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The Journal of Immunology, 2000, 164: 273-282.
Copyright © 2000 by The American Association of Immunologists

Importance of the Membrane-Proximal Extracellular Domains for Activation of the Signal Transducer Glycoprotein 1301

Ingo Kurth, Ursula Horsten2, Stefan Pflanz, Andreas Timmermann, Andrea Küster, Heike Dahmen, Ingrid Tacken, Peter C. Heinrich3 and Gerhard Müller-Newen

Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1–D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4–D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4–D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane-bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants {Delta}4 and, to a lesser extent, {Delta}6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant {Delta}5 does not respond. Because membrane-bound {Delta}5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overexpression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The coordination and regulation of immune responses is mainly mediated by cytokines that specifically bind to cell surface receptors. Cytokine receptors are usually classified due to structural similarities. Class I cytokine receptors are characterized by the presence of at least one cytokine binding module (CBM)4 that consists of two fibronectin-type III-like (FNIII) domains. The N-terminal domain contains a set of four conserved cysteine residues, and the C-terminal domain contains a WSXWS motif or a closely related sequence. Receptors belonging to this family are engaged by helical cytokines consisting of four tightly packed {alpha}-helices (1). The cytoplasmic parts of cytokine receptors do not contain a kinase domain, but constitutively associate with Janus tyrosine kinases (Jaks). Binding of the ligand leads to receptor dimerization and juxtaposition of the associated Jaks, which results in their activation by tyrosine phosphorylation. Subsequently, the activated Jaks phosphorylate tyrosine residues of the receptor. These phosphotyrosine residues are docking sites for latent transcription factors of the STAT family, which also become phosphorylated at the receptor. The phosphorylated STATs dimerize and translocate into the nucleus to regulate target gene expression (2).

Whereas the ectodomains of the receptors for growth hormone (GH), erythropoietin (Epo), or prolactin solely consist of a single CBM, other members of the class I cytokine receptor family, such as gp130, G-CSFR, or the leptin receptor show a more complex architecture, which in respect to their biological functions is not understood (3). The glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and cardiotrophin-1 (4). The extracellular part of gp130 contains an Ig-like domain (D1) followed by a single CBM (D2 and D3) and three FNIII domains (D4, D5, and D6) (5). Signal transduction is achieved by either homodimerization of gp130 in response to IL-6 (6) and IL-11 (7) or heterodimerization of gp130 with the LIFR in response to LIF (8), CNTF (9), OSM (8), or CT1 (10). Alternatively, OSM induces the heterodimerization of gp130 with the recently cloned OSMR (11). The cytokines IL-6, IL-11, and CNTF alone do not efficiently engage the signal transducing receptor chains. They first bind to their specific {alpha} receptors that can functionally be replaced by the respective soluble counterparts lacking the transmembrane and cytoplasmic regions (12). In addition to at least one CBM, all receptors involved in IL-6-type cytokine signaling contain an Ig-like domain located N-terminally of the most membrane-proximal CBM. Besides the Ig-like domain and CBM(s), those receptors triggering the cytoplasmic signal transduction cascade (gp130, LIFR, and OSMR) all contain three additional membrane-proximal FNIII domains whose function is still unknown. Mutagenesis studies using deletion mutants of gp130 revealed that the CBM of gp130 as well as the Ig-like domain are required for the interaction with IL-6/IL-6R (13, 14, 15), IL-11/IL-11R (16), and OSM (17). A more detailed analysis of the binding epitope revealed that the CBM of gp130 binds the ligand in a way similar to the GH/GHR interaction (15, 18). These findings were supported by the crystal structure of the gp130 CBM (19) as well as the solution structure of gp130-D3 (20).

Ligand-induced dimerization of cytokine receptors is widely accepted as a prerequisite for receptor activation (21, 22). In this respect, the functional role of the membrane-proximal domains of gp130 (D4–D6) is not understood. Because these three domains represent a common structural feature of the signal transducing IL-6-type cytokine receptors, D4–D6 of gp130 may be essential for receptor activation. The studies presented here using deletion mutants of gp130 reveal that the membrane-proximal domains play an essential role in the coupling of ligand binding and signal transduction. Our findings suggest a mechanism of receptor activation that differs from the mechanism of activation established for short cytokine receptors such as GHR, EpoR, or prolactin receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Enzymes, proteins, Abs, chemicals, and cell culture

Enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany), and protein A-Sepharose was obtained from Pharmacia (Freiburg, Germany). DMEM and antibiotics were obtained from Life Technologies (Eggenstein, Germany), and FCS from Seromed (Munich, Germany). Radiochemicals were purchased from Amersham International (Aylesbury, U.K.). Recombinant human IL-6 was expressed in Escherichia coli, refolded, and purified as described by Arcone et al. (23). Soluble IL-6R (sIL-6R) (24), soluble gp130 (25), as well as soluble IL-11R (16) were expressed in insect cells as described previously. The monoclonal gp130 Abs B-R3, B-S12-G7, B-P4, and B-P8 were generated as described previously (26). All other Abs were purchased from Dako (Hamburg, Germany). The PBS buffer contained 200 mM NaCl, 2.5 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4. Simian monkey kidney cells (COS-7) were cultured in DMEM, and Ba/F3 cells and Ba/F3 transfectants were cultured in DMEM containing 5% (v/v) conditioned medium from X63Ag8-653 BPV-mIL-3 myeloma cells (as a source of IL-3) in a water-saturated atmosphere containing 5% CO2. These media were supplemented with 10% (v/v) FCS, streptomycin (100 µg/ml), and penicillin (60 µg/ml). Ba/F3 transfectants were cultured in the presence of 1 mg/ml G418. HepG2 cells were cultivated in DMEM/F12.

Plasmid construction

The starting point for cloning of {Delta}4, {Delta}5, and {Delta}6 was the full-length human gp130 cDNA cloned into the XhoI and BamHI sites of the eukaryotic expression vector pSVL lacking the EcoRI site (gp130-pSVL{Delta}Eco). Using this vector as template, for each deletion mutant two fragments were amplified. The first fragment encodes the N-terminal region of gp130 down to the deletion. The sense primer (5'-GTGTT ACTTC TGCTC T-3') anneals upstream of the gp130 cDNA within the vector. The respective antisense primer introduces an XmaI site (underlined) that determines the deletion 5'-CTTGG TGCTT TCCCG GGTCT ATCTT CATAG G-3' ({Delta}4), 5'-TTTAA GATCC ATCCC GGGGT GAGTA G-3' ({Delta}5), or 5'-GTAGG TCCTT TCCCG GGTGG AGCTT GTTTA AGG-3' ({Delta}6). The second fragment encodes the C-terminal part of gp130 following the deleted domain. Again, the respective sense primer introduces an XmaI site (underlined) 5'-GCTAC TCACC CCGGG ATGGA TCTTA AAG-3' ({Delta}4), 5'-CCACC TTCCC CCGGG CCTAC TGTTC GG-3' ({Delta}5), or 5'-CCAAA GTTTG CTCCC GGGGA AATTG AAG-3' ({Delta}6). The antisense primer (5'-TCTAG TTGTG GTTTG T-3') anneals within the vector. The first fragment was digested with XhoI and XmaI; the second fragment was digested with XmaI and BstEII. Together, both fragments were cloned in the XhoI- and BstEII-digested gp130-pSVL{Delta}Eco, yielding the construct encoding the deletion mutant. To construct the D5-GCSFR chimera the corresponding domain of the G-CSFR was amplified by PCR and cloned into the newly introduced XmaI site of the vector encoding the deletion mutant {Delta}5. The human G-CSFR cDNA served as a template. The following oligonucleotides were used to amplify the DNA encoding domain 5 of G-CSFR (XmaI site underlined): 5'-GGCCC AGCTC TGACC CCCGG GCATG CCATG GCCCG-3' and 5'-TAGAT GCAGC TCTGG CCCGG GGGAG GGAGC CATTT C-3'. PCRs were performed applying standard procedures. All plasmids were sequenced using an ABI Prism Automated sequencer (Perkin-Elmer, Norwalk, CT). For expression in HepG2 cells, the cDNAs were subcloned into the expression vector pRcCMV.

