The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine
2001; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês
10.1093/emboj/20.7.1692
ISSN1460-2075
Autores Tópico(s)Immune Cell Function and Interaction
ResumoArticle2 April 2001free access The ciliary neurotrophic factor receptor α component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine Hélène Plun-Favreau Hélène Plun-Favreau INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Greg Elson Greg Elson Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III, 74164 Saint Julien-en-Genevois, France Search for more papers by this author Marie Chabbert Marie Chabbert INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Josy Froger Josy Froger INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Odile deLapeyrière Odile deLapeyrière INSERM U382, IBDM (CNRS-INSERM-Univ.Mediterranée), Campus de Luminy, case postale 907, 13288 Marseille, France Search for more papers by this author Eric Lelièvre Eric Lelièvre INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Catherine Guillet Catherine Guillet INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Jacques Hermann Jacques Hermann INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Jean-François Gauchat Jean-François Gauchat Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III, 74164 Saint Julien-en-Genevois, France Search for more papers by this author Hugues Gascan Corresponding Author Hugues Gascan INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Sylvie Chevalier Sylvie Chevalier INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Hélène Plun-Favreau Hélène Plun-Favreau INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Greg Elson Greg Elson Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III, 74164 Saint Julien-en-Genevois, France Search for more papers by this author Marie Chabbert Marie Chabbert INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Josy Froger Josy Froger INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Odile deLapeyrière Odile deLapeyrière INSERM U382, IBDM (CNRS-INSERM-Univ.Mediterranée), Campus de Luminy, case postale 907, 13288 Marseille, France Search for more papers by this author Eric Lelièvre Eric Lelièvre INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Catherine Guillet Catherine Guillet INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Jacques Hermann Jacques Hermann INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Jean-François Gauchat Jean-François Gauchat Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III, 74164 Saint Julien-en-Genevois, France Search for more papers by this author Hugues Gascan Corresponding Author Hugues Gascan INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Sylvie Chevalier Sylvie Chevalier INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France Search for more papers by this author Author Information Hélène Plun-Favreau1, Greg Elson2, Marie Chabbert1, Josy Froger1, Odile deLapeyrière3, Eric Lelièvre1, Catherine Guillet1, Jacques Hermann1, Jean-François Gauchat2, Hugues Gascan 1 and Sylvie Chevalier1 1INSERM EMI 9928, CHU d'Angers, 4 Rue Larrey, 49033 Angers, Cedex, France 2Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III, 74164 Saint Julien-en-Genevois, France 3INSERM U382, IBDM (CNRS-INSERM-Univ.Mediterranée), Campus de Luminy, case postale 907, 13288 Marseille, France ‡H.Plun-Favreau and G.Elson contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1692-1703https://doi.org/10.1093/emboj/20.7.1692 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor α component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130–LIFR complex on their surface. Correspondingly, CLC–CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC. Introduction Ciliary neurotrophic factor (CNTF) uses a multimeric receptor composed of the gp130 signal-transducing protein associated with leukaemia inhibitory factor receptor (LIFR) and a specific binding subunit, the CNTF binding receptor α component (CNTFR), which is anchored to the membrane through a glycophosphatidylinositol linkage (Hibi et al., 1990; Davis et al., 1991, 1993a,b; Gearing et al., 1991; Taga and Kishimoto, 1997). Binding of CNTF to CNTFR leads to gp130–LIFR dimerization and downstream signalling events involving, among others, the recruitment of the JAK1–STAT3 signalling pathway (Lütticken et al., 1994; Stahl et al., 1994, 1995; Heinrich et al., 1998). Gp130 and LIFR are also present in the receptor complexes for the cytokines LIF (Gearing et al., 1992), cardiotrophin-1 (CT-1) (Pennica et al., 1995), oncostatin M (OSM) (Gearing et al., 1992) and