLTNF-R75 was calculated at 654 pM for H1 and 395 pM for H2, which is in agreement with previous reports of transfected, wild-type EPO-R.29,30 This finding confirms that the transmembrane and intracellular parts of EPO-R do not modulate the affinity of ligand binding.31 Since hTNF-R75 is constitutively phosphorylated,15 we made use of this property to label EPOR/TNF-R75 H1 and H2 proteins. Immunoprecipitation of 32P-phosphorylated hybrid receptors revealed a major and a minor protein band with an apparent molecular mass of 60 kDa and 58 kDa, respectively, both for H1 and for H2 EPO-R/TNF-R75 fusions (Fig. 3). The discrepancy between the sizes of the hybrid receptor polypeptides and the predicted 45 kDa can be ? ? ? ? ? ?@ ? ? ?@h? ? ? ? ?W26KO@? ? ?7@@@@@? ? ?@@?@@@? ? ?3@@@@@? ? ?@@? @@@@@@f?N@@?@@? ? 3@@@@@f?J@@@@@? ? ?@ ?@(Mg?7@@@@@? ?W2@@@@?e? 7@h?@@@@@@? ?7@@@@@Le? @@)Kg?3@@@@@? ?3@?@@@,e? @@@@@@g@@@?@? ?V'@@@(Ye? @@XI'@f?7@@@@@? ?@@@f? @?2@6X @@)XN@f?3@W@@@? ?W&@@@)Xe? @@@@@1 @@@)X@f?V40R4@? ?7@@@?@1e? @@@?@5 @@?@@@ ?3@@@X@5e? @@@@@? 3@@@@@f?@@@@@@? ?V40R@(Ye? @@@?@1 V40?'@f?@@@@@@? ?@Y?e? @@@@@@ N@g?@@@@? @@@@@@e? @W@@@@ ?@g?@@@@? 3@@@f? @0R'@@ @@@6X@f?@@@@@@? V4@@f? ?V@@ @@@@@@f?@@@@@@? ?@L?e? ?O2@@@ W@ ?W2@@@)Xe? @@@@ '@6Xe?W&@ ?7@@@?@1e? 3@@@ V'@)X??7@5 ?@@@@?@5e? V4@@@@ ?V'@)K?@(Y ?@@@@@(Ye? ?W@@ V@@@@(Y? W@H?e? ?7@@?f@@R'@@ ?V40R@5?e? 7@@?@1 @@@
?@

Figure 1. Schematic representation of mature H1- and H2-type hybrid receptors.

The first and last amino acids of mEPO-R and hTNF-R75-derived sequences are indicated; the numbers refer to the position of the corresponding amino acids on mEPO-R and hTNF-R75 cDNAs.4,32 Extracellular (EC), transmembrane (TM) and intracellular (IC) domains are delineated.

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N@@@@5 ?3@@@@5? ?V4@@@0Y @@@@@@ @?gV4@@@@@?e ?J@@@@@L J@@@@? @@@@f?@ @? @@ ?@ ?7@@@@@1 7@@@@1 ?7@@@@1? @@?@@@@@6X ?W2@@@@@ @@@@@@@?e @@f?@@?e@@hf@?f?@@?@@e@@ ?@?@@??@@?@@@?hf@?g?@@@@@@1 ?7@@@@@@L? @@@@@@@?e @??@ ?@ ?3@@@@@5 ?J@@@@@@ ?@@@@@@? ?@@@@@@@ ?@@?J@@@@@@@)Khf@@?)X?e@@@?@@@?e ?@?V4@@@0Y?@ ?@@@@@@@@@ ?@@@@@@?@? ?@@@@@@@ .R4@@@@0R4@?hf?@1?e@@@@@@@=e @? @@g@? ?@ @@W5 @@ ?@@?e?W@@@XI4@? @? ?@@@@@@@ @@@@@@ ?@@@@@@? ?@@@@@@U ?@@@@@@@ W&@@@)X?e ?@g?@@@@@@@ @@@@@@ ?@@@@@@? ?@@@@@@1 ?@@@@@@@ 7@@@@@1?e ?@@@@@@@ @@@?@@e?@ ?@@@?@@L ?@@??@@5 ?@@@@?@@e?@ @@e@@@?e @? ?@@@@@@5 @@@@@5 ?@@@@@@@ ?3@@@@@H ?@@@@@@5 3@@@@@5?e ?@ W@@? @@@? @?eW@@0M? ?N@@@@@?g@? W@@@@? V'@@@@H?e @?f?W2@@Y@1 @?e@@@@@1 @@@Y ?J@@@@@?he?@he7@@?@1?@ ?V@@@@L?e ?7@@@@@@ @@@@@@ N@@@6K ?7@@@@@L ?J@@@@@@ @@@@@@1?e ?@@@@@@@ @@@?@@ ?@@@@@6X ?@@??@@@ ?7@@@@@@ @@@@@@@?e ?@ ?@@@V'@@ 3@@@@@ ?@@V4@@) ?@@@@@@Hhf?@g?3@@V'@@?@ @@@@@@5?e @??@ V4@@@@ ?@@L ?@@@@@@? @??V4@?N@@ @@@@@0Y?e @?I@?V4@f?@ ?@ I/ @@ @? ?@ ?@f@? ?@@@@@@@ ?@@@@@@@ @@@@@@@?e @? @?e?3@@@@@@f@? ?3@@@@@@ 3@@@@@@?e @? @? ?@e@? ?V40M?h?@hf?V40M?f?@ S@0Mf@? @? ?@ ?@ .Mh ?@?@f@? ?@ ?@ @? ?@e?@ ?@ ?@f @? @? ?@ @? @? ?@ ?@ ?@ ?@ @@ @? ?@he?@ ?@K?eO@K? ?J@@ ?3@@@@@@@@@@@@@@e@@@@@@h?W2@@@@@6XfW&@5 @@@?@@@@ @?hf ?@ @? ?N@@@@@@V'@@@@@@?J@@@@@@h?&@@@@@@@1f7@@U 3@@?3@@@g?@ @? @@@@@5?N@@e@@?7@@e@@he?@@??@@@e?J@@S,?@@@@6X?@@@@@?N@@?N@@@f?@h?@f?@ @? ?@ @@@@@He@@@@@@?@@@e@@e@6?2@@@??@@@@@@@e?7@@0YJ@@@@@)X@@@@@L?@@??@@@ @?h@@@@@Le@@@@@@?@@@e@@e@@@@@@@??@@@@@fJ@@?e7@@?@@@@@@@@@1?@@??@@@ @?@? @@@@@1?J@@X?e?3@@e@@fI4@@@?J@@?@@@@?W&@5?e3@@?@@W@@@@@@@?@@??@@@L?he@? ?J@@@@@@W&@@1?e?N@@@@@5he7@@@@@@@?7@@H?eN@@@@@(Y@@@@@@@@@@@@@@1? ?@ @? ?@ ?@@@@@@@@@@@@?f@@@@0Yhe@@@0?4@@X@@5f?@@@@0Y?@@@@0?4@@@@@@@@? @?e?@ ?B@@(Y ?I@Mhe?@ ?@ ?@ @@H? @? @@ @? ?@ ?@ ?@ ?@ @?f?@ ?@ @? @? @? @? ?@ ?@ @?he?@ ?@ @??@ @?he @? W. .Y @? ?@ @?g ?@ @?hf?@ ?@ ?@ @?@? ?@ ?@ @? @? @? ?@ /X @?hf?@ V/ @? @? @? @?@?