To express the deletion constructs and chimera in the context of gp130{gamma}, respective cDNAs were subcloned into the vector pSVLEg-Y{gamma}440 (27) using the restriction endonucleases XhoI and BstEII. The resulting vector encodes the extracellular domain, the transmembrane, and a truncated intracellular part of gp130 (containing boxes 1 and 2) fused to the IFN-{gamma} receptor motif YDKPH, which predominantly activates STAT1. For construction of plasmids encoding the soluble gp130 proteins the deletion constructs were subcloned into a vector (pSVLsgp130-Flag) (18) encoding the extracellular domain of gp130 fused to a Flag epitope at the C-terminus.

Transfection of cells

Plasmid DNA was transfected into Ba/F3 cells by electroporation. Twenty-eight micrograms of the gp130 expression vector were coelectroporated with 2 µg of pSV2neo into 3.5 x 106 cells in 0.8 ml of medium applying a single 70-ms pulse at 200 V. Selection with G418 (3 mg/ml) was initiated 24 h after transfection. Selected Ba/F3 clones were screened for the presence of membrane-bound gp130 proteins by flow cytometry. COS-7 cells were transiently transfected using the DEAE-dextran method. The efficiency of transfection was analyzed by FACS. HepG2 cells were transfected by the calcium phosphate coprecipitation method as described previously (28).

Soluble radioactive binding assay

The binding assay was performed using IL-6 radiolabeled with 125I according to the procedure of Bolton and Hunter (29). Sixty nanograms of sgp130 in 500 µl of TNET buffer (20 mM Tris-HCl (pH 7.5), 140 mM NaCl, 5 mM Na2EDTA, 1% Triton X-100, 2 mM methionine, and 0.01% NaN3) was incubated with 0.5 µg of the respective Ab (B-P4 or B-R3). Five nanograms of 125I-labeled IL-6 and 100 ng of sIL-6R were added. After incubation for 3 h at room temperature, the complexes formed were immunoprecipitated by the addition of protein A-Sepharose. Coprecipitated 125I-labeled IL-6 was quantified using a gamma counter.

Immunofluorescence staining

COS-7 cells were transiently transfected with the respective expression vectors (13) encoding gp130 deletion mutants. Forty-eight hours after transfection, the cells were fixed with paraformaldehyde, incubated with the gp130 mAbs B-P4 and B-R3, respectively, and stained with a rhodamine-conjugated secondary Ab. Subsequently, the cells were mounted with Mowiol and analyzed by fluorescence microscopy.

Flow cytometry

Cells were collected, washed, and resuspended in cold PBS containing 5% FCS and 0.1% sodium azide. Subsequently, cells were incubated on ice with 4 µg/ml gp130 Abs B-S12-G7 or B-P8. Cells were washed with cold PBS/azide and incubated with PE-conjugated anti-mouse IgG Fab at a 1/50 dilution. Again, cells were washed with cold PBS/azide and then resuspended in 400 µl of PBS/azide followed by flow cytometric analysis using a FACScalibur (Becton Dickinson, Mountain View, CA).

Electrophoretic mobility shift assay

Cells were incubated at 37°C for 15 min or for the periods of time indicated in the figures in the presence of IL-6/sIL-6R or IL-11/sIL-11R or were left unstimulated. COS-7 cells were stimulated with 12.5 ng/ml IL-6 and 500 ng/ml sIL-6R. Ba/F3 cells were stimulated with 33 ng/ml IL-6 and 500 ng/ml sIL-6R or 150 ng/ml IL-11 and 700 ng/ml sIL-11R or µg/ml BS-12-G7 and µg/ml B-P8. Preparation of nuclear extracts and EMSAs were performed as previously described (30). A double-stranded sis-inducible element (SIE) oligonucleotide derived from the c-Fos promoter (m67SIE; 5'-GATCC GGGAG GGATT TACGG GGAAA TGCTG-3') was used as the 32P-labeled probe (31). The protein-DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol. Electrophoresis was performed using 0.25x TBE buffer at 72 V/cm.

Proliferation assay

Ba/F3 cells (20,000 cells/well) were plated on 96-well plates and stimulated with varying amounts of IL-6 in the presence of 500 ng/ml of sIL-6R or with Abs as indicated in the diagrams. After 72 h of incubation, viable and metabolically active cells were quantified using a colorimetric assay based on the Cell Proliferation Kit II (XTT; Roche Molecular Biochemicals).

Soluble ternary complex formation assay

Soluble gp130-Flag proteins from supernatants of transfected COS-7 cells were incubated with 1 µg/ml of flag-Ab and subsequently 1 mg/ml protein A-Sepharose was added. After an overnight incubation at 4°C, immunoprecipitates were washed twice with TNET (20 mM Tris-HCl (pH 7.5), 140 mM NaCl, 5 mM EDTA, and 1%Triton X-100) and incubated with 4, 10, or 30 nM IL-6 and 70 nM sIL-6R. After 16 h the complexes were washed twice with TNET, resuspended in Laemmli buffer, incubated at 95°C for 5 min and separated on a 12.5% SDS-polyacrylamide gel under reducing conditions followed by electroblotting.

Binding of 125I-labeled IL-6 to stably transfected Ba/F3 cells

Ba/F3 cells (5 x 106) stably transfected with gp130, {Delta}4, {Delta}5, or {Delta}6 were incubated in the presence of sIL-6R (200 nM) with varying concentrations of 125I-labeled IL-6 (1340 cpm/fmol) ranging from 0.15–10 nM. After incubation for 16 h at 4°C the cells were centrifuged through a mixture of dinonyl- and dibutylphthalate oil (1.020 g/ml). Cell-associated and free radioactivity were measured using a gamma counter. Specific binding was obtained by subtracting the radioactivity associated with untransfected Ba/F3 cells.