cardiotrophin-like cytokine (CLC) (Senaldi et al., 1999; Shi et al., 1999). CNTF promotes the differentiation and survival of a wide range of cell types in the nervous system. In particular, CNTF maintains motor neuron viability in vitro, prevents the degeneration of axotomized motor neurons and attenuates motor deficits in different strains of mice with neuromuscular deficiencies (Lin et al., 1989; Stöckli et al., 1989; Oppenheim et al., 1991; Sendtner et al., 1992; Curtis et al., 1993; Mitsumoto et al., 1994). Besides its activities in the nervous system, CNTF has trophic effects on denervated skeletal muscle and is a regulator of muscular strength in aging (Helgren et al., 1994; Guillet et al., 1999). Only a mild loss of motor neurons leading to minor muscle weakness is observed in adult CNTF−/− mice (Masu et al., 1993). Moreover, a null mutation in the human CNTF gene does not lead to neurological disease (Takahashi et al., 1994). In contrast, the CNTFR subunit is essential for survival of nearly half of all motor neurons during development, and mice harbouring a homozygous CNTFR−/− mutation die shortly after birth (DeChiara et al., 1995). These dramatic viability differences suggest that CNTFR plays a critical role during development, at a period when CNTF is almost absent, by serving as a receptor for a second, developmentally important ligand. CLC is a new member of the CNTF/LIF family of cytokines isolated by expressed sequence tag database screening, and is also referred to as novel neurotrophin-1/B-cell stimulating factor-3 (Senaldi et al., 1999; Shi et al., 1999). CLC expressed in Escherichia coli has been shown to recruit the gp130–LIFR pathway in a neuroblastoma cell line and is able to activate NF-κB and SRE reporter constructs in myeloid cells. It also supports the survival of chicken embryonic motor and sympathetic neurons. We recently demonstrated that CLC associates with the soluble receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine. CLF expression is required for CLC secretion, and the CLC–CLF heterocomplex displays activities only on those cells expressing the functional CNTF receptor (Elson et al., 1998, 2000). In the present study, we show that the cellular release of functional CLC can also be mediated by CNTFR to generate a composite cytokine displaying functional activities on cells expressing the gp130 and LIFR components on their surface. Results Modelling of CLC A CLC model was built based on the alignment of its sequence with those of human CNTF and murine LIF, whose crystallographic structures have been resolved (Robinson et al., 1994; McDonald et al., 1995). The alignment was obtained from multiple alignment of the interleukin (IL)-6 cytokine family using Clustal_W with only minor manual adjustments (Thompson et al., 1994) (Figure 1). The identity score between CLC and CNTF was low (15%), with only 27 residues conserved in the modelled structure (residues 7–181) (Figures 1 and 2A). However, helix D was highly conserved between CLC and CNTF, with a 14-residue-long segment (residues 160–174) displaying 50% identity. These conserved residues in CNTF are also maintained among different species (Saggio et al., 1995). Figure 1.Alignment of human CLC (CLCh) with human CNTF (CNTFh) and murine LIF (LIFm). Conserved residues are in bold. The residues of CNTFh and LIFm corresponding to helices A–D are in red. The residues of CNTF whose coordinates are not resolved in the crystal structure are in blue. The sequences of the leader peptides have been removed. Download figure Download PowerPoint Figure 2.Overall structure of CLC. (A) Ribbon drawing of modelled CLC with the side chains of residues that are conserved in CLC and CNTF. Conserved residues corresponding to the signature of LIFR binding (Tyr34, Gly39, Phe151 and Lys154) are shown in orange. Conserved leucines are shown in green. Other conserved residues are shown in red. (B) The solvent-accessible surface of CLC. The surfaces of the conserved residues located in helix D are coloured red and that of Thr166 is coloured cyan. The two views correspond to the same protein orientation. Download figure Download PowerPoint The 27 conserved residues were not randomly located within the tri-dimensional protein structure (Figure 2A). Ten of these residues were leucines, principally located within the hydrophobic core of the protein. The four residues (Tyr34, Gly39, Phe151 and Lys154) that correspond to the 'signature' of LIF receptor binding, and that are conserved in CLC, LIF, CNTF, CT-1 and OSM (Grötzinger et al., 1999; Bravo and Heath, 2000), were spatially close in the tri-dimensional