Interleukin-6 signal transducer gp130 mediates oncostatin M signaling.

Oncostatin M (OM) is a multifunctional cytokine that is structurally and functionally related to interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). The specific receptor for OM has been demonstrated (by chemical cross-linking) to be a 150-kDa protein in a number of cell lines. The IL-6 signal transducer, gp130, is also an affinity converter for the LIF receptor. It does not bind to either IL-6 or LIF, but associates with the alpha subunits of the receptors and transduces the signals. We examined the possible involvement of gp130 in OM binding and signaling. We demonstrate that: (a) anti-gp130 monoclonal antibodies (mAbs) block the inhibitory effect of OM on A375 cell growth, (b) the binding and cross-linking of 125I-OM to H2981 cells are completely abolished by anti-gp130 mAbs, (c) the cross-linked OM-receptor complex is immunoprecipitated by anti-gp130 mAbs, and (d) COS-7 cells transfected with the full-length cDNA encoding gp130 exhibit increased OM binding and cross-linking, which are also blocked by anti-gp130 mAbs. Therefore, we conclude that the 150-kDa OM binding protein previously characterized in a variety of cell lines is gp130. OM is the natural ligand for gp130 and gp130 mediates the biological responses of OM.     

Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130.

Leukemia inhibitory factor (LIF) is a cytokine with a broad range of activities that in many cases parallel those of interleukin-6 (IL-6) although LIF and IL-6 appear to be structurally unrelated. A cDNA clone encoding the human LIF receptor was isolated by expression screening of a human placental cDNA library. The LIF receptor is related to the gp130 'signal-transducing' component of the IL-6 receptor and to the G-CSF receptor, with the transmembrane and cytoplasmic regions of the LIF receptor and gp130 being most closely related. This relationship suggests a common signal transduction pathway for the two receptors and may help to explain similar biological effects of the two ligands. Murine cDNAs encoding soluble LIF receptors were isolated by cross-hybridization and share 70% amino acid sequence identity to the human sequence.     

Molecular cloning and expression of an IL-6 signal transducer, gp130

Interleukin-6 (IL-6) signal is transduced through a membrane glycoprotein, gp130, which associates with IL-6 receptor (IL-6-R). A cDNA encoding human gp130 has been cloned, revealing that it consists of 918 amino acids with a single transmembrane domain. The extracellular region comprises six units of a fibronectin type III module, and part of this region of approximately 200 amino acids has features typical of a cytokine receptor family. A cDNA-expressed gp130 showed no binding property to IL-6 or several other cytokines. Although a transfectant with an IL-6-R cDNA expressed mainly low affinity IL-6 binding sites, an increase in high affinity binding sites was observed after cotransfection with a gp130 cDNA. This confirmed that a gp130 is involved in the formation of high affinity IL-6 binding sites. A cloned gp130 could associate with a complex of IL-6 and soluble IL-6-R and transduce the growth signal when expressed in a murine IL-3-dependent cell line.     

Two different epitopes of the signal transducer gp130 sequentially cooperate on IL-6-induced receptor activation

Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (DeltaD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with DeltaD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of DeltaD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.     

Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.     

Studies on the interleukin-6-type cytokine signal transducer gp130 reveal a novel mechanism of receptor activation by monoclonal antibodies

The transmembrane glycoprotein gp130 belongs to the family of hematopoietic cytokine receptors. It represents the common signal transducing receptor component of the so called interleukin-6-type cytokines. For several cytokine receptors including gp130 it has been shown that receptor activation cannot only be achieved by the natural ligand but also by single monoclonal antibodies raised against the receptor ectodomain. These findings have been interpreted in a way that dimerization of cytokine receptors is sufficient for receptor activation. Here we show that the recently described gp130-activating antibody B-S12 actually consists of two different monoclonal antibodies. By subcloning of B-S12 the monoclonal antibodies B-S12-A5 and B-S12-G7 were obtained. The individual antibodies are biologically inactive, in combination they exert B-S12-like activity on hepatoma cells. On Ba/F3 cells stably transfected with gp130 a combination of B-S12-G7 with another monoclonal gp130 antibody, B-P8, is required to stimulate proliferation. Using gp130 deletion mutants we show that all three antibodies map to domains 2 and 3 of gp130 which constitute the cytokine binding module. The individual antibodies inhibit activation of the signal transducer by interleukin-6 and interfere with binding of interleukin-6 to gp130. Interestingly, the combination of B-S12-G7 and a Fab fragment of B-P8 retains biological activity. We conclude from our data that (i) the monoclonal antibodies activate gp130 by mimicking the natural ligand and (ii) enforcement of gp130 dimerization is not sufficient for receptor activation but additional conformational requirements have to be fulfilled.     

