Importance of the membrane-proximal extracellular domains for activation of the signal transducer glycoprotein 130

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 Delta4 and, to a lesser extent, Delta6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant Delta5 does not respond. Because membrane-bound Delta5 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.     

LIF receptor-gp130 interaction investigated by homology modeling: implications for LIF binding.

Leukemia inhibitory factor (LIF), a member of the gp130 family of helical cytokines, is involved in the hemopoietic and neural systems. The LIF signal transducing complex contains two receptor molecules, the LIF receptor (LIFR) and gp130. The extracellular region of the LIFR is unique in that it includes three membrane-proximal fibronectin type III domains and two cytokine binding domains (CBDs) separated by an immunoglobulin-like domain. Although some mutagenesis data on LIF are available, it is not yet known which regions of LIFR or gp130 bind LIF. Nor is it known whether LIFR contacts gp130 in a manner similar to the growth hormone receptor dimer and, if so, through which of its CBDs. To attempt to elucidate these matters and to investigate the receptor complex, models of the CBDs of LIFR and the CBD of gp130 were constructed. Analyses of the electrostatic isopotential surfaces of the CBD models suggest that gp130 and the membrane-proximal CBD of LIFR hetero-dimerize and bind LIF through contacts similar to those seen in the growth hormone receptor dimer. This work further demonstrates the utility of electrostatic analyses of homology models and suggests a strategy for biochemical investigations of the LIF-receptor complex.     

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.     

Novel inhibitors for murine and human leukemia inhibitory factor based on fused soluble receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.     

Gene expression of receptors for IL-6, LIF, and CNTF in regenerating skeletal muscles.

The biological actions of interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) are mediated via respective functional receptor complexes consisting of a common signal-transducing component, gp130, and other specific receptor components, IL-6 receptor alpha (IL-6R), LIF receptor beta (LIFR), and CNTF receptor alpha (CNTFR). IL-6, LIF, and CNTF are implicated in skeletal muscle regeneration. However, the cell populations that express these receptor components in regenerating muscles are unknown. Using in situ hybridization histochemistry, we examined spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles after muscle contusion. At the early stages of regeneration (from 3 hr to Day 2 post contusion), significant signals for gp130 and LIFR mRNAs were detected in myonuclei and/or nuclei of muscle precursor cells (mpcs) and in mononuclear cells located in extracellular spaces between myofibers after muscle contusion, but IL-6R mRNA was expressed only in mononuclear cells. At Day 7 post contusion, signals for gp130, LIFR, and IL-6R mRNAs were not detected in newly formed myotubes, whereas the CNTFR mRNA level was upregulated in myotubes. These findings suggest that the upregulation of receptor subunits in distinct cell populations plays an important role in the effective regeneration of both myofibers and motor neurons. (J Histochem Cytochem 48:1203-1213, 2000)     

Receptor subunit-specific action of oncostatin M in hepatic cells and its modulation by leukemia inhibitory factor

The related cytokines, interleukin-6 (IL-6), oncostatin M (OSM), and leukemia inhibitory factor (LIF) direct the formation of specific heteromeric receptor complexes to achieve signaling. Each complex includes the common signal-transducing subunit gp130. OSM and LIF also recruit the signaling competent, but structurally distinct OSMRbeta and LIFRalpha subunits, respectively. To test the hypothesis that the particularly prominent cell regulation by OSM is due to signals contributed by OSMRbeta, we introduced stable expression of human or mouse OSMRbeta in rat hepatoma cells which have endogenous receptors for IL-6 and LIF, but not OSM. Both mouse and human OSM engaged gp130 with their respective OSMRbeta subunits, but only human OSM also acted through LIFR. Signaling by OSMRbeta-containing receptors was characterized by highest activation of STAT5 and ERK, recruitment of the insulin receptor substrate and Jun-N-terminal kinase pathways, and induction of a characteristic pattern of acute phase proteins. Since LIF together with LIFRalpha appear to form a more stable complex with gp130 than OSM with gp130 and OSMRbeta, co-activation of LIFR and OSMR resulted in a predominant LIF-like response. These results suggest that signaling by IL-6 cytokines is not identical, and that a hierarchical order of cytokine receptor action exists in which LIFR ranks as dominant member.     