model of CLC. The residues equivalent to Phe151 and Lys154 in LIF (Phe156 and Lys159) and in CNTF (Phe152 and Lys155) correspond to the 'hot spot' residues of the site III interaction with LIFR (Inoue et al., 1995; Di Marco et al., 1996; Hudson et al., 1996). A cluster of conserved residues was located at or near site I corresponding to the interaction site of the cytokine with its specific receptor α-chain. For CNTF, site I of CNTFR binding corresponds to the C-terminal part of helix D and the C-terminus of the AB loop (Panayotatos et al., 1995). Conserved Glu163, Trp167, Arg170 and Asp174 made a continuous stripe on the surface of helix D (Figure 2B). This stripe was partly shielded from solvent by the AB loop. Alternative loop conformations obtained by simulated annealing did not lead to significantly increased solvent exposure of the conserved residue cluster of helix D (data not shown). Receptor binding might, however, dramatically alter the positioning of the loop. Such a conformational change has been observed for human growth hormone, which belongs to the same class of four-helix-bundle cytokines (Bazan, 1990, 1991). In an unbound state, the AB loop lies on the surface of the D helix, whereas in complex with the receptor, a structural reorganization of the AB loop with an 8 Å shift in its positioning allows the direct interaction of helix D side chains with the receptor (De Vos et al., 1992). The CNTF residue equivalent to Thr166 of CLC (Gln167) has been shown to be important for the binding of CNTF to CNTFR. When this residue was mutated to Thr, the affinity of CNTF for CNTFR was increased 10-fold (Saggio et al., 1995). This residue is located on the surface of helix D, close to the conserved cluster. The CNTF residues equivalent to Arg170 and Asp174 (Arg171 and Asp175) were crucial for the correct binding to the CNTFR chain (Inoue et al., 1997). These residues are involved in electrostatic interactions with each other, and with Glu75 and Arg72 on helix B. These electrostatic interactions might be essential for the proper positioning of the AB loop, receptor recognition, protein folding and/or structural stability of human CNTF (McDonald et al., 1995; Inoue et al., 1997). The CLC model indicates that Arg170 and Asp174 may be involved in electrostatic interactions with Asp72 and Arg168, respectively. This striking similarity between CLC and CNTF in a region corresponding to the CNTFR binding site of CNTF (site I) prompted us to investigate the possibility that the interaction of CLC with the soluble CNTFR might allow the secretion of CLC. CLC associates with CNTFR to form a secreted composite cytokine CLC cDNA was tagged with a protein C epitope coding sequence, subcloned in a mammalian expression vector and expressed in Cos-7 cells (Figure 3). Whereas the protein was readily detectable in cell lysates, secretion of the cytokine into the culture medium was not observed, in accordance with previous observations (Elson et al., 2000). Since CLC contains a putative contact site for CNTFR, the cytokine was co-expressed together with a soluble truncated form of CNTFR (sCNTFR, leading to a mature polypeptide of 316 amino acids). CLC was found to be present in the culture medium as detected by western blotting, indicating that CLC secretion can be mediated by CNTFR (Figure 3A). Interestingly, similar results were observed when co-expressing CLC together with CNTFR anchored to the membrane through a GPI linkage (Davis et al., 1991, 1993b), indicating that post-transcriptional or enzymatic processes led to the release of a soluble form of the receptor associated to CLC. In this latter case, the apparent molecular weight of the receptor portion was in the range of 75 kDa, in agreement with that described previously (Davis et al., 1993b). Similarly, CLC was secreted outside the cells when co-expressed with CLF, as we reported previously (Elson et al., 2000). In contrast, co-expression of a soluble form of IL-6 receptor (IL-6R) with CLC failed to induce secretion of the cytokine, underlining the specificity of the interaction between CLC and either CLF or CNTFR. Figure 3.CNTFR and CLC interact to form a secreted complex. (A) Cos-7 cells were transfected with the indicated cDNAs. (a) Expression of CLC-protC in transfected Cos-7 cell lysates detected by western blot analysis with a peroxidase-coupled anti-protC mAb. (b) Expression of CNTFR in transfected Cos-7 cell lysates detected with the biotinylated AN-E4 anti-CNTFR mAb. (c) Expression of CNTFR in transfected Cos-7 cell culture medium. Culture media were immunoprecipitated with the AN-C2 anti-CNTFR