Figure 2. Radiolabelled EPO binding to PC55EPO75 cells expressing H1 or H2 chimeric EPO-R/TNF-R75.

Cells derived from PC55EPO75 clones expressing either H1 (s, cl 412/2) or H2 (d, cl 3-9/2) EPO-R/TNF-R75 hybrids were incubated with various concentrations of recombinant 125I-EPO for 10 h at 4°C. Specific binding was plotted by the Scatchard method. The inset shows saturation curves.

accounted for by secondary modifications.32 The 2 kDa difference in size between the major and the minor EPO-R/TNF-R75 band is probably due to differences in N-linked glycosylation.33 Control PC60hTNF-R55 cl 8 cells showed no EPO-R nor hybrid EPO-R/TNFR75 expression.

@? ?@@@@@?W2@@6X??@@@@? @@ @@@@@?@@@@6KO2@@@6X?@@@@6?2@ ?@@@@@?7@@@@1?J@@@@??@@@@??@ @5 @?f@@V'@@@@(?'@1?@@@@@@@@ ?@e?@?@(MI4@?7@fJ@f?@ @? @@6Xe@@?V@@@@H?V'@??W@(Y@@@ ?@@@@@?@H?f@@@@6?&@@@6X?@ @1 @@@)e@@@@0Y@@f@??7@H?@@@ ?@@@@@?@L??W&?@@V'@@@@V'@1?@ @@ @Xf@(M?e@@L?W&@??@@??@X? ?@f?@)KO&@?@@?V@@@@?V@@?@ @)K?e@Hf3@)?&@5?J@5??@)X@@ ?@f?@@@@@5?3@@@@V'@@@@5?@ @5 ?@gI4@0Y?V4@@@?V4@@0Y?@@@@@@?@?fV4@@@0Y?@0Y??3@@ ?V4@0Y ?@@@@@6K @@@@@@@@@@@@@@6K @@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K @@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@ ?I@M ?@@? @@ ?@@? @@ ?@@? ?@e?@@? ?@e?@@?W2@? ?@e?@@?&@@? ?@@@@@@?e@?e?@ ?@@@@@@?e@? J@e?@@?e@? 7@e?@@?e@? @@e?@@?e@?