Immunoprecipitation of Jak1

Transfected COS-7 cells were stimulated for 15 min with 12.5 ng/ml IL-6 and 500 ng/ml sIL-6R as indicated in the figure. Cells were treated with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM sodium orthovanadate, and 1 mM NaF in 15% glycerol) in the presence of the protease inhibitors pepstatin, leupeptin, aprotinin, and PMSF for 30 min at 4°C. After centrifugation the supernatants were incubated with 4 µg/ml Jak1 Ab and 5 mg/ml protein A-Sepharose.

Reporter gene assay using transfected HepG2 cells

HepG2 cells were transiently transfected with expression vectors encoding wild-type gp130 or D5-GCSFR, an {alpha}2-macrogloblulin promotor luciferase gene reporter construct (28), and a ß-galactosidase control vector. Twenty-four hours after transfection cells were stimulated with IL-6 (10 ng/ml) or left unstimulated. After an additional 16 h, luciferase activity was measured using the luciferase kit from Promega (Madison, WI) and normalized to ß-galactosidase activity to correct for transfection efficiency.

Immunoblotting and enhanced chemiluminescence detection

Immunoprecipitated proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane by a semidry electroblotting procedure (32). Polyvinylidene difluoride membranes were blocked in a solution of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Nonidet-P40 containing 10% BSA and probed with Ab, followed by incubation with HRP-conjugated secondary Ab. Immunoreactive proteins were detected by chemiluminescence using the enhanced chemiluminescence kit (Amersham) following the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The IL-6 and IL-11 neutralizing gp130 mAb B-P4 maps to the membrane-proximal part of gp130

In previous studies several mAbs directed against the ectodomain of gp130 were characterized. Some of these Abs act agonistically, such as the combination of B-S12 and B-P8, while others, such as B-R3, were neutralizing (26). The mAb B-P4 has been described to specifically inhibit IL-11 (26) and, depending on the cells investigated, IL-6 responses (33). The antagonistic mAbs B-P4 and B-R3 were used as precipitating Abs in a ternary complex formation assay. Soluble gp130 (sgp130) lacking the transmembrane and cytoplasmic parts was incubated with B-P4 or B-R3. Subsequently, iodinated IL-6 was added in the absence or presence of soluble IL-6R (sIL-6R). Precipitation of sgp130 bound to B-P4 via protein A-Sepharose in the presence of sIL-6R leads to coprecipitation of IL-6, whereas B-R3 fails to coprecipitate significant amounts of IL-6 (Fig. 1GoA) indicating that, in contrast to B-R3, binding of B-P4 to gp130 does not interfere with ligand binding. To confine the epitopes of B-P4 and B-R3, COS-7 cells were transfected with deletion constructs encoding either the membrane-distal or the membrane-proximal half of the ectodomain fused to the transmembrane and cytoplasmic regions of human gp130. Staining of the cells by immunofluorescence revealed that B-P4 maps to the membrane-proximal domains (D4–D6), whereas B-R3 recognizes the membrane-distal part of gp130 (Fig. 1GoB). Because B-P4 interferes with activation of gp130, but not with ligand binding, and maps to the membrane-proximal domains, we conclude that these domains are involved in activation of the signal transducer.



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  		FIGURE 1. The antagonistic mAb B-P4 does not interfere with ligand binding and maps to the membrane-proximal half of gp130. A, Soluble gp130 was first incubated with the mAbs B-P4 or B-R3 and subsequently with 125I-labeled IL-6 and sIL-6R as indicated. Complexes formed (see scheme) were immunoprecipitated, and coprecipitated radioactivity was measured. B, COS-7 cells were transfected with expression vectors encoding gp130 deletion constructs as indicated schematically on the left. Forty-eight hours after transfection, cells were fixed and incubated with mAb B-P4 or B-R3. Bound Abs were visualized by fluorescence microscopy using a rhodamine-conjugated secondary Ab, and stained cells were photographed at x100 magnification.

 
The membrane-proximal domains are not required for soluble ternary complex formation, but contribute to binding of IL-6/sIL-6R complexes to membrane-bound gp130

To further assess their functional role in ligand binding and receptor activation, the domains D4, D5, and D6 of human gp130 were deleted individually, applying PCR-based methodology. Domain borders were chosen according to the suggestions of Hibi et al. (5) (schematic representation of deletion mutants in Fig. 2Go). To perform ternary complex formation assays using soluble receptor proteins, the transmembrane and cytoplasmic parts of wild-type gp130 and of the deletion mutants were replaced by a flag epitope (sgp130-flag). From supernatants of transfected COS-7 cells the soluble recombinant receptor proteins were collected by immunoprecipitation using a flag-Ab. After addition of sIL-6R in combination with varying amounts of IL-6, ternary complexes formed were precipitated via the Sepharose-bound sgp130-flag. The complexes were separated by SDS-PAGE followed by immunoblotting using IL-6 and flag Abs. The different electrophoretic mobilities of sgp130-flag and the deletion mutants (Fig. 3GoA, upper panel) roughly mirror the loss of molecular mass due to deletion of the individual domains and covalently linked N-glycans. Deletion of domains 4 (101 aa) and 6 (96 aa) is accompanied by a loss of three and two potential N-glycosylation sites, respectively, whereas domain 5 (95 aa) lacks potential N-glycosylation sites. The s{Delta}6-flag appears as a broader band upon SDS-PAGE. This microheterogeneity is probably due to uneven glycosylation. In the presence of IL-6/sIL-6R complexes, coprecipitation of IL-6 is achieved with all the deletion constructs as detected by immunoblotting (Fig. 3GoA, lower panel). The IL-6 coprecipitation is indicative of ternary complex formation, because incubation of wild-type sgp130 with IL-6 in the absence of sIL-6R as well as precipitation using supernatants from untransfected cells does not lead to coprecipitation of IL-6 (control lanes). The amount of coprecipitated IL-6 does not substantially differ between sgp130-flag and the deletion constructs. Even at low IL-6 concentrations no significant difference in ternary complex formation is observed, indicating that the soluble gp130 deletion mutants bind the ligand with comparable affinities. Thus, the ligand binding capability of soluble gp130 is not affected by deletion of individual membrane-proximal domains.



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  		FIGURE 2. Schematic representation of gp130 and the deletion mutants {Delta}4, {Delta}5, and {Delta}6. The domains of the gp130 extracellular part (D1–D6) are depicted followed by the transmembrane (black bar) and cytoplasmic part. Sites of deletions of individual domains in the mutants {Delta}4, {Delta}5, and {Delta}6 are marked by arrowheads.

