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 (original) (raw)
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In Vitro Reconstitution of Recognition and Activation Complexes between Interleukin-6 and gp130
Biochemistry, 2001
Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific R-receptor (RR) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6RR/ gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its RR, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-RR and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6RR, and 1 gp130-CHR. Addition of the N-terminal (D1) Iglike domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6RR, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.
The EMBO journal, 1993
Here, we report the analysis of the structure-function relationship of the extracellular region of human interleukin 6 receptor (IL-6R). Upon binding of IL-6, IL-6R becomes associated extracellularly with a non-IL-6-binding but signal transducing molecule, gp130, and the IL-6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin-like domain, was responsible both for IL-6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL-6R (sIL-6R) could bind IL-6 and mediate IL-6 functions through gp130, amino acid substitutions were introduced into sIL-6R by site-directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL-6 binding have a tendency to be distributed to the hinge region between the two 'barrel'-like fibronectin type III modules and to the same side of these two 'barrels'. Amino aci...
Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130
Cell, 1989
Interleukin-6 mediates pleiotropic functions in various types of cells through its specific receptor (IL-6-R) the cDNA of which has already been cloned. We report here that an 60 kd single polypeptide chain (IL-6-R) is involved in IL-6 binding and that IL-6 triggers the association of this receptor with a non-ligand-binding membrane glycoprotein, gp130. The association takes place at 37°C within 5 min and is stable for at least 40 min in the presence of IL-6, but does not occur at 0%. Human IL-6-R can associate with a murine gpl30 homolog and is functional in murine cells. Mutant IL-6-R lacking the intracytoplasmic portion is functional, suggesting that the two polypeptide chains interact to involve their extracellular portion. In fact, a soluble IL-6-R lacking the transmembrane and intracytoplasmic domains can associate with gp130 in the presence of IL-6 and mediate its function. These findings indicate that the complex of IL-6 and IL-6-R can interact with a non-ligand-binding membrane glycoprotein, gp130, extracellularly and can provide the IL-6 signal.
Cytokine, 1995
Interleukin 6 (IL-6) is a cytokine with a myriad of biological activities and has been implicated in the pathogenesis of autoimmune, inflammatory and neoplastic diseases (for reviews, see Refs 1-3). The biological activities of IL-6 result from interaction of the cytokine with a complex receptor consisting of two transmembrane glycoproteins. IL-6 first binds with low affinity to an 80 kDa receptor chain (IL-6Rα). The IL-6/IL-6Rα complex then binds with high affinity to the 130 kDa signal transducing chain (gp130 or IL-6R), leading to disulphide linked homodimerization and tyrosine phosphorylation of this molecule and signal transduction. 4 The IL-6 signal transduction pathways are just beginning to be unravelled. Recently it was shown that gp130 can associate with various signal transducing molecules including the transcription factor APRF/STAT3, 5-7 the JAK-Tyk family of protein kinases 4,5,8,9 and the Src-related tyrosine kinase Hck. 10 The IL-6Rα is specific for IL-6, while gp130 is also part of the high affinity receptors for oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and IL-11, explaining the functional redundancy of this family of cytokines. 11-16 Also at the structural level similarities between these cytokines can be observed: they all belong to the group of long chain cytokines with a similar antiparallel four ␣-helical bundle core structure as growth hormone. 17,18 At the amino acid sequence level these proteins have two regions with notable similarities. 19,20 One of these regions is called the D1-motif and for IL-6 is formed by the amino acids Asn156-Thr164. It is intriguing to speculate that this region is involved in contact formation to the protein gp130 which is common to the receptor complexes of both IL-6, LIF, OM, CNTF and IL-11.
Journal of Biological Chemistry, 2000
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 gp130activating 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.
Journal of Biological Chemistry, 1998
The transmembrane protein gp130 is a shared component of the receptor complexes for the interleukin-6 (IL-6)-type cytokines, which include IL-6, leukemia inhibitory factor (LIF) and oncostatin M (OSM). In addition to its role in the generation of high affinity receptors, gp130 is required for signal transduction by these cytokines. In the present study we have examined the role of the N-terminal located, extracellular immunoglobulin (Ig)-like module of gp130 in signal transduction by IL-6 and LIF. We have expressed wild-type human gp130 or three mutants in murine myeloid M1-UR21 cells that lack functional endogenous gp130 but express the IL-6 receptor (IL-6R) and the LIF receptor (LIFR). By measuring cellular responses, such as morphological changes upon differentiation, soft agar colony formation, and induction of tyrosine phosphorylation of the signal transducer and activator of transcription, STAT3, we show that signaling by IL-6, but not LIF, is significantly reduced by mutations in the Ig-like module of gp130. However, the binding of 125 I-labeled IL-6 or LIF is not affected by these mutations. We also present evidence that the Ig-like module forms part of the epitope of an anti-gp130 monoclonal antibody that neutralizes the bioactivity of IL-6, but not of LIF or OSM. The data suggest that gp130-activation by IL-6 and LIF requires different regions of gp130, that the Ig-like module of gp130 may be required for IL-6-induced gp130 dimerization, and that the stoichiometry of the high affinity IL-6 receptor-complex differs from those of the receptorcomplexes for LIF and OSM.