@@e@@eW2@6X? @He@@e7@@@1? @?e@@e@MW@@? @@@@@@e?W&@5? @?e@@eW&@0Y? @?e@@e7@ @?e@@e@@@@@? @?e@@

@@@@@@@@@@@@@@@@@@@@@@@@6K @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@h@@@@@@@@@@@@@@@6K? @@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@ I4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@h@@@@@@@@@@@@ @@h@?hI4@@@@@@ ?@@? @@ @@g?J@? ?@@? @@ @@g?7@? ?@@? @@ ?@@? ?@@@6X?@ ?@@@@)X@ O@K? @?hf@@@@@@@? ?@@@@@@@hf?@?W@@@@ @@@@@@@@he@??W2@@@?@ @?hf3@@@@@@? ?N@?eI@hf?@@@Y@@@ N@@@@@@@hf?7@V'@?@ ?@@@?@@@ ?W26K? ?3@LN@@@@?hfV+M? W2@@@?@@ hf@? ?@@@?@ ?7@@@@?@hf@@6X?@@? ?N@1?3@@@? 7@@@@?@@ I'@@ ?@@@@@@@ ?@@V'@?@he?J@@@)X@@? @@?V4@@? @@ he?@@@@@@@hf ?V4@ ?'@@@@@@@?hf@@X?@@ ?3@@@@@@ ?@@LN@@@he?7@X?@@@@? @@ he?3@@@@@@hf ?V'@@@@@@?hf3@)T@@ ?V'M ?3@1?3@@he?3@)T@@@@? ?'@6K? e?N@? V4@0R4@@ '@6K S(M? @5?V4@hf@@0R'@@?h ?V4@@6K? V4@@6K ?7@?eV4@?he?J@L ?@6K6K ?7@Y ?I4@@@hf?@@Y ?'@)K? '@6K I4@@@@ ?@@@6Khf?@@@6K I@hf?@@@6K f?V4@@6K?hf?@@@ V4@@@6K? ?W@@ I4@@6K I4@@6X I4@@6XhfI4@@6Kh I4@@@@ ?@@@U? I4@@@?hf?I4@@@ I4@@@? I4@) I4@) ?W@5 ?I4@ @6X@@@)Xhf?@@@@@?@ I4@? I4@? O&@? @@@@e@1hf?@@V'@?@ ?@?@@@6X ?@@@@@6X O)X? he?@@@@@@1hf @@@@e@5hf?@@LN@@@ ?@?@@@@1 ?@@@@@@1he?W2@@@?@@? hf?@@6X? @@@@@@)X he?@@@@V'@hf @@@@@@@Hhf?3@1?3@@ ?@?@X?@@ ?@X?@@@@he?7@V'@?@@? he?@?@@?V@hf I@?4@?hf?V4@?V4@hf?@@@)K ?@@@@@@6X?hf3@@@@@@) ?@)X@@@@he?@@LN@@@@? he?@@@@@@@hf?@@@)X@@ ?@@@@@@@he?3@1?3@@@? hf?@@?I4@@)?hfV'@@?@ ?V4@@@ @@@@@@@?hf?@@@@@@@ ?V4@?V4@@?he?@@@@@@@hf?@@@@@@@ ?N@@V4@@hf?@@? ?I4@ N@@? @@ @@9? @? ?@@= @@ @? @@ ?B@@ @? @@ B@@? @@ ?@@? @? @@ @@ @? @@ ?@@? @@ ?@@? @? @@ @@ @? @@ ?@@? ?@5? 3@L? @@ ?3@? ?@ ?@@@@@@@he?@@@@@@@ V@)X @@ ?V@? ?@)K @@6X ?@@@@@@@he?@@@@@@@ f?@@@@@@@hf@@@@ f@@@@@@@@ @@@@@@@?hf?@@@@@@1 @@@) ?@W@0MW@he?@W@@?W@h hf?@@@@@@@@?h f?3@@@@@@hf3@@@ 3@@@@@@?hf?3@@@@@5 @?hf3@@@@@@@ N@@? ?@@U?W&@he?@@>@T&@h ?V'@@@@Hhf?3@@@@@@ f?V+M V'@@@@ V'@@@? ?@@? ?@@)?7@@he?@@0R@@5h V'@@@?hf?V4@@@@? ?V4@@@ ?V'@@? ?@0Y ?I'@@? ?@M? ?V4@@? ?I4@ V4@? V4@?

?O2@ ?W2@@@ W&@@@@ *@@@@@ V4@@@@ W@@@ ?O&@@@ @@@@@@ 3@@@@@ V4@@@@ I4@@ I@

Figure 3. cells.

?@6?2@@6X? ?@@@@@@@1? ?@@@@??@@? ?@@@@??@5? ?@@@@@@0Y? ?@6?2@@6X? ?@@@@@@@1? ?@@@@??@@? ?@@@@@@@5? ?@@@@0Y? ?@ ?@@@@@@@@? ?@@?@@(M ?@@?@@H? ?@@?@@ ?@@@@@@@@? ?@@@@@@@@? @@ @@ @@ ?@@? ?@@?@? ?@@?@? ?@@?@? ?@@@@@@@ ?@@@@6K? ?@@@@@@@ ?W@@@5 O&@@0Y ?@@@@? ?@@@@@@@ ?@@@@@@@ ?@ ?@ ?@@@@@@@ ?@e?I4@ ?@ ?@?W2@@@ ?7@@@@ ?@X? ?@X@)K ?@@@@@@@ ?W2@@@6X ?7@@@@@1 ?@@?eW@ ?@@??O&@ ?3@@@@@5 ?N@@@@@H ?J@@@@@L ?7@@@@@1 ?@@@@?W@ ?@@@@W&@ ?@@@@@@5 W@@@@H ?W&
?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?W26X??O26X??@f@@@6K?g ? W&@@)?2@@@1??@f@@@@@@g 6X? 7@e@@@@?@@??@W2@?@@?I'@?O2@ N@@@@@@1? ?W2@@?@@@@@?@@?@@??@@@5?@@e ?@@@@@@@? ?&@@@?@@e@@@@@@@??@@@e@@e @@? @@e@@@@@@@??@@@1?@@e?@@@@@ @@L 3@@@@V'@@@5??@V'@?@@@@@(Y@@@ V4@@@?V4@0Y??@?V4@@@@@0Y?@@@@@@? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?W2@e@@@6X??@f?@@@@6X?f 6X? ?7@@e@@@@1??@f?@@@@@1?W2 @1? J@@@?J@@@@@??@W2@??@e?@@?7@ @?@@? ?W&@@@W&@@@@@??@@@H??@e?@@? J@@?@@@@? @@@@?7@@@@@@@X?@@?J@@@L??@e &@5?@?@@L @@@@?@@@@@(Y@)X@@?7@?@)X?@?O @@@ ?I40Y?@@@@@?@@?3@,?@@@@0Y?@@ ?V+Yhf?? ?