 


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  		FIGURE 3. Binding of soluble and membrane-bound wild-type gp130 and deletion mutants to IL-6/sIL-6R complexes. A, The gp130 and the deletion mutants {Delta}4, {Delta}5, and {Delta}6 were secreted by transfected COS-7 cells as soluble proteins containing the ectodomain fused to a C-terminal Flag epitope (sgp130-Flag, s{Delta}4-Flag, s{Delta}5-Flag, and s{Delta}6-Flag). Seventy-two hours after transfection the recombinant proteins were immunoprecipitated from cell supernatants using a Flag Ab. Precipitates were incubated with an excess of sIL-6R (70 nM) and varying concentrations of IL-6 as indicated in the figure. Ternary complexes formed (see scheme) were coprecipitated and separated by SDS-PAGE. Subsequently, the proteins were transferred to a polyvinylidene difluoride membrane for immunoblot analysis. Proteins were visualized by enhanced chemiluminescence using the Abs indicated. Supernatants from untransfected COS-7 cells served as a control (co). B, Cell surface expression of gp130 and the deletion constructs {Delta}4, {Delta}5, and {Delta}6 in stably transfected Ba/F3 cells was analyzed by FACS. Untransfected cells served as a control (co). Cells were incubated with the gp130 mAbs B-P8 or B-S12-G7, respectively, and stained with a PE-labeled secondary Ab (open peaks). The filled peaks correspond to cells incubated with secondary Ab alone. C, Ba/F3 cells (5 x 106) stably transfected with gp130 or the deletion mutants {Delta}4, {Delta}5, and {Delta}6 as well as untransfected cells were incubated with 200 nM sIL-6R and different concentrations of 125I-labeled IL-6 (1340 cpm/fmol) for 16 h at 4°C. Radioactivity bound to untransfected cells was subtracted from radioactivity bound to transfected cells to obtain specific binding. Specifically bound radioactivity is presented as a function of the 125I-labeled IL-6 concentration.

 
The deletion mutants {Delta}4, {Delta}5, and {Delta}6 were stably transfected into Ba/F3 cells, a mouse pro-B cell line that does not express gp130 endogenously. FACS analyses using the two gp130 mAbs B-P8 and B-S12-G7, which map to the membrane-distal part of gp130, revealed that all deletion mutants are expressed on the cell surface to a similar extent compared with wild-type gp130 (Fig. 3GoB). The binding capabilities of the cell surface expressed receptor mutants were studied by an equilibrium binding assay using radiolabeled IL-6 (125I-labeled IL-6). In the presence of a large excess of sIL-6R, the transfected cells were incubated with 125I-labeled IL-6 for 16 h at 4°C. Subsequently, cell-bound and free radioactivity were separated by centrifugation of the cells through a dinonylphthalate/dibutylphthalate cushion (18). Unspecific binding was determined by incubation of untransfected Ba/F3 cells with 125I-labeled IL-6/sIL-6R. As in the soluble ternary complex formation assay, binding of 125I-labeled IL-6 to {Delta}5 was indistinguishable from binding to wild-type gp130 (Fig. 3GoC). Analysis of the binding data by Scatchard transformation revealed that the affinities of both wild-type gp130 and {Delta}5 ranged from 2.5–4 nM. This value is in agreement with those determined previously for Ba/F3 cells expressing gp130 (18). The mutants {Delta}4 and {Delta}6, however, showed reduced binding of 125I-labeled IL-6. For practical reasons saturating concentrations of 125I-labeled IL-6 could not be applied. Therefore, analysis of the data for {Delta}4 and {Delta}6 by Scatchard transformation was not possible. Because reduced binding capability of {Delta}4 and {Delta}6 is not due to reduced surface expression (see Fig. 3GoB), we conclude that these domains are required for optimal ligand binding by membrane-bound gp130.

Each membrane-proximal domain is required for activation of gp130 by IL-6 as well as IL-11

The ability of the receptor mutants to activate STAT3 was analyzed by EMSA after stimulation of the stably transfected Ba/F3 cells with IL-6/sIL-6R or IL-11/sIL-11R (Fig. 4GoA). Cells transfected with wild-type gp130 responded with a strong activation of STAT3, whereas all deletion mutants were unable to induce STAT activation. Ba/F3 cells, which normally proliferate IL-3-dependently, are known to grow in response to various cytokines after transfection of the corresponding receptor chains. Therefore, cells transfected with wild-type gp130 proliferate after IL-6/sIL-6R stimulation (Fig. 4GoB). Again, the response to IL-6/sIL-6R is totally abolished in cells stably transfected with the deletion mutants (Fig. 4GoB), although these cells still proliferate in response to IL-3 (not shown).



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  		FIGURE 4. Biological activity of gp130 and the deletion mutants {Delta}4, {Delta}5, and {Delta}6. A, Ba/F3 cells stably transfected with gp130 or the deletion mutants {Delta}4, {Delta}5, or {Delta}6 were stimulated with IL-6/sIL-6R or IL-11/sIL-11R, respectively, as indicated. After 15 min, nuclear extracts were prepared, and the protein concentrations were quantified. Ten micrograms of nuclear protein was analyzed by EMSA using a 32P-labeled m67SIE probe derived from the c-Fos promotor providing a binding site for STAT1 and STAT3. Protein-DNA complexes were separated by PAGE and visualized by autoradiography. STAT3 and STAT1 homodimers as well as STAT3/1 heterodimers are indicated by arrowheads. B, Ba/F3 cells (4 x 105/ml) stably transfected with gp130 or the deletion mutants {Delta}4, {Delta}5, and {Delta}6 were seeded in a 96-well plate and incubated with 0.5 µg/ml sIL-6R and increasing amounts of IL-6 as indicated in the diagram. After 72 h a tetrazolium compound was added as a substrate and incubated for 5 h at 37°C. Subsequently, the absorbances at 450 and 690 nm were measured. The difference in absorbances corresponds to the number of metabolically active cells (XTT proliferation assay). C, COS-7 cells were transfected with expression vectors encoding gp130{gamma}, {Delta}4{gamma}, {Delta}5{gamma}, and {Delta}6{gamma}, respectively. Forty-eight hours after transfection cells were stimulated with IL-6/sIL-6R for 15 min (+) or were left unstimulated (-). Nuclear extracts were prepared and analyzed for STAT activation as described above. Untransfected cells served as a control (co). The arrowhead indicates the STAT1 homodimer.

 
In previous studies we established a system that allows the analysis of STAT activation induced by gp130 mutants in transiently transfected COS-7 cells even in the presence of endogenous gp130 (15, 18). Because endogenous gp130 preferentially activates STAT3 (see Fig. 4GoA), the STAT recruiting modules of gp130 mutants were replaced by the strongly and specifically STAT1 activating module YDKPH derived from the IFN-{gamma} receptor (gp130{gamma}). The use of gp130{gamma} constructs enabled us to discriminate between STAT activation of the endogenous and the transfected gp130 in COS-7 cells. Even under conditions in which the gp130{gamma} deletion mutants were overexpressed in COS-7 cells, no significant STAT1 activation was observed in response to IL-6/sIL-6R (Fig. 4GoC). Thus, each of the membrane-proximal domains of gp130 is required for signal transduction by IL-6-type cytokines that induce homodimerization of gp130.