mAb (10 μg/ml). Western blot analysis of the immunoprecipitates was performed with the biotinylated AN-E4 anti-CNTFR mAb. (d) Expression of CLC-protC in transfected Cos-7 cell culture media. Culture media were immunoprecipitated with an anti-myc mAb. Western blot analysis of the immunoprecipitates was performed with the peroxidase-coupled anti-protC mAb. (e) CLC-protC is present in the culture medium as a complex with CNTFR. Culture media were immunoprecipitated with the AN-C2 anti-CNTFR mAb (10 μg/ml). Western blot analysis of the immunoprecipitates was performed with the peroxidase-coupled anti-protC mAb. (B) Physical interaction between site I CLC mutants and CLF or soluble CNTFR. Cos-7 cells were transfected with cDNAs encoding the indicated CLC mutants, wild-type CLC (positive control) or β-galactosidase (negative control), together with cDNAs encoding either CLF or sCNTFR. After 72 h, supernatants were harvested and immunoprecipitated with an anti-CLF mAb or with an anti-CNTFR mAb. Western blotting was performed with an anti-protein C mAb recognizing the wild-type as well as the mutated forms of CLC. The two differentially glycosylated forms of CLC are indicated with arrows. Download figure Download PowerPoint To test whether CLC and CNTFR were released as a composite cytokine, cell supernatants from Cos-7 transfected cells were subjected to immunoprecipitation with an anti-CNTFR antibody. The presence of CLC in the immunopurified fraction was revealed by western blotting using an anti-protein C antibody (Figure 3A, e). CNTFR and CLC specifically associated and could be co-precipitated, thus forming a secreted heterodimer. Similar results were also observed when the same experiments were performed in the human embryonic kidney (HEK) 293 fibroblast cell line (data not shown). The relative amount of sCNTFR engaged in the formation of the composite cytokine was determined by high-performance liquid chromatography (HPLC) gel filtration analysis of culture supernatants containing CLC–sCNTFR (see Figure 4B). Western blot analysis of the eluted fractions showed that ∼30% of soluble CNTFR was associated with CLC (an estimate of the composite cytokine concentration present in transfected fibroblast cell culture supernatants was 1–5 nM), whereas the majority of secreted CLC was retained in a complex with sCNTFR (lane 7). Figure 4.Stoichiometry and composition of CLC–sCNTFR cytokine. (A) Upper panel: Cos-7 cells were co-transfected with CLC-protC, CLC-myc and either sCNTFR (+CNTFR) or an empty control vector (−CNTFR). After 72 h, supernatants were immunoprecipitated with an isotype control mAb, an anti-myc mAb, an anti-protC mAb or an anti-CNTFR mAb. Western blotting analysis was performed with a biotinylated anti-protC mAb. Lower panel: Cos-7 cells were co-transfected with CNTFR-HA, CNTFR-Flag and either CLC-protC (+CLCprotC) or with an empty control vector (−CLCprotC). Supernatants were immunoprecipitated with an isotype control mAb, an anti-HA mAb, an anti-Flag mAb or an anti-protC mAb. Western blotting was performed with a peroxidase-coupled anti-HA mAb. (B) The culture supernatant of an HEK 293 cell line stably transfected with CLC–sCNTFR was size-fractionated on a Superose 12 column. Fractions were analysed by western blotting using an anti-protC mAb to detect CLC, or the AN-E4 antibody to detect sCNTFR. Column calibration was performed using standard purified proteins. (C) SDS–PAGE analysis of purified CLC–sCNTFR. CLC–sCNTFR was purified with an anti-protC column followed by QAE HPLC. Gels were silver stained and protein concentration determined using known concentrations of BSA. Western blotting analysis of a companion lane was performed using both anti-protC and anti-CNTFR mAbs for visualization. Download figure Download PowerPoint CLC contacts CNTFR through a site I binding Mutants of CLC were engineered to determine the binding site(s) to CNTFR. Mutations were designed to impair the putative site I of interaction with the specific receptor α-chain(s). Positions were selected based on analogy with CNTF. Two residues located at the C-terminal part of the CNTF AB loop (positions 63 and 64), equivalent to CLC Ala60 and Thr61, are known to be essential for CNTFR binding. Mutation of Gln63 of CNTF to Lys or Glu impairs CNTFR binding (Panayotatos et al., 1995), whereas mutation of the same residue to Arg creates a protein with increased activity (Panayotatos et al., 1995). Mutation of the neighbouring Trp64 to Ala also impairs CNTFR binding (Panayotatos et al., 1995). Ala60 of CLC was thus mutated to Arg, Lys and Glu, or Thr61 to Ala. Arg171 and