Expression of hybrid EPO-R/TNF-R75 in PC55EPO75

Different clones expressing transfected H1 or H2 chimeric receptors and the parental PC60hTNF-R55 cl 8 were metabolically labelled with 32Pi and lysed. The receptors were immunoprecipitated with an anti-EPO-R antiserum and loaded on a 10% SDS-polyacrylamide gel. The migration of EPO-R/TNF-R75 proteins is indicated by arrowheads.

EPO-mediated GM-CSF induction in PC60 cells transfected with hybrid EPO-R/TNF-R75 constructs Table 1A shows the induction of GM-CSF secretion in PC60 cells expressing both hTNF-R55 and a hybrid EPO-R/TNF-R upon stimulation with recombinant human EPO and/or R32WS86T, the hTNF-R55specific hTNF mutein. The data result from a representative triplicate experiment on several independent clones. R32WS86T clustering induced in all transfectants levels of GM-CSF comparable with the one of their parental clone, PC60TNF-R55 cl 8 cells. This indicates that hTNF-R55 function was not impaired in any of the cells coexpressing the EPOR/TNF-R75 hybrids. Remarkably, EPO stimulation was only functional in cells expressing the EPO-R/TNFR75 H2 fusion, in which the transmembrane region of TNF-R75 was included. Triggering with EPO of the H2type cells resulted in GM-CSF levels approximately 5 times lower than obtained with an hTNF-R55 agonist. As expected, D143F, an hTNF-R75 agonist, had no effect on GM-CSF induction (data not shown). Cell lines expressing H1 were unresponsive to EPO treatment. A trivial explanation for this differential response of H1 and H2 transfectants might be found in the receptor numbers or affinities, but Scatchard analysis did not reveal any significant difference (Fig. 2). The distinct behaviour of the two chimeric receptors was also observed after combined addition of EPO and R32WS86T: PC55EPO75 H2 cells produced about 4 to 8 times more GM-CSF upon EPO and R32WS86T stimulation as compared to the level of induction with R32WS86T alone, while H1 cells did not produce more GM-CSF when EPO was added in addition to the TNFR55 agonist (Table 1). Taken together, these data point to an important role of the transmembrane region in hTNF-R75 signalling. To compare the GM-CSF-inducing capacity of dimerized EPO-R/TNF-R75 H2 and trimerized wildtype hTNF-R75, PC60hTNF-R55 cl 8/hTNF-R75 cells were treated with R32WS86T, D143F or a combination of both hTNF muteins (Table 1B). EPO addition to these cultures did not influence the levels of GM-CSF secretion (data not shown). In contrast to EPO stimulation in H2 cells, D143F-mediated hTNF-R75 triggering of PC60hTNF-R55 cl 8/hTNF-R75 cells was as effective as R32WS86T in GM-CSF induction. This indicates that dimerization is far less effective for initiating signal transduction than trimerization, especially if one takes into account the much higher receptor number in H2 cells. Induction of apoptosis by the chimeric receptors TNF induces apoptosis in several cell lines,34,35 a process of cell death accompanied by chromatin condensation, invagination of the nuclear membrane and

704 / Declercq et al.

CYTOKINE, Vol. 7, No. 7 (October 1995: 701–709)

TABLE 1. EPO-mediated GM-CSF induction in PC60hTNF-R55 cells transfected with EPOR/TNF-R75 hybrids GM-CSF activity (ng/ml)

A PC60hTNF-R55 cl 8 PC60hTNF-R55 cl 8 neo PC60hTNF-R55 cl 8/hTNF-R75 PC55EPO75 H1 cl 4-4/2 cl 4-7/11 cl 4-12/2 cl 4-15/4 PC55EPO75 H2 cl 1-5/12 cl 1-8/11 cl 3-2/2 cl 3-9/2

B PC60hTNF-R55 cl 8 PC60hTNF-R55 cl 8 neo PC60hTNF-R55 cl 8/hTNF-R75

Blank

R32WS86T

EPO

R32WS86T 1 EPO

,0.1 (2) ,0.1 (2) 0.3 (2) ,0.1 (2) 0.3 (2) 0.4 (2) ,0.1 (2) ,0.1 (2) ,0.1 (2) ,0.1 (2) 0.5 (2)

