Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

Abstract

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL‐6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution‐phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL‐6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL‐6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL‐6 variants complexed with IL‐6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL‐6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL‐6, two IL‐6R alpha and two gp130 molecules. We propose here a model representing the IL‐6 receptor complex as hexameric, which might be common to other helical cytokines.

Interleukin‐6: Structure‐function relationships

“…This assumes that the receptors follow the structural pattern that has been observed in the GHR, PRLR, and EpoR, the class 1 cytokine receptors for which structures are known (de Vos et al, 1992;Livnah et al, 1996). For IL-6 and CNTF, such a three-site cytokine/receptor binding model is consistent with mutagenesis data and the finding that the ternary receptor-complexes of IL-6 (Ward et al, 1994b;Paonessa et al, 1995) and CNTF are hexameric (cf. The ternary complex).…”

“…Based on the assumption that the complex formation of IL-6 with its cellular receptors can be mimicked by soluble forms of IL-6R and gp130, we (Ward et al, 1994b) and, more recently, Paonessa et al (1995) showed that IL-6 mediates its actions on target cells through the formation of a hexameric complex comprising two molecules each of IL-6, ILdR, and gp130. While there are models of the formation and topology of the hexameric complex Ward et al, 1996), a detailed understanding of the interactions between IL-6 and its receptor molecules at a highresolution structural level is lacking.…”

“…7). As discussed below, IL-6 (Ward et al, 1994b;Paonessa et al, 1995) and CNTF form a hexameric receptor-complex consisting of two ligands, two ligand-binding receptors, and two signaling receptors.…”

“…This finding was conhomodimer gp130 f m e d by careful immunoprecipitation studies using differentially tagged wild-type and mutated IL-6 and sIL-6R as well as sgpl30 . The model proposed by Paonessa et al (1995), shown in Figure 8A, suggests that several interaction surfaces hold the complex together.…”

Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer

“…One of the key cytokines is IL-6, which was originally identified as B-cell stimulatory factor-2 and was shown to affect hybridoma growth and also stimulate antibody productivity (Makishima et al 1992). In the proposed mechanism, IL-6 binds to IL-6R and gp130 (Bravo et al 1998;Hibi et al 1990;Muller-Newen et al 2000) to form hexameric complex composed of two molecules of each component (Paonessa et al 1995;Skiniotis et al 2005;Ward et al 1996). This in turn triggers homodimerization and activation of gp130, leading to activation of the Janus kinase family (JAK1, JAK2, TYK2) of signal transducers, which further activate transcription activators (STAT1, STAT3) and mitogen-activated protein kinases (MAP-KKK, MAPKK, MAPK) (Matsuda et al 1999).…”