Asp175 of CNTF, located at the C-terminal half of helix D, are also crucial for the correct binding to the CNTFR α-chain (Panayotatos et al., 1995; Inoue et al., 1997). The corresponding residues, conserved in CLC (Arg170 and Asp174), were mutated to Ala. Mutants were co-expressed in Cos-7 cells together with sCNTFR. Their ability to bind CLC and to permit the release of the CLC–sCNTFR composite cytokine was investigated (Figure 3B). Mutations introduced in the C-terminal part of helix D (positions 170 and 174) and in position 60 of the AB loop entirely abrogated the secretion of CLC when co-expressed with sCNTFR. In contrast to that observed for CNTF, the Thr61→Ala mutant conserved its capacity to bind sCNTFR and to be secreted. These results show that CLC can associate with CNTFR through a site I binding to generate a secreted composite cytokine. Co-expression of the same set of CLC mutants with its alternate subunit, CLF (Elson et al., 2000), affected neither the CLF–CLC contact nor the secretion of the heterodimer (Figure 3B). This clearly demonstrates that CLF and sCNTFR interact with two different regions on CLC. One is identified as site I for CLC interaction with sCNTFR and the second, which remains to be identified, is required to generate the CLC–CLF composite cytokine. Interestingly, we consistently observed a preferential association of sCNTFR to a higher molecular weight form of CLC (25 kDa), whereas CLF could indifferently bind both 22 or 25 kDa forms of the molecule (Figure 3A and B). A possible explanation would be differences in the glycosylation states of CLC and in the receptor accessibility to the different glycosylated forms. Stoichiometry of the CLC–sCNTFR cytokine We next examined the stoichiometry and composition of the heterodimeric cytokine. In order to facilitate the detection of putative multimeric associations, CLC was tagged with either the myc or protein C (protC) epitope. Modified sCNTFR containing a Flag or haemagglutinin (HA) motif at the C-terminus were also generated. cDNAs encoding for CLC-myc and CLC-protC were simultaneously co-transfected in Cos-7 cells together with an expression vector encoding untagged sCNTFR. The culture medium was immunoprecipitated with antibodies directed against the CLC tagged motifs or CNTFR (Figure 4A). Western blotting experiments using an anti-protC monoclonal antibody (mAb) revealed that CLC-myc could be released in a dimeric form associated to CLC-protC. Additionally, the release of CLC tagged proteins depended on their co-expression with CNTFR. Similar experiments were carried out using sCNTFR labelled with Flag or HA epitopes. In contrast to that observed for CLC, secretion of the soluble receptor also occurred in the absence of CLC. Interestingly, when expressed alone, a proportion of sCNTFR spontaneously formed a stable dimer (Figure 4A and B). This was unlikely to be due to aggregate formation since protein determination by western blotting or ELISA detection (data not shown) indicated an sCNTFR concentration in the range 1–5 nM. In order to determine the molecular weight of the CLC–sCNTFR complex, the culture supernatant of the 293 cell line stably transfected with CLC–sCNTFR was size fractionated on a Superose 12 column. Retarded fractions were studied by western blotting using mAbs directed against CNTFR or the protC epitope (Figure 4B). CLC eluted from the size-exclusion column peaked in fraction 7, corresponding to an apparent mol. wt of 120–150 kDa. With the exception of fractions surrounding fraction 7, no other significant signal could be detected in the gel filtration eluate. This indicated that fraction 7 contained the vast majority of CLC. Soluble CNTFR was recovered in fractions ranging from 50 to 150 kDa, indicating that only ∼30% of the soluble receptor was engaged in composite cytokine formation. Size-exclusion chromatography and immunoprecipitation experiments using tagged proteins (see above) support a model where at least a portion of the CLC–sCNTFR complex consists of two molecules of both CLC (mol. wt 22–25 kDa) and sCNTFR (mol. wt 50 kDa), leading to an apparent mol. wt of 140–150 kDa. The CLC–sCNTFR complex was purified to apparent homogeneity with an anti-protein C affinity column followed by a QAE HPLC step. SDS–PAGE analysis revealed two prominent bands with apparent mol. wts of 25 and 50 kDa, respectively, identified as CLC and sCNTFR by western blotting using anti-protC and anti-CNTFR mAbs (Figure 4C). CNTFR is required for biological responses to CLC Functional properties of CLC and CLC–sCNTFR were tested in proliferation assays using derivatives