Separate functions for the two modules of the membrane-proximal cytokine binding domain of glycoprotein 190, the leukemia inhibitory factor low affinity receptor, in ligand binding and receptor activation

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130. Both are members of the hematopoietic receptors family characterized by the cytokine receptor homology (CRH) domain, which consists of two barrel-like modules of around 100 amino acids each. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an immunoglobulin-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin-like repeats. A minimal D1IgD2 fragment is required for binding LIF. By using transmembrane forms of deletion mutants in gp190 ectodomain, we demonstrated that removal of D1 led to spontaneous activation of the receptor and that this property was devoted to a peptidic sequence localized within the last 42 amino acids of the carboxyl-terminal module of D2. By using soluble forms of deletion mutants made by progressive truncations from the end of the D1IgD2 fragment, we demonstrated that the carboxyl-terminal module of D2 was dispensable for LIF binding and that the correct conformation of the D1Ig fragment required a full amino-terminal module of D2. Therefore, the two constitutive modules of the membrane-proximal CRH domain D2 of gp190 fulfill two distinct roles in gp190 function, i.e. in stabilizing the conformation of gp190 allowing LIF binding and in activating the receptor.     

The upper cytokine-binding module and the Ig-like domain of the leukaemia inhibitory factor (LIF) receptor are sufficient for a functional LIF receptor complex.

To elucidate the function of the two cytokine-binding modules (CBM) of the leukemia inhibitory factor receptor (LIFR), receptor chimeras of LIFR and the interleukin-6 receptor (IL-6R) were constructed. Either the NH(2)-terminal (chimera RILLIFdeltaI) or the COOH-terminal LIFR CBM (chimera RILLIFdeltaII) were replaced by the structurally related CBM of the IL-6R which does not bind LIF. Chimera RILLIFdeltaI is functionally inactive, whereas RILLIFdeltaII binds LIF and mediates signalling as efficiently as the wild-type LIFR. Deletion mutants of the LIFR revealed that both the NH(2)-terminal CBM and the Ig-like domain of the LIFR are involved in LIF binding, presumably via the LIF site III epitope. The main function of the COOH-terminal CBM of the LIFR is to position the NH(2)-terminal CBM and the Ig-like domain, so that these can bind to LIF. In analogy to a recently published model of the IL-6R complex, a model of the active LIFR complex is suggested which positions the COOH-terminal CBM at LIF site I and the NH(2)-terminal CBM and the Ig-like domain at site III. An additional contact is postulated between the Ig-like domain of gp130 and the NH(2)-terminal CBM of the LIFR.     

Analysis of the leukemia inhibitory factor receptor functional domains by chimeric receptors and cytokines

In contrast to other hematopoietic cytokine receptors, the leukemia inhibitory factor receptor (LIFR) possesses two cytokine binding modules (CBMs). Previous studies suggested that the NH(2)-terminal CBM and the Ig-like domain of the LIFR are most important for LIF binding and activity. Using the recently engineered designer cytokine IC7, which induces an active heterodimer of the LIFR and gp130 after binding to the IL-6R, and several receptor chimeras of the LIFR and the interleukin-6 receptor (IL-6R) carrying the CBM of the IL-6R in place of the COOH-terminal LIFR CBM, we could assign individual receptor subdomains to individual binding sites of the ligand. The NH(2)-terminal CBM and the Ig-like domain of the LIFR bind to ligand site III, whereas the COOH-terminal CBM contacts site I. Furthermore, we show that LIFR mutants carrying the IL-6R CBM instead of the COOH-terminal CBM can replace the IL-6R by acting as an alpha-receptor for IL-6. However, in situations where a signaling competent receptor is bound at IL-6 site I, ligand binding to site III is an absolute requirement for participation of the receptor in a signaling heterodimer with gp130; i.e., a functional receptor complex of IL-6 type cytokines cannot be assembled solely via site I and II as in the growth hormone receptor complex.     

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.     

Crystal Structure of the Entire Ectodomain of gp130: INSIGHTS INTO THE MOLECULAR ASSEMBLY OF THE TALL CYTOKINE RECEPTOR COMPLEXES*

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1–6, D1–D6) at 3.6 Å resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4–D6) at 1.9 Å. This represents the first atomic resolution structure of the complete ectodomain of any “tall” cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.     