Functional properties of extracellular domains of transducer receptor gp130

Cytokine receptor molecules have been shown to have extracellular domains of complex structure and a multistep activation system. Glycoprotein gp130 is a typical transducer of cytokine signal; it functions by forming multicomponent receptor complexes and transferring signals of tens of cytokines from the IL-6 family. Structural organization and basic functioning principles of gp130 are well known, as well as related signal pathways, which function during normal differentiation and are involved in pathogenesis of many tumors. The role of gp130 in IL-6-dependent tumors is best studied. In this review, based on extensive accumulated data, we examine the functional significance of certain parts of gp130 extracellular domains. Potentials of a recently developed method for estimation of receptor activation at the level of epitope structure are discussed.     

The structure of the unliganded extracellular domain of the interleukin-6 signal transducer gp130 in solution

Interleukin-6 (IL-6) plays an important role in immune responses and signals via two different pathways. When IL-6 binds to its non-signalling membrane-bound receptor (IL-6R), a non-covalent dimer of the ubiquitous interleukin-6 signal transducer gp130 is recruited to initiate intracellular signalling cascades. This so-called classical signalling pathway is restricted to cells expressing the membrane-bound IL-6R, such as hepatocytes and certain leukocytes. In addition, an alternative trans-signalling pathway uses soluble forms of IL-6R (sIL-6R) in complex with IL-6 to activate cells expressing gp130, but not membrane-bound IL-6R. In both cases, a tetrameric or hexameric signalling complex consisting of two gp130 molecules and one or two molecules each of IL-6 and (s)IL-6R is formed. The structure of the hexameric complex of the ligand-binding domains of gp130 (D1-D3) with IL-6 and sIL-6R has been solved by X-ray crystallography as well as the full-length extracellular part of gp130 (D1-D6) as a monomer. Since gp130 exists as a preformed dimer on the cell surface, we used a sgp130Fc fusion protein - consisting of two extracellular gp130 regions (D1-D6) dimerised by an IgG1-Fc part - to study the structure of unliganded gp130 extracellular domains in solution by small-angle X-ray scattering (SAXS). The SAXS data indicated that sgp130Fc forms a rigid molecule in solution. The low resolution structural model reveals an elongated assembly with an Fc base and two gp130 arms, whereby the orientation of the ligand-binding domains D1-D3 with respect to the membrane-proximal domains D4-D6 differs from that in the crystallographic monomer. Functional implications of these findings are discussed.     

The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.     

Functional and biochemical association of Hck with the LIF/IL-6 receptor signal transducing subunit gp130 in embryonic stem cells.

The role played by the Src-related tyrosine kinase, Hck, in embryonic stem (ES) cell differentiation was investigated by replacing a conserved C-terminally located tyrosine with phenylalanine by gene targeting. Targeted ES cells display a 7- to 9-fold elevation in constitutive Hck kinase activity and require approximately 15 times less leukaemia inhibitory factor (LIF) than parental ES cells to maintain their stem cell character in vitro. We also demonstrate a rapid and transient increase in Hck tyrosine kinase activity in parental ES cells stimulated by LIF and, finally, show that Hck is physically associated with gp130, an affinity converter and signal transducing component of the LIF receptor. Thus, these results provide biological and biochemical evidence that Hck participates in signal transduction from the LIF receptor.     

A non-conservative polymorphism in the IL-6 signal transducer (IL6ST)/gp130 is associated with myocardial infarction in a hypertensive population

Inflammation is a key component in the development of atherosclerosis, and myocardial infarction (MI); therefore we investigated the association between an interleukin-6 signal transducer (IL6ST)/gp130 polymorphism, gp130 function and risk of MI. Structural modeling suggested that a non-conservative single nucleotide polymorphism in the gp130, Gly148Arg, can change the stability and functional properties of the molecule. In vitro studies were done with BAF/3 cells lacking endogenous gp130. Cells stably transfected with the gp130 148Arg variant proliferated less and showed slightly lower STAT-3 phosphorylation in response to gp130 stimulation as compared to cells transfected with gp130 148Gly. In a prospectively followed hypertensive cohort we identified 167 patients who suffered a MI during the study and compared them to matched controls (mean age 57 years, 73% males, n=482). Carriers of the 148Arg variant (f(Arg)=0.12) of the gp130 receptor had decreased odds ratio for MI in univariate analysis (0.56, 95% CI 0.34-0.91, p=0.02). In conclusion, a genetically determined structural variant of the IL-6 receptor subunit gp130 is, independently of other known risk factors, associated with decreased risk of MI. The variant is also associated with decreased IL-6 responsiveness and could lead to a configuration change in the gp130 receptor.     