of the IL-3-dependent Ba/F3 cell line (Kallen et al., 1999). Purified CLC from cell lysates or purified CLC–sCNTFR was added to cultures of Ba/F3 cell lines engineered to express the appropriate combinations of receptor subunits leading to functional IL-6-type cytokine responses (Figure 5A). The purified CLC–sCNTFR complex induced a robust proliferation of Ba/F3 cells expressing on their surface either the functional LIF receptor (gp130 and LIFR; hereafter called Ba/F3 GL) or the tripartite CNTF receptor (gp130, LIFR and CNTFR; hereafter abbreviated to Ba/F3 GLC). The respective specific activities were 2 × 105 and 5 × 107 U/mg of protein. The observed difference in specific activities was possibly due to a partial dissociation of the composite cytokine during the biological assay, rendering CLC less efficient on Ba/F GL cells, but fully active on Ba/F3 GLC cells, with the membrane-expressed tripartite CNTF receptor. In line with this observation, we recently made a fusion protein consisting of sCNTFR covalently associated to CLC and displaying a specific activity of 3 × 107 U/mg (C.Guillet, manuscript in preparation). Importantly, CLC purified from Cos-7 cell lysates triggered the proliferation of Ba/F3 GLC cells, but not of Ba/F3 GL cells, demonstrating the absolute requirement of CNTFR for generating a functional response to CLC (Figure 5B). Figure 5.Proliferative response of transfected Ba/F3 cell lines to the CLC–sCNTFR complex. (A) Effect of CLC–sCNTFR on parental Ba/F3 cells and cells transfected with gp130, gp130–LIFR or gp130–LIFR–CNTFR. Cells were cultured in triplicate with 3-fold dilutions of appropriate positive controls, purified CLC–sCNTFR (filled squares) or IL-2 (open circles) used as an irrelevant cytokine. After a 72 h culture period, a [3H]Tdr pulse was performed and the amount of incorporated radioactivity was determined using a β-counter. Vertical bars indicate the SEM. (B) Effect of purified CLC from Cos-7 transfected cell lysates on Ba/F3 cells transfected with gp130–LIFR and gp130–LIFR–CNTFR. Cells were cultured in triplicate with 3-fold dilutions of the indicated cytokines. (C) The AN-HH1 anti-gp130 mAb prevents the proliferative response of the Ba/F3 gp130/LIFR/CNTFR cell line to the CLC–sCNTFR complex. Transfected Ba/F3 cells were incubated in triplicate in culture medium (marked as 0) containing 1 ng/ml CNTF, CLC–sCNTFR or IL-3. The AN-HH1 antibody (hatched bars) or a control IgG2a antibody (open bars) was added at a final concentration of 30 μg/ml. After a 72 h culture period, [3H]Tdr was added for 4 h, and the incorporated radioactivity determined. Download figure Download PowerPoint The involvement of gp130 in CLC–sCNTFR signalling was confirmed by the inhibition of proliferation following addition of a neutralizing anti-gp130 mAb to the Ba/F3 GLC cell culture (or to Ba/F3 GL cells) (Figure 5C). Furthermore, there was no detectable response to CLC–sCNTFR when Ba/F3 cells expressed gp130 alone, implicating LIFR in the functional receptor complex (Figure 5A). Membrane binding of CLC–sCNTFR and recruitment of gp130, LIFR and STAT3 signalling proteins We further determined the affinity of CLC–sCNTFR for its receptor complex. Since iodination of CLC–sCNTFR completely inactivated the biological activity of the composite cytokine, affinity values were obtained by competing for CNTF binding to its functional receptor. Radiolabelled CNTF bound to its tripartite receptor expressed on the SK-N-GP neuroblastoma cell line with an affinity of 50–60 pM (data not shown), which is in agreement with our previous studies (Robledo et al., 1996, 1997). In the displacement experiments, cells were incubated in the presence of 0.6 nM iodinated CNTF and increasing concentrations of putative unlabelled competitors to reach a 100- to 200-fold excess concentration (Figure 6A). Determination of the CLC–sCNTFR affinity constant(s) from the competition experiments gave a Kd of 300 or 150 pM when considering a dimeric or a tetrameric form of the composite cytokine, respectively. Figure 6.Competition of CLC–sCNTFR for the binding of radiolabelled CNTF to its receptor complex. (A) The SK-N-GP cell line was incubated in the presence of 0.6 nM iodinated CNTF and increasing concentrations of unlabelled CNTF (closed circles), CLC–sCNTFR (closed squares) or IL-2 (open squares). After a 2 h incubation period at 4°C, the bound radioactivity was separated by centrifugation through an oil layer. Tyrosine phosphorylation of gp130, LIFR and STAT3 occurred in SK-N-GP neuroblastoma (gp130+LIFR+CNTFR+) (B) and
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