Membrane distal cytokine binding domain of LIFR interacts with soluble CNTFR in vitro

Ciliary neurotrophic factor (CNTF) is a member of the gp130 family of cytokines. The functional receptor complex of CNTF is composed of the CNTF receptor alpha (CNTFR), gp130 and the leukemia inhibitory factor receptor (LIFR). Three regions on CNTF have been identified as binding sites for its receptors. The ligand-receptor interactions are mediated through the cytokine binding domains (CBDs) and/or the immunoglobulin-like domains of the receptors. However, in the case of CNTF, the precise nature of the protein-protein contacts in the signaling complex has not yet been resolved. In this study, we provide the first demonstration that the membrane distal CBD (CBD1) of LIFR associates in vitro with soluble CNTFR in the absence of CNTF. Moreover, purified CBD1 partially blocks CNTF signaling, but not that of interleukin-6 or LIF, in human embryonal carcinoma cell line Ntera/D1 cells. These data raise the possibility that LIFR has the capability to form a ligand-free complex with CNTFR.     

GP130/OSMR is the only LIF/IL-6 family receptor complex to promote osteoblast differentiation of calvaria progenitors

Leukemia inhibitory factor (LIF) and its receptor (LIFR) are "twins" of Oncostatin M (OSM) and OSMR, respectively, likely having arisen through gene duplications. We compared their effects in a bone nodule-forming model of in vitro osteogenesis, rat calvaria (RC) cell cultures. Using a dominant-negative LIF mutant (hLIF-05), we showed that in RC cell cultures mouse OSM (mOSM) activates exclusively glycoprotein 130 (gp130)/OSMR. In treatments starting at early nodule formation stage, LIF, mOSM, IL-11, and IL-6 + sIL-6R inhibit bone nodule formation, that is, osteoprogenitor differentiation. Treatment with mOSM, and no other cytokine of the family, in early cultures (day 1-3 or 1-4) increases bone colony numbers. hLIF-05 also dose dependently stimulates bone nodule formation, confirming the inhibitory action of gp130/LIFR on osteogenesis. In pulse treatments at successive stages of bone nodule formation and maturation, LIF blocks osteocalcin (OCN) expression by differentiated osteoblasts, but has no effect on bone sialoprotein (BSP) expression. Mouse OSM inhibits OCN and BSP expression in preconfluent cultures with no or progressively reduced effects at later stages, reflecting the disruption of early nodules, possibly due to the strong apoptotic action of mOSM in RC cell cultures. In summary, LIFR and OSMR display differential effects on differentiation and phenotypic expression of osteogenic cells, most likely through different signal transduction pathways. In particular, gp130/OSMR is the only receptor complex of the family to stimulate osteoprogenitor differentiation in the RC cell culture model.     

Differential regulation of leukemia inhibitory factor-stimulated neuronal gene expression by protein phosphatases SHP-1 and SHP-2 through mitogen-activated protein kinase-dependent and -independent pathways

The neurally active cytokine leukemia inhibitory factor (LIF) signals through a bipartite receptor complex composed of LIF receptor alpha (LIFR) and gp130. gp130 and LIFR contain consensus binding motifs for the protein tyrosine phosphatase SHP-2 surrounding tyrosines 118 and 115 (Y118 and Y115) of their cytoplasmic domains, respectively. These sites are necessary for maximal activation of mitogen-activated protein kinase (MAPK). Coexpression of catalytically inactive, but not wild-type, SHP-2 reduced LIFR- and gp130-mediated activation of MAPK up to 75%. Conversely, coexpression of the wild-type, but not catalytically inactive, SHP-1, a related phosphatase, reduced activity up to 80%, demonstrating that SHP-2 and SHP-1 have opposing effects on the MAPK pathway. Mutation of Y115 of the cytoplasmic domain of LIFR eliminates receptor-mediated tyrosine phosphorylation of SHP-2. In contrast, SHP-1 association with gp130 and LIFR is constitutive and independent of Y118 and Y115, respectively. SHP-1 has a positive regulatory role on LIF-stimulated vasoactive intestinal peptide (VIP) reporter gene expression in neuronal cells, whereas the effect of SHP-2 is negative. Furthermore, LIF-stimulated MAPK activation negatively regulates this VIP reporter gene induction. SHP-2 also negatively regulates LIF-dependent expression of choline acetyltransferase, but this regulation could be dissociated from its effects on MAPK activation. These data indicate that SHP-1 and SHP-2 are important regulators of LIF-dependent neuronal gene expression via both MAPK-dependent and -independent pathways.     

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.