2.1 (0.1) 1.9 (0.2) 11.9 (0.7) 14.1 (0.4) 3.0 (0.5) 5.9 (0.2) 0.9 (2) 4.6 (0.6) 1.9 (0.1) 7.1 (0.2) 8.4 (0.6)

,0.1 (2) ,0.1 (2) 0.3 (2) ,0.1 (2) 0.5 (0.1) 0.5 (2) ,0.1 (2) 1.5 (0.3) 0.2 (2) 0.8 (0.1) 3.3 (0.5)

2.2 (0.2) 1.7 (0.3) 11.5 (0.9) 12.3 (0.6) 4.2 (0.2) 5.9 (0.2) 0.9 (0.1) 32.6 (1.1) 15.4 (0.9) 43.6 (1.2) 27.3 (3.0)

Blank

R32WS86T

D143F

R32WS86T 1 D143F

,0.1 (2) ,0.1 (2) 0.3 (2)

2.9 (0.1) 1.9 (0.2) 11.9 (0.7)

,0.1 (2) ,0.1 (2) 8.1 (0.6)

1.8 (0.1) 1.5 (0.1) 74.7 (8.0)

Cells (5 3 104/well) were incubated in the presence of medium (blank), EPO (100 ng/ml), the hTNF-R55-specific mutein R32WS86T (500 ng/ml), the hTNF-R75-specific mutein D143F (500 ng/ml) or combinations. These concentrations induced maximal levels of GM-CSF induction (data not shown). After 48 h, GM-CSF activity in the supernatant was determined and expressed as ng/ml per 106 cells. The mean value of triplicate cultures is also indicated (SD in brackets).

internucleosomal DNA fragmentation.36 We reported that PC60 cells coexpressing the two types of hTNF-Rs undergo TNF-mediated apoptosis, in contrast to cells expressing only one kind of TNF-R.26 We further showed that, although the presence of both receptor types was required, triggering of only one type was sufficient to induce intermediate levels of apoptosis. We have analysed now the cooperation between hTNFR55 and one of the chimeric EPO-R/TNF-R. The results, presented in Table 2A, indicate that the presence of only the intracellular domain of TNF-R75 was sufficient to render PC60hTNF-R55 cl 8-derived cells susceptible to TNF-R55-induced cell death, since both PC55EPO75 H1 and H2 cells showed R32WS86Tinduced cell killing as PC60hTNF-R55 cl 8/hTNF-R75 cells do, but not the parental PC60hTNF-R55 cl 8 cell line. The hTNF-R75 transmembrane region was apparently not essential to render PC55EPO75 cells sensitive to R32WS86T-mediated apoptosis. EPO triggering of H1-R or H2-R did not induce apoptosis, indicating that dimerization of either receptor type was not sufficient for signal transduction leading to apoptosis. Surprisingly, when both R32WS86T and EPO were added to H2 cell cultures, EPO could augment twofold the R32WS86T-induced apoptosis; this was not observed for H1 transfectants. These results are in agreement with the data for GM-CSF induction, in which the combined addition of EPO and R32WS86T

was only synergistic when the intracellular domain of the TNF-R75 remained linked to its own transmembrane region. As expected, D143F had no effect on cell death in H1- or H2-type cultures (data not shown). Stimulation of control PC60hTNF-R55 cl 8/hTNFR75 cells with R32WS86T or D143F, which trimerize wild-type hTNF-R55 or hTNF-R75, respectively, caused apoptosis in these cultures (Table 2B). Combined triggering of hTNF-R55 and hTNFR75 resulted in additive levels of apoptosis. EPO addition to these cells had no effect (data not shown).

DISCUSSION Several reports have shown that clustering of the TNF-R by monoclonal or polyclonal antibodies is able to induce a biological response, indicating that the TNF molecule per se is not required for signalling.11,37,38 We reported previously that the rat/mouse T-cell hybridoma PC60 transfected with hTNF-R75 could be triggered by agonistic anti-hTNF-R75 antibodies leading to the induction of several cytokines, e.g. GM-CSF.12 But only in double-transfected PC60 cells, which expressed both hTNF-R55 and hTNF-R75, did triggering of the latter lead to apoptosis.26 To analyse the effect of hTNF-R75 dimerization rather than trimerization for the induction of GM-CSF and apoptosis, we constructed two chimeric EPO-R/TNF-R75, which both

Signalling by chimeric TNF receptors / 705

TABLE 2. TNF-R75

Induction of apoptosis in PC60hTNF-R55 cells transfected with chimeric EPO-R/

Percentage of propidium iodide-positive cells

A PC60hTNF-R55 cl 8 PC60hTNF-R55 cl 8 neo PC60hTNF-R55 cl 8/hTNF-R75 PC55EPO75 H1 cl 4-4/2 cl 4-7/11 cl 4-12/2 PC55EPO75 H2 cl 1-8/11 cl 3-2/2 cl 3-9/2

B PC60hTNF-R55 cl 8 PC60hTNF-R55 cl 8 neo PC60hTNF-R55 cl 8/hTNF-R75

Blank

R32WS86T

EPO

R32WS86T 1 EPO

4.0 (0.2) 4.2 (0.2) 3.7 (0.3) 5.4 (0.3) 7.1 (0.3) 2.5 (0.1) 5.4 (0.4) 2.3 (0.2) 4.9 (0.4)