The deletion mutants show differential responses upon stimulation by agonistic gp130 mAbs

gp130 can efficiently be activated by combination of the mAbs B-S12-G7 and B-P8 (26) (unpublished observations). To investigate how the gp130 deletion mutants respond to stimulation by the agonistic gp130 Abs, Ba/F3 cells stably expressing the deletion mutants were incubated with equimolar concentrations of B-S12-G7 and B-P8. Interestingly, the mutants {Delta}4 and, to a lesser extent, {Delta}6 respond to the mAbs with activation of STAT3, whereas the {Delta}5 mutant did not induce significant STAT activation (Fig. 5GoA). Furthermore, the proliferative response of the transfected Ba/F3 cells upon stimulation by agonistic Abs was analyzed. {Delta}4 transduces a proliferative signal, although much less pronounced than that of wild-type gp130 (Fig. 5GoB). Even at high Ab concentrations no maximal response was achieved. The weak STAT activation induced by {Delta}6 shown in Fig. 5GoA was not sufficient to induce proliferation of Ba/F3 cells. In line with the observed lack of STAT activation, {Delta}5 did not induce a proliferative response. In summary, domains 4 and 6 are strictly required for activation of gp130 by IL-6 and IL-11, but a partial activation can be achieved by stimulation with agonistic Abs. In the absence of D5, neither treatment with agonistic mAbs nor stimulation by cytokines leads to the formation of an active gp130 homodimer.



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  		FIGURE 5. Activation of gp130 and deletion mutants by agonistic mAbs. A, Ba/F3 cells stably transfected with gp130 and the deletion mutants {Delta}4, {Delta}5, and {Delta}6 were stimulated with equimolar amounts of the agonistic mAbs B-S12-G7 and B-P8 (1 µg/ml each) for 15 min. Subsequently, nuclear extracts were prepared, and STAT activation was analyzed by EMSA. B, Ba/F3 cells (4 x 105/ml) were seeded in a 96-well plate and incubated with increasing equimolar amounts of the agonistic mAbs B-S12-G7 and B-P8 as indicated in the diagram. After 72 h proliferation of the cells was measured as described in Fig. 3GoB (XTT assay).

 
Replacement of D5 of gp130 by the corresponding domain of the G-CSFR

Although ligand binding capability is fully retained, the {Delta}5 deletion mutant is unable to transduce a signal in response to various stimuli. Thus, D5 plays a central role in coupling ligand binding to the membrane-distal part of gp130 with intracellular signal transduction. To assess the specificity of D5 function in the formation of the active gp130 homodimer, D5 of gp130 was replaced by the corresponding domain of the G-CSFR. The G-CSFR was chosen because its domain architecture is identical with that of gp130, and moreover, this receptor shares 46% sequence similarity with gp130. A fragment of the human G-CSFR cDNA encoding the corresponding domain was amplified by PCR and cloned into the {Delta}5 deletion constructs to obtain D5-GCSFR (schematically represented in Fig. 6GoA) and D5-GCSFR{gamma}. The chimera D5-GCSFR{gamma} was analyzed using the STAT1 activation assay in COS-7 cells, because several attempts to stably transfect Ba/F3 cells with the D5-GCSFR construct failed. Unexpectedly, transfection of D5-GCSFR{gamma} resulted in an activation of STAT1 that is independent from cytokine stimulation, suggesting a constitutive activation of this gp130/G-CSFR chimera (Fig. 6GoB, upper panel). Analysis of the time course of constitutive receptor activation revealed that STAT activation reaches its maximal level within 40 h after transfection and is sustained for at least 65 h (Fig. 6GoB, lower panel). Surface expression of D5-GCSFR{gamma} and gp130{gamma} was compared by FACS analysis. Cells transfected with gp130{gamma} or D5-GCSFR{gamma} show a partial shift of the peak to higher fluorescence intensity compared with untransfected cells, indicating that the chimeric protein appears at the cell surface (Fig. 6GoC). Tyrosine phosphorylation of Jak1 is the most upstream event in gp130-dependent activation of the Jak/STAT pathway. As shown in Fig. 6GoD, Jak1 is constitutively phosphorylated in cells transfected with D5-GCSFR{gamma}, whereas phosphorylation of Jak1 in gp130{gamma}-transfected cells is observed only after IL-6 stimulation.



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  		FIGURE 6. Functional characterization of the gp130/G-CSFR chimera. A, Schematic representation of the chimera D5-GCSFR in comparison to gp130 (white) and G-CSFR (gray). B, COS-7 cells were transfected with expression vectors encoding gp130{gamma} or D5-GCSFR{gamma}. Forty-eight hours after transfection cells were stimulated with IL-6/sIL-6R for 15 min (+) or were left unstimulated (-, upper panel). Time course of constitutive STAT activation was analyzed by preparation of nuclear extracts at the time points after transfection indicated (lower panel). Nuclear extracts were prepared and analyzed by EMSA as described in Fig. 4Go. C, COS-7 cells were transfected with expression vectors encoding gp130{gamma} or D5-GCSFR{gamma}. For FACS analyses, 48 h after transfection COS-7 cells were incubated with the gp130 mAb B-S12 and stained with a PE-labeled secondary Ab. Untransfected cells served as a control (co). Fluorescence in the range of marker 1 (M1) indicates gp130 overexpression due to transfection of cells. D, COS-7 cells were transfected with expression vectors encoding gp130{gamma} or D5-GCSFR{gamma}. Forty-eight hours after transfection cells were stimulated with IL-6/sIL-6R for 15 min (+) or were left unstimulated (-). Jak1 was immunoprecipitated from cell lysates, and precipitated proteins were separated by SDS-PAGE followed by immunoblotting. Tyrosine phosphorylation was detected using a phosphotyrosine Ab (upper panel). The blot was reprobed using a Jak1 Ab to control equal loading of the lanes (lower panel).

 
To analyze whether the observed constitutive activation of the chimera depends on the cellular environment, HepG2 cells were transiently transfected with wild-type gp130 or D5-GCSFR. To measure induction of the acute phase protein {alpha}2-macroglobulin, a reporter gene construct was cotransfected containing the {alpha}2-macroglobulin promoter followed by the luciferase gene. Luciferase activity was measured in stimulated as well as unstimulated cells (Fig. 7Go). Cells were stimulated with low amounts of IL-6 to prevent stimulation of the reporter gene by endogenous gp130 (control lanes). Cells additionally transfected with gp130 are more sensitive to IL-6 stimulation, and therefore, strong induction of the luciferase gene is observed. In cells transfected with D5-GCSFR no constitutive activation of the reporter gene is detectable. However, these cells respond to IL-6 comparably to cells transfected with wild-type gp130. Therefore, in HepG2 cells replacement of D5 of gp130 by the corresponding domain of the GCSFR retains the functionality of gp130.



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  		FIGURE 7. Induction of the {alpha}2-macrogloblulin promotor in HepG2 cells transfected with wild-type gp130 or D5-GCSFR. HepG2 cells were transiently transfected with expression vectors encoding wild-type gp130 or D5-GCSFR. Cells were cotransfected with an {alpha}2-macroglobulin promotor luciferase gene reporter construct and a ß-galactosidase control vector. Twenty-four hours after transfection cells were stimulated (+) with IL-6 at suboptimal concentration (10 ng/ml) or were left unstimulated (-). After 16 h luciferase activity was measured and normalized to ß-galactosidase activity to correct for transfection efficiency. Error bars show mean deviations derived from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. Signal transduction is triggered either by gp130 homodimerization in response to IL-6 and IL-11 or heterodimerization of gp130 with a second signal transducing receptor (LIFR or OSMR) in response to LIF, CNTF, cardiotrophin-1, or OSM. An intriguing structural feature of the signal transducing receptor subunits is the presence of three membrane-proximal FNIII domains in addition to the Ig-like domain and the CBM(s). In the case of gp130 (13) and LIFR (34) it has been shown that the ligands bind to the CBMs and Ig-like domains of the receptors. To date, the functional relevance of the membrane-proximal domains of gp130 was unclear. Mapping of a gp130 neutralizing Ab to the membrane-proximal part of gp130 suggested a functional role of these domains in receptor activation.

