Influence of Interleukin-6 (IL-6) Dimerization on Formation of the High Affinity Hexameric IL-6·Receptor Complex

Abstract

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6 M ) and dimeric (IL-6 D ) forms of Escherichia coli-derived human IL-6 and the extracellular ("soluble") portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6 D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6 M is more potent than IL-6 D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37°C of IL-6 dimers into active monomers. The increased binding affinity of IL-6 D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6 D resulted from a decreased ability of the IL-6 D ⅐(sIL-6R) 2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6⅐IL-6R⅐gp130) 2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6 M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.

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).…”

Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry

“…Recombinant IL-6 D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6 D (sIL-6R) 2 complex (16). In contrast to the binary IL-6⅐sIL-6R complex, IL-6 D (sIL-6R) 2 binds gp130 weakly and does not show significant biological activity in the signal transducer and activator of transcription 3 (STAT3) phosphorylation assay (16).…”

“…Previously, we have shown that a dimeric form of recombinant IL-6 (IL-6 D ) is a potent antagonist for IL-6 signaling (16). Recombinant IL-6 D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6 D (sIL-6R) 2 complex (16).…”

“…Recombinant IL-6 D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6 D (sIL-6R) 2 complex (16). In contrast to the binary IL-6⅐sIL-6R complex, IL-6 D (sIL-6R) 2 binds gp130 weakly and does not show significant biological activity in the signal transducer and activator of transcription 3 (STAT3) phosphorylation assay (16). Natural (glycosylated) human IL-6 is also known to form a dimer that makes up a substantial part of IL-6 in blood or fibroblast secretions (17)(18)(19) and has also been shown to interact with membrane-bound IL-6R (15,20,59).…”

Function and Molecular Modeling of the Interaction between Human Interleukin 6 and Its HNK-1 Oligosaccharide Ligands

“…The question remained to explain how the lectin activity could participate to the formation of these hexamers. The recombinant soluble form of gp130 used in the previous studies for detecting the formation of hexamers (73)(74)(75)(76) was produced in Chinese hamster ovary cells. This point seems of importance, since these cells are (as HepG2 cancer cell types) competent for the synthesis of the HNK-1 epitope.…”