4.5 (0.4) 3.7 (0.3) 15.7 (0.3) 39.3 (0.3) 16.4 (0.8) 27.9 (0.7) 16.7 (1.1) 14.9 (0.6) 24.1 (1.1)

4.0 (0.5) 3.5 (0.2) 4.2 (0.2) 5.0 (0.5) 6.6 (0.6) 2.6 (0.3) 5.5 (0.6) 2.4 (0.1) 6.4 (0.5)

5.1 (0.8) 4.2 (0.7) 15.4 (0.7) 42.9 (1.8) 17.3 (0.3) 34.5 (0.4) 33.8 (1.7) 36.5 (1.0) 46.8 (1.0)

Blank

R32WS86T

D143F

R32WS86T 1 D143F

5.0 (0.7) 4.6 (0.2) 3.7 (0.3)

5.9 (0.3) 3.7 (0.3) 15.7 (0.3)

5.9 (0.6) 4.3 (0.2) 15.1 (0.2)

6.2 (0.4) 4.5 (0.6) 38.0 (0.7)

Cells (5 3 104/well) were incubated for 20 h in the presence of medium (blank), EPO (100 ng/ml), hTNF-R55-specific mutein R32WS86T (500 ng/ml), hTNF-R75-specific mutein D143F (500 ng/ml) or combinations. These concentrations induced maximal levels of apoptosis (data not shown). After staining with propidium iodide, the number of apoptotic cells was analysed on a fluorometer and shown as percentage of propidium iodide-positive cells. The mean value of triplicate cultures is given (SD in brackets).

consisted of the extracellular mEPO-R domain and the intracellular hTNF-R75 domain, but H1 carried the transmembrane region of mEPO-R and H2 that of hTNF-R75. It is well-established that signal transduction by EPO is initiated by dimerization of the extracellular receptor domain, which also brings together the corresponding intracellular domains.27,28 Truncation of the intracellular region of the hTNF-R75 results in a receptor which has lost its GM-CSF-inducing capacity in PC60 cells (our unpublished results) and its proliferation and NF-κB-inducing property in CT6 cells,39 indicating that this part is essential for signal transduction. PC60hTNF-R55 cl 8 cells transfected with the hybrid EPO-R/TNF-R75 constructs revealed for both H1 and H2 transfectants a single class of low-affinity EPO-R with kDa values of 654 pM and 395 pM, respectively. These affinities were similar to those reported for transfected wild-type EPO-R in Ba/F3, FDCp1 and 32D cells, and were shown to transduce normal growthstimulatory effects in these cells.28–30 H1 and H2 cells expressed similar receptor numbers on their surface, namely 125 000 and 106 000 receptors/cell, respectively (Fig. 2); this has to be compared with about 5000 hTNFR75 molecules on PC60hTNF-R55 cl 8/hTNF-R75 double-transfectants.26 This considerable difference in efficiency of expression is remarkable as both constructs involve the same promoter; possibly, the cysteine-rich repeats in the extracellular domain of hTNF-R75 hamper translation.

Structure-function analysis of TNF indicates that this trimer acts by clustering three receptor molecules.13 Biophysical methods established a 1:3 stoichiometry for TNF binding to the TNF-R55 and TNF-R75 extracellular domains.14,16,40 Trimerization was confirmed by the X-ray structure data of the complex between lymphotoxin and soluble TNF-R55.17 The data here reported indicate that EPO-mediated TNF-R75 intracellular domain dimerization was sufficient to transduce a signal within the cell. When in addition hTNF-R55 was triggered with the hTNF-R55-specific agonist R32WS86T, maximal GM-CSF production and induction of apoptosis was observed upon EPO stimulation of H2 cells (Table 1). The fact that H1 cells did not respond in this way, indicates that the hTNF-R75 intracellular domain only functions efficiently when linked to its natural transmembrane domain. This observation is reminiscent of a report describing that the transmembrane region of the human low-affinity nerve growth factor receptor contains functional information for signalling.41 Since both TNF-R75 and NGFR75 belong to the same receptor superfamily, it is tempting to believe this might be a common feature for the members of this family. In addition, it has been demonstrated that transmembrane interactions can mediate oligomerization of the T-cell receptor complex42 and of glycophorin A.43 At present, the exact role of the hTNF-R75 transmembrane region in EPO-R/ TNF-R75-mediated signalling is unclear. Perhaps it is

706 / Declercq et al.