To achieve a first assessment of the importance of these domains for receptor activation, three deletion mutants of human gp130 were generated, each lacking one individual FNIII domain ({Delta}4, {Delta}5, and {Delta}6). As shown by FACS analysis, each of the deletion mutants was expressed in amounts comparable to that of wild-type gp130. Therefore, deletion of the domains does not lead to structural alterations that would impair expression of the mutants. As soluble proteins all deletion mutants retained the ability to bind IL-6/sIL-6R complexes to the same extent as gp130, confirming that the membrane-proximal part of wild-type gp130 is not involved in ligand binding. Furthermore, the functionality of the mutants with respect to ligand binding shows that deletion of single domains of gp130 does not lead to disintegration of gp130 structure. However, upon expression of the mutants as transmembrane proteins on the cell surface, deletion of the membrane-proximal domains differentially interfered with ligand binding. Deletions of D4 and D6 reduced the affinity of gp130 to IL-6/sIL-6R complexes, whereas deletion of D5 had no significant effect on ligand binding. The soluble receptor proteins are not restricted with respect to their relative orientations. For membrane-bound proteins only two of three translational and one of three rotational degrees of freedom are available, leading to strong restrictions of the relative orientation of the two gp130 molecules in the gp130 dimer. Reduced binding of membrane-bound {Delta}4 and {Delta}6 may therefore originate from the inability of these mutants to adjust the correct relative orientation required for high affinity ligand binding. Direct involvement in ligand binding in a sense that D4 and D6 contact the ligand is unlikely, because as soluble proteins both mutants bind IL-6/sIL-6R complexes indistinguishably from wild-type gp130. Moreover, previous work defined the ligand-binding epitopes in the membrane-distal domains of gp130 (13, 15).

Each deletion mutant was unable to induce signal transduction in response to stimulation by both IL-6 and IL-11 suggesting a functional role of each of the membrane-proximal domains in receptor activation. Intriguingly, using different cellular systems no activation of {Delta}5 was observed, although this deletion mutant, even in its membrane-bound form, binds IL-6/sIL-6R comparable to wild-type gp130. In the case of membrane-bound {Delta}4 and {Delta}6 the lack of biological activity may partially be due to the reduced affinity to IL-6/sIL-6R complexes. Therefore, responsiveness of the deletion mutants to agonistic mAbs was studied. The Abs are believed to activate cytokine receptors by virtue of their bivalency that enables them to dimerize the receptor. Whereas deletion of D5 led to a loss of activity even by stimulation with the Abs, the mutants {Delta}4 and, to a lesser extent, {Delta}6 retained residual activity. The total loss of activity of {Delta}5 is not due to participation of D5 in the Ab epitope because this deletion mutant is recognized by both agonistic Abs in FACS analysis. Furthermore, in a previous study the epitopes of the agonistic Abs were mapped to the membrane-distal part of gp130 (26). Thus, there seems to exist an absolute requirement for domain 5 to achieve activation of gp130. None of the deletion mutants is stimulated by the Abs to an extent that is achieved with wild-type gp130. We conclude from these findings that dimerization of gp130 by the natural ligand or by mAbs is not sufficient for receptor activation. A well-defined active conformation of the receptor has to be adjusted to allow a productive juxtaposition of the cytoplasmic parts that leads to activation of the associated kinases and finally to cytoplasmic signal transduction. These conformational requirements cannot be fulfilled in the absence of D5.

To decide whether there is either a specific functional requirement for D5 of gp130 or whether it plays a rather unspecific role (e.g., as a spacer), this domain was replaced by the corresponding domain of the homologous G-CSFR. Most surprisingly, upon overexpression the D5 chimera was ligand independently activated, suggesting that the membrane-proximal half of receptors such as gp130 and G-CSFR has an intrinsic propensity to dimerize that might normally be activated upon ligand binding, possibly by enhancing the accessibility of dimer interface(s). Our assumption is that in the D5-GCSFR chimera a kind of disturbance has been introduced that leads to ligand-independent interaction of these proposed dimer interfaces. This idea is supported by the fact that chimeric receptors consisting of the ectodomain of the G-CSFR fused to the transmembrane and cytoplasmic parts of gp130 are only activated in response to G-CSF (35). Thus, D5 of G-CSFR in the context of the complete G-CSFR ectodomain does not lead to ligand-independent activation of gp130. It was not possible to stably transfect this chimera into Ba/F3 cells to test its mitogenic activity, suggesting that expression of this chimera is inconsistent with viability of the cells. We therefore transiently expressed the chimera in HepG2 cells and assessed its activity using a reporter gene assay. In these cells, no constitutive activation of D5-GCSFR was observed, but the chimera responded normally to IL-6 stimulation. Thus, in HepG2 cells replacement of gp130 D5 by the corresponding domain of the G-CSFR restores a functional receptor. Possibly, constitutive activation of this mutant is only observed after strong overexpression, as seen in transfected COS-7 cells (see FACS, Fig. 6GoC) or critically depends on the cell type. Cell type-dependent constitutive activation has also been described for activating mutations in ßc, the common ß-chain of the receptors for IL-3, IL-5, and GM-CSF (36).

We conclude from our studies that gp130 is activated by a mechanism that differs from the activation mechanism of short cytokine receptors such as the EpoR or GHR (Fig. 8GoA, left panel). From the structure of the GH/sGHR complex it can be deduced that receptor activation is achieved by binding of two receptor molecules to a single bivalent ligand (37). Most of the dimerization energy is contributed by the receptor ligand interaction, which is to some extent supported by receptor/receptor interactions. In the case of gp130 we observe a functional dichotomy of the ectodomain. The membrane-distal half binds the ligand, whereas the membrane-proximal half mediates receptor activation. Both parts of the molecule must be functionally coupled to ensure that in the wild-type proteins receptor activation occurs only after ligand binding. We present a model of gp130 activation that is in line with the findings described above (Fig. 8Go, central panel). D5 adjusts the correct spacing of the cytoplasmic parts required for signaling. In the absence of D5, the ligand can still be bound, but a nonproductive conformation is adjusted (Fig. 8Go, right panel). Consequently, partial restoration is achieved by adding back D5 of the G-CSFR. Our model proposes an interaction of domains 5 of two gp130 molecules. Possibly, in the homologous G-CSFR this interaction is somewhat stronger, leading to constitutive activation of the chimera in COS-7 cells. All members of the cytokine receptor family that contain additional membrane-proximal domains (LIFR, OSMR, G-CSFR, IL-12R, and leptin receptor) may be activated by a similar mechanism. Indeed, a role of the membrane-proximal domains of the G-CSFR in receptor activation has been established by showing that simultaneous deletion of the three membrane-proximal domains strongly impairs signal transduction in response to G-CSF, whereas ligand binding is largely unaffected (38).