A functional role of the membrane-proximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor.

gp130 is the common signal transducing receptor subunit of interleukin (IL)-6-type cytokines. gp130 either homodimerizes in response to IL-6 and IL-11 or forms heterodimers with the leukemia inhibitory factor (LIF) receptor (LIFR) in response to LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) or cardiotrophin-like cytokine resulting in the onset of cytoplasmic tyrosine phosphorylation cascades. The extracellular parts of both gp130 and LIFR consist of several Ig-like and fibronectin type III-like domains. The role of the membrane-distal domains of gp130 (D1, D2, D3) and LIFR in ligand binding is well established. In this study we investigated the functional significance of the membrane-proximal domains of gp130 (D4, D5, D6) in respect to heterodimerization with LIFR. Deletion of each of the membrane-proximal domains of gp130 (Delta 4, Delta 5 and Delta 6) leads to LIF unresponsiveness. Replacement of the gp130 domains by the corresponding domains of the related GCSF receptor either restores weak LIF responsiveness (D4-GCSFR), leads to constitutive activation of gp130 (D5-GCSFR) or results in an inactive receptor (D6-GCSFR). Mutation of a specific cysteine in D5 of gp130 (C458A) leads to constitutive heterodimerization with the LIFR and increased sensitivity towards LIF stimulation. Based on these findings, a functional model of the gp130-LIFR heterodimer is proposed that includes contacts between D5 of gp130 and the corresponding domain D7 of the LIFR and highlights the requirement for both receptor dimerization and adequate receptor orientation as a prerequisite for signal transduction.     

Caspase-mediated cleavage of the signal-transducing IL-6 receptor subunit gp130

The present study characterizes the molecular mechanisms of CD95L-induced inhibition of IL-6 signaling, which is known to mediate hepatoprotective effects in response to various toxins. CD95L-induced caspase activation leads to degradation of gp130, thereby suppressing IL-6-induced phosphorylation of STAT3 (Tyr⁷⁰⁵) and of tyrosine phosphatase SHP2 (Tyr⁵⁸⁰). Degradation of gp130 protein in response to CD95L was largely prevented after inhibition of caspase 3 or 8. Introduction of a point mutation into a newly identified caspase cleavage site located within position 800–806 (DHVDGGD) of the cytoplasmic tail of gp130 leads to cleavage resistance of the respective receptor in an in vitro assay with recombinant active caspase 3. Correspondingly, the release of a C-terminal gp130-cleavage product of approximately 18 kDa was also inhibited after mutagenesis of this cleavage motif. In conclusion, this study demonstrates that caspase activation by CD95L antagonizes IL-6 signaling by a caspase-mediated cleavage of gp130 thereby probably counteracting hepatoprotective effects of IL-6.     

Identification of amino acid residues of gp130 signal transducer and gp80 alpha receptor subunit that are involved in ligand binding and signaling by human herpesvirus 8-encoded interleukin-6

Human herpesvirus 8-encoded interleukin-6 (vIL-6) signals through the gp130 signal transducer but is not dependent on the IL-6 receptor alpha subunit (IL-6R, gp80) that is required for signaling by endogenous IL-6 proteins; however, IL-6R can enhance vIL-6 activity and can enable signaling through a gp130 variant, gp130.PM5, that is itself unable to support vIL-6 signaling. These findings suggest that the vIL-6-gp130 interactions are qualitatively different from those of human IL-6 (hIL-6) and that vIL-6 signaling may be more promiscuous than that of hIL-6 but that IL-6R may play a role in vIL-6 signaling in vivo. To examine the receptor binding requirements of vIL-6, we have undertaken mutational analyses of regions of gp130 and IL-6R potentially involved in interactions with ligand or in functional complex formation and used these variants in functional, ligand-binding, and receptor dimerization assays. The data presented identify positions within two interstrand loops of the gp130 cytokine-receptor homology domain that are important for vIL-6 signaling and vIL-6-induced receptor dimerization and show that vIL-6, like hIL-6, can form complexes with IL-6R and gp130 but that the roles of putative cytokine-binding residues of IL-6R in ligand-induced functional complex formation are qualitatively different in the case of vIL-6 and hIL-6.     

Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor

gp130 is a shared cytokine signaling receptor and the founding member of the 'tall' class of cytokine receptors. A crystal structure of the ligand-binding domains of gp130 in complex with human interleukin-6 (IL-6) and its a-receptor (IL-6Ralpha) revealed a hexameric architecture in which the gp130 membrane-distal regions were approximately 100 A apart, in contrast to the close apposition seen between short cytokine receptor complexes. Here we used single-particle EM to visualize the entire extracellular hexameric IL-6-IL-6Ralpha-gp130 complex, containing all six gp130 domains. The structure reveals that gp130 is bent such that the membrane-proximal domains of gp130 are close together at the cell surface, enabling activation of intracellular signaling. Variation in the receptor bend angles suggests a possible conformational transition from open to closed states upon ligand binding; this transition is probably representative of the other tall cytokine receptors.     

IL-6 receptor independent stimulation of human gp130 by viral IL-6

The genome of human herpes virus 8, which is associated with Kaposi's sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.