necessary for appropriate clustering of the intracellular domains, or a molecule involved in signal transduction may be associated with this part of the receptor. The transmembrane region of EPO-R can be replaced without implications for EPO binding or EPO-transducing activity, showing that EPO-mediated signalling occurs irrespective of the EPO-R transmembrane region.31 Introduction of either H1 or H2 in PC60hTNF-R55 cells rendered these cells sensitive to TNF-R55-mediated apoptosis (Table 2). This finding confirms our previous results which showed that the presence of hTNF-R75 (even without triggering) allowed R32WS86T-dependent induction of apoptosis in PC60 transfectants.26 The results reported here provide further evidence for the existence of a cooperation between hTNF-R55 and the untriggered, intracellular domain of TNF-R75. Remarkably for this process, the transmembrane part of hTNF-R75 was not required (Table 2). At present, the mechanism of this cooperation is not known; one possibility is that TNF-R75-associated molecules positively affect TNF-R55 signal transduction. Recently, two TNF-R75-associated molecules involved in receptor signalling, TRAF1 and TRAF2, were described and their cDNAs isolated.39 Together with a novel serine/threonine protein kinase that associates with the cytoplasmic domain of the TNFR75 and phosphorylates both TNF-R types,44 TRAF1 and TRAF2 are potential candidates for modulating TNF-R55 function. Alternatively, a spontaneous, but low-level aggregation of the abundant hTNF-R75 may cause continuous signalling which positively affects hTNF-R55 functions. Dimerization of H2-R in the presence of R32WS86T stimulated GM-CSF production and apoptosis to the same extent as the combined addition of R32WS86T and D143F in PC60 cells expressing both hTNF-Rs. Nevertheless, dimeric stimulation with EPO alone was less efficient than trimeric activation with D143F. Treatment of H2 transfectants with EPO only induced modest amounts of GM-CSF and was not able to elicit apoptosis. These data may indicate a qualitative or quantitative difference between the intracellular signal transduced by receptor dimerization and the signal derived from hTNF-R75 trimerization. Furthermore, the number of receptors was considerably different: H2 cells expressed about 100 000 EPO-R/TNF-R75 per cell, while PC60hTNFR55 cl 8/hTNF-R75 cells had only about 5000 hTNFR75 per cell. However, clustering of the latter by D143F was sufficient to induce apoptosis.26 Although the signal generated by dimerization of the TNF-R75 intracellular domain was not sufficient to trigger apoptosis or efficient in GM-CSF induction, it was able to amplify the TNF-R55 signal. Either the single EPO-R/TNFR75 H2 dimer signal is lacking an accessory

CYTOKINE, Vol. 7, No. 7 (October 1995: 701–709)

function/interaction in comparison with TNF-R75 trimerized receptors, or it is simply too weak for a strong biological response. For example, a trimeric intracellular domain may provide specific docking for an intermediary factor, while a dimer may allow only a weak interaction. In summary, we show here that dimerization of chimeric EPO-R/TNF-R75, consisting of the extracellular domain of mEPO-R and the cytoplasmic region of hTNF-R75, was functional in PC60 cells. More specifically, dimerized EPO-R/TNF-R75 H2 were capable of synergizing with hTNF-R55-induced cytokine production and apoptosis. However, dimeric triggering of these chimeric receptors with EPO alone was less efficient, despite a 20-fold higher receptor number, than trimerization of hTNF-R75 by the specific agonist D143F. The chimeric receptors only responded to EPO in H2 cells, i.e. when the matching transmembrane region of TNF-R75 was present. Our results also prove that the hTNF-R75 extracellular part per se is not required for signalling. Furthermore, the expression of chimeric EPO-R/TNF-R75, regardless the presence of the hTNF-R75 transmembrane region, facilitated TNF-R55-dependent signal transduction leading to apoptosis. This means that the introduction of the intracellular domain of hTNF-R75, even without triggering, was sufficient to contribute to hTNF-R55-mediated activities in PC60 cells.

MATERIALS AND METHODS Cytokines, cytokine assays and antibodies Purified, E. coli-derived hTNF-R55-specific mutein R32WS86T and D143F, a hTNF-R75-specific hTNF mutein,21,45 were prepared in our laboratory. R32WS86T retained about 50% of the hTNF-R55-mediated cytotoxic activity on Hep2 or on KYM 39A6 cells, and did not show any hTNF-R75-mediated GM-CSF induction on hTNF-R75transfected PC60 cells. The mutein D143F retained about 10% of its hTNF-R75 bioactivity, but lost completely the ability to interact with hTNF-R55. At the concentrations used in our experiments, the hTNF muteins do not trigger mTNF-R.45 Recombinant human EPO was generously provided by CILAG (Schaffhausen, Switzerland). 125I-labelled human EPO with a specific radioactivity of 850 Ci/mmole was purchased from Amersham International (Amersham, UK). Rabbit antiserum against an NH2-terminal peptide of the mEPO-R was a kind gift of Dr H. F. Lodish.33 GM-CSF was quantified, relative to the murine GM-CSF reference preparation of the National Institute for Biological Standards (Potters Bar, UK), in an FDCpl proliferation assay.46

Cells The mouse/rat T-cell hybridoma PC60.21.14.4 (PC6047) was provided by Dr M. Nabholz (ISREC, Epalinges, Switzerland). PC60hTNF-R55 cl 8 cells were derived from

Signalling by chimeric TNF receptors / 707

transfection of PC60 cells with the hTNF-R55 gene as reported; PC60hTNF-R55 cl 8/hTNF-R75 cells were generated by transfection with the pSV25SHTNFR75 vector and are described elsewhere.26