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  		FIGURE 8. Proposed mechanism for activation of cytokine receptors. In the case of short cytokine receptors such as EpoR or GHR (left panel) binding of the natural ligand to the CBMs adjusts the active conformation of the homodimer. High affinity binding of the ligand to the CBM and Ig-like domain of gp130 is not sufficient for receptor activation, because in the case of {Delta}5 (right panel) this does not lead to signal transducing receptor complexes. The gp130 membrane-proximal domains are necessary for adjustment of the active dimer (central panel). The scheme highlights the functional role of domain 5 (striped) for productive spacing of the cytoplasmic parts of gp130.

 
Even after overexpression in COS-7 cells no constitutive activation of wild-type gp130 is observed, although this could be expected due to formation of gp130 dimers as a result of random interactions. It is tempting to speculate that in addition to the rigid model presented in Fig. 8Go, a conformational change in the gp130 ectodomain necessary for receptor activation is induced upon ligand binding. By this hypothetical mechanism, signal transduction in the absence of the ligand is prevented. The insertion of the analogous domain of the G-CSFR into the completely inactive deletion mutant {Delta}5 leads to a ligand-independent mechanism of receptor activation in COS-7 cells. In the family of cytokine receptors constitutively active variants of the EpoR (39), thrombopoietin receptor (40), and ßc (36) have been described (for review, see Ref. 41). The ectodomains of these receptors consist solely of one (EpoR) or two (thrombopoietin receptor, ßc) CBMs and lack additional FNIII domains. For gp130 and receptors of similar ectodomain architecture (LIFR, OSMR, G-CSFR, IL-12R, and the leptin receptor), the membrane-proximal domains are promising targets for rational design of constitutively active cytokine receptor variants that can be used in basic research on the physiological consequences of constitutive receptor activation. In the previously described cases of constitutively activated cytokine receptors the oncogenic potential of ligand independent receptor activation has been established. A role for constitutively active gp130 in the development of human malignancies can also be imagined.


   Acknowledgments
 
We thank John Wijdenes (Diaclone, Besançon, France) for providing the gp130 mAbs used in this study, and Drs. Iris Behrmann and Lutz Graeve for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Fonds der Chemischen Industrie (Frankfurt am Main, Germany). Back

2 Current address: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universität Erlangen-Nürnberg, Universitätsstrasse 22, D-91054 Erlangen, Germany. Back

3 Address correspondence and reprint requests to Dr. Peter C. Heinrich, Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany. E-mail address: Back

4 Abbreviations used in this paper: CBM, cytokine binding module; FNIII, fibronectin type III-like; Jak, Janus kinase; GH, growth hormone; GHR, GH receptor; Epo, erythropoietin; EpoR, Epo receptor; G-CSFR, G-CSF receptor; LIF, leukemia inhibitory factor; LIFR, LIF receptor; CNTF, ciliary neurotrophic factor; OSM, oncostatin M; OSMR, OSM receptor; SIE, sis-inducible element; sIL-6, soluble IL-6; sgp130, soluble gp130. Back

