Abstract
The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.
Highlights
In most instances, growth factors and cytokines drive the sequential assembly of multiprotein receptor complexes [1, 2]
Site-directed mutagenesis, and in vitro receptor binding assays have synergistically contributed during the last few years to defining the functional and structural determinants of the IL-6 molecule and the mechanism governing the assembly of a multi-subunit receptor complex
This process is mediated by interactions that two IL-61⁄7IL-6R␣ subcomplexes establish simultaneously with two gp130 receptor subunits
Summary
Growth factors and cytokines drive the sequential assembly of multiprotein receptor complexes [1, 2]. No three-dimensional structure has as yet been obtained, the receptor chains of the IL-6 family have all been shown to contain a conserved 220amino acid-long region called the cytokine binding domain, which is responsible for binding the cytokine and is predicted to fold as a tandem repeat of immunoglobulin-like -barrels, like the GH receptor (GHbp) [9, 12] Starting from these considerations, IL-6 interaction with one IL-6R␣ and one gp130 chain has been postulated by molecular modeling studies [13,14,15,16] as being similar to the binding of GH to the cytokine binding domains of its two receptor chains [9]. We used site 2, site 3, and site 2/3 mutants to prove that two gp130 molecules associate to the IL-61⁄7IL-6R␣ subcomplex with two topologically distinct orientations, which can be identified through suitable in vitro receptor binding and immunoprecipitation assays [18] Another IL-6 region, called 2a, which spans residues Thr43–Glu, has been found to take part in signaling [20]. Site 3 is defined as a composite gp130 binding site, and we discuss its functional and structural equivalence with a topologically conserved gp130 or LIFR binding site present in other cytokines of the same family
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