Plasmid constructions We constructed two types of EPO-R/TNF-R75 hybrids: H1, in which the extracellular and transmembrane parts of mEPO-R were fused to the intracellular region of hTNF-R75; H2, in which the extracellular domain of mEPO-R was linked to the transmembrane and intracellular regions of hTNF-R75 (Fig. 1). For the H1-type mutant, the extracellular and transmembrane parts of EPO-R were generated using polymerasechain reaction (PCR) with the following primer pair: 59-ACCAAGCTTAAGCTAGGGCTGCATCATGG-39, containing an extra HindIII site and overlapping with ATG, and 59-CGCTCATGAGCAGGGCCAGAACCGTCAGC39, in which a BspHI site was introduced. The fragment was amplified and digested with HindIII and BspHI, and fused to the BspHI/SaII fragment derived from plasmid pSV25SHTNF-R7512 in a HindIII/SaII-opened pSV25S vector.48 The extracellular domain in the H2-type chimeric receptor was generated using the same forward primer as in the H1 construct and 59-GAGGATCCAGGTCGCTAGCGGTCAG-39, containing a BamHI site, as reverse primer. This HindIII/BamHI fragment was ligated to a BamHI/BstX1 TNF-R75 fragment, cut out from PCR-generated DNA (primer pair 59-CTGGATCCTTTCGCTCTTCCAGTTGGAC-39 and 59TCATCCAGCATCAGGCACTCC-39), and a BstX1 /SaII TNF-R75 fragment isolated from pSV25SHTNF-R75, into the HindIII/Sall-opened pSV25S vector. Vent polymerase (New England Biolabs, Beverly, MA) was used for all PCR reactions in a commercially supplied buffer. To confirm the sequence of these chimeric receptors, mEPOR/hTNF-R75 constructs were submitted to double-stranded DNA sequencing. The cDNA encoding mEPO-R was kindly provided by Dr H. F. Lodish.32

Transfections PC60hTNF-R55 cl 8 cells were transfected by electroporation. Briefly, exponentially growing cells were washed once with growth medium supplemented with fetal calf serum. 5 3 106 cells were resuspended in 800 µl medium. EcoRI-linearized plasmid containing the cDNA for the EPOR/TNF-R75 hybrid (10 µg) was added together with the selection plasmid pSV2Neo (1 µg).49 The mixture was exposed to a single voltage pulse (1500 µF, 300 V) and immediately transferred to culture. Two days later G418 (Gibco Bio-Cult) was added at a final concentration of 1.5 mg/ml. Then cells were cloned by limiting dilution and analysed for receptor expression.

Scatchard analysis 3 3 105 cells were incubated for 10 h at 4°C with varying concentrations of 125I-EPO ranging from 20 to 2000 pM in 200 µl culture medium as described.29 To measure cellassociated radioactivity, the cells were sedimented through a phthalate oil cushion, and the cell pellets were counted in a γ-counter. Specific binding was calculated as the difference between the cell-associated counts in the absence and presence of a 100-fold excess of unlabelled ligand. Aspecific binding was less than 0.1% of the total binding.

Apoptosis assay The number of apoptotic cells in a given culture was measured by a propidium iodide exclusion assay,50 which is a valid and quantitative reflection of the extent of apoptosis in PC60 cells.26 Propidium iodide was added to the cells at a final concentration of 30 µM and analysed on an EPICS 753 fluorometer (Coulter Electronics, Luton, UK). The propidium iodide dye was excited at 488 nm and the fluorescence was measured with a 610 nm long-pass filter.

Acknowledgements Receptor labelling and immunoprecipitation PC60 cells expressing hybrid EPO-R/TNF-R75 were washed once with phosphate-free RPMI 1640 medium (Gibco Bio-Cult, Paisley, UK) supplemented with 1% fetal calf serum (dialysed against 0.9% saline) and followed by a 30 min starvation period at 37°C. Cells were resuspended in 1 ml of fresh medium containing 200 µCi 32pi and incubated for 3 h at 37°C. Then cells were washed twice with ice-cold phosphatebuffered saline and lysed in 1 ml of lysis buffer (20 mM TrisHCl pH 7.3, 150 mM NaCl, 0.5% Nonidet P40, 0.5% Na deoxycholate, 1 mM NH41 vanadate, 1 mM EDTA) supplemented with the protease inhibitors PMSF (100 µg/ml) and aprotinin (0.27 trypsin-inhibitory units/ml), and left for 15 min on ice. After centrifugation at 14 000 3 g for 10 min, antibodies were added to the supernatant (1/500 anti-EPO-R final concentration), and the mixture was left rotating for 1.5 h at 4°C, followed by incubation with protein A-Sepharose for another 1.5 h. Proteins were eluted with Laemmli gel-loading buffer containing 2-mercaptoethanol and loaded on a 10% SDS-polyacrylamide gel.

The authors are grateful to CILAG (Schaffhausen, Switzerland) for their gift of recombinant human EPO, to Dr H. F. Lodish (Whitehead Institute for Biomedical Research, Cambridge, MA) for providing mEPO-R cDNA and anti-EPO-R antibodies, as well as to Dr M. Nabholz (ISREC, Epalinges, Switzerland) for making available the T-cell hybridoma PC60.21.14.4. They also thank W. Burm and D. Ginneberge for technical assistance, F. Molemans for DNA sequencing and A. Raeymaekers for recombinant cytokine purification. P. V. is a postdoctoral research assistant with the NFWO. Research was supported by the IUAP, the FGWO and an EC Biotech Programme on ‘In vitro immunotoxicology’.

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