Received for publication April 15, 1999. Accepted for publication October 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bazan, J. F.. 1990. Structural design and molecular evolution of a cytokine receptor superfamily. Proc. Natl. Acad. Sci. USA 87:6934.[Abstract/Free Full Text]
  2. Ihle, J. N.. 1995. Cytokine receptor signaling. Nature 377:591.[Medline]
  3. Wells, J. A., A. M. de Vos. 1996. Hematopoietic receptor complexes. Annu. Rev. Biochem. 65:609.[Medline]
  4. Heinrich, P. C., I. Behrmann, G. Müller-Newen, F. Schaper, L. Graeve. 1998. IL-6-type cytokine signaling through the gp130/JAK/STAT pathway. Biochem. J. 334:297.
  5. Hibi, M., M. Murakami, M. Saito, T. Hirano, T. Taga, T. Kishimoto. 1990. Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell 63:1149.[Medline]
  6. Murakami, M., M. Hibi, N. Nakagawa, T. Nakawaga, K. Yasukawa, K. Yamanishi, T. Taga, T. Kishimoto. 1993. IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase. Science 260:1808.[Abstract/Free Full Text]
  7. Yin, T., T. Taga, M. L. Tsang, K. Yasukawa, T. Kishimoto, Y. C. Yang. 1993. Involvement of IL-6 signal transducer gp130 in IL-11-mediated signal transduction. J. Immunol. 151:2555.[Abstract]
  8. Gearing, D. P., M. R. Comeau, D. J. Friend, S. D. Gimpel, C. J. Thut, J. McGourty, K. K. Brasher, J. A. King, S. Gillis, B. Mosley, et al 1992. The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor. Science 255:1434.[Abstract/Free Full Text]
  9. Davis, S., T. H. Aldrich, N. Stahl, L. Pan, T. Taga, T. Kishimoto, N. Y. Ip, G. D. Yancopoulos. 1993. LIFRß and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor. Science 260:1805.[Abstract/Free Full Text]
  10. Pennica, D., K. J. Shaw, T. A. Swanson, M. W. Moore, D. L. Shelton, K. A. Zioncheck, A. Rosenthal, T. Taga, N. F. Paoni, W. I. Wood. 1995. Cardiotrophin-1: biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex. J. Biol. Chem. 270:10915.[Abstract/Free Full Text]
  11. Mosley, B., C. De Imus, D. Friend, N. Boiani, B. Thoma, L. S. Park, D. Cosman. 1996. Dual oncostatin M (OSM) receptors: cloning and characterization of an alternative signaling subunit conferring OSM-specific receptor activation. J. Biol. Chem. 271:32635.[Abstract/Free Full Text]
  12. Taga, T., M. Hibi, Y. Hirata, K. Yamasaki, K. Yasukawa, T. Matsuda, T. Hirano, T. Kishimoto. 1989. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 58:573.[Medline]
  13. Horsten, U., H. Schmitz-Van de Leur, J. Müllberg, P. C. Heinrich, S. Rose-John. 1995. The membrane distal half of gp130 is responsible for the formation of a ternary complex with IL-6 and the IL-6 receptor. FEBS Lett. 360:43.[Medline]
  14. Hammacher, A., R. T. Richardson, J. E. Layton, D. K. Smith, L. J. L. Angus, D. J. Hilton, N. A. Nicola, J. Wijdenes, and R. J. Simpson. 1998. The immunoglobulin-like module of gp130 is required for signaling by interleukin-6 but not by leukemia inhibitory factor. J. Biol. Chem. 273.
  15. Kurth, I., U. Horsten, S. Pflanz, H. Dahmen, A. Küster, J. Grötzinger, P. C. Heinrich, G. Müller-Newen. 1999. Activation of the signal transducer gp130 by both interleukin-6 and interleukin-11 requires two distinct binding epitopes. J. Immunol. 162:1480.[Abstract/Free Full Text]
  16. Dahmen, H., U. Horsten, A. Küster, Y. Jacques, S. Minvielle, I. M. Kerr, G. Ciliberto, G. Paonessa, P. C. Heinrich, G. Müller-Newen. 1998. Activation of the signal transducer gp130 by interleukin-11 and interleukin-6 is mediated by similar molecular interactions. Biochem. J. 331:695.
  17. Staunton, D., K. R. Hudson, J. K. Heath. 1998. The interactions of the cytokine-binding homology region and immunoglobulin-like domains of gp130 with oncostatin M: implications for receptor complex formation. Protein Eng. 11:1093.[Abstract/Free Full Text]
  18. Horsten, U., G. Müller-Newen, C. Gerhartz, A. Wollmer, J. Wijdenes, P. C. Heinrich, J. Grötzinger. 1997. Molecular modeling-guided mutagenesis of the extracellular part of gp130 leads to the identification of contact sites in the interleukin-6 (IL-6)/IL-6 receptor/gp130 complex. J. Biol. Chem. 272:23748.[Abstract/Free Full Text]
  19. Bravo, J., D. Staunton, J. K. Heath, E. Y. Jones. 1998. Crystal structure of a cytokine-binding region of gp130. EMBO J. 17:1665.[Medline]
  20. Kernebeck, T., S. Pflanz, G. Müller-Newen, G. Kurapkat, R. M. Scheek, K. Dijkstra, P. C. Heinrich, A. Wollmer, S. Grzesiek, J. Grötzinger. 1999. The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region. Protein Sci. 8:1.
  21. Lemmon, M. A., J. Schlessinger. 1994. Regulation of signal transduction and signal diversity by receptor oligomerization. Trends Biochem. Sci. 19:459.[Medline]
  22. Heldin, C.-H.. 1995. Dimerization of cell surface receptors in signal transduction. Cell 80:213.[Medline]
  23. Arcone, R., P. Pucci, F. Zappacosta, V. Fontaine, A. Malorni, G. Marino, G. Ciliberto. 1991. Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. Eur. J. Biochem. 198:541.[Medline]
  24. Weiergräber, O., U. Hemmann, A. Küster, G. Müller-Newen, J. Schneider, S. Rose-John, P. Kurschat, J. P. J. Brakenhoff, M. H. L. Hart, S. Stabel, et al 1995. Soluble human interleukin-6 receptor: expression in insect cells, purification and characterization. Eur. J. Biochem. 234:661.[Medline]
  25. Müller-Newen, G., A. Küster, U. Hemmann, R. Keul, U. Horsten, A. Martens, L. Graeve, J. Wijdenes, P. C. Heinrich. 1998. Soluble interleukin-6 receptor potentiates the antagonistic activity of soluble gp130 on interleukin-6 responses. J. Immunol. 161:6347.[Abstract/Free Full Text]
  26. Wijdenes, J., P. C. Heinrich, G. Müller-Newen, C. Roche, G. Zong-Jiang, C. Clement, B. Klein. 1995. Interleukin-6 signal transducer gp130 has specific binding sites for different cytokines as determined by antagonistic and agonistic anti-gp130 mAbs. Eur. J. Immunol. 25:3474.[Medline]
  27. Gerhartz, C., B. Heesel, J. Sasse, U. Hemmann, C. Landgraf, J. Schneider-Mergener, F. Horn, P. C. Heinrich, L. Graeve. 1996. Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the interleukin-6 signal transducer gp130. I. Definition of a novel phosphotyrosine motif mediating STAT1 activation. J. Biol. Chem. 271:12991.[Abstract/Free Full Text]
  28. Yuan, J., U. M. Wegenka, C. Lütticken, J. Buschmann, T. Decker, C. Schindler, P. C. Heinrich, F. Horn. 1994. The signaling pathways of interleukin-6 and interferon-{gamma} converge by the activation of different transcription factors which bind to common responsive DNA elements. Mol. Cel. Biol. 14:1657.[Abstract/Free Full Text]
  29. Bolton, A. E., W. M. Hunter. 1973. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133:529.[Medline]
  30. Wegenka, U. M., C. Lütticken, J. Buschmann, J. Yuan, F. Lottspeich, W. Müller-Esterl, C. Schindler, E. Roeb, P. C. Heinrich, F. Horn. 1994. The interleukin-6-activated acute-phase response factor is antigenically and functionally related to members of the signal transduction and transcription (Stat) factor family. Mol. Cell. Biol. 14:3186.[Abstract/Free Full Text]
  31. Wagner, B. J., T. E. Hayes, C. J. Hoban, B. H. Cochran. 1990. The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO J. 9:4477.[Medline]
  32. Kyhse-Anderson, J.. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins form polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10:203.[Medline]
  33. Lebeau, B., F. A. Montero-Julian, J. Wijdenes, G. Müller-Newen, H. Dahmen, M. Chérel, P. C. Heinrich, H. Brailly, M.-M. Hallet, A. Godard, et al 1997. Reconstitution of two isoforms of the human interleukin-11 receptor and comparison of their functional properties. FEBS Lett. 407:141.[Medline]
  34. Owczarek, C. M., Y. Zhang, M. J. Layton, D. Metcalf, B. Roberts, N. A. Nicola. 1997. The unusual species cross-reactivity of the leukemia inhibitory factor receptor {alpha}-chain is determined primarily by the immunoglobulin-like domain. J. Biol. Chem. 272:23976.[Abstract/Free Full Text]
  35. Baumann, H., A. J. Symes, M. R. Comeau, K. K. Morella, Y. Wang, D. Friend, S. F. Ziegler, J. S. Fink, D. P. Gearing. 1993. Multiple regions within the cytoplasmic domains of leukemia inhibitory factor receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells. Mol. Cell. Biol. 14:138.[Abstract/Free Full Text]
  36. Jenkins, B. J., R. J. D‘Andrea, T. J. Gonda. 1995. Activating point mutations in the common ß-subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of ß-subunit dimerisation and cell type-specific molecules in signaling. EMBO J. 14:4276.[Medline]
  37. de Vos, A. M., M. Ultsch, A. A. Kossiakoff. 1992. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255:306.[Abstract/Free Full Text]
  38. Fukunaga, R., E. Ishizaka-Ikeda, C.-X. Pan, Y. Seto, S. Nagata. 1991. Functional domains of the granulocyte colony-stimulating factor receptor. EMBO J. 10:2855.[Medline]
  39. Yoshimura, A., G. Longmore, H. F. Lodish. 1990. Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity. Nature 348:647.[Medline]
  40. Souyri, M., I. Vigon, J. Pencioletti, J. Heard, P. Tambourin, F. Wendling. 1990. A putative truncated cytokine receptor gene transduced by the myeloproliferative leukaemia virus immortalises hematopoietic progenitors. Cell 63:1137.[Medline]
  41. Gonda, T. J., R. J. D’Andrea. 1997. Activating mutations in cytokine receptors: implications for receptor function and role in disease. Blood 89:355.[Free Full Text]



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