Dynamics of the gp130 cytokine complex: a model for assembly on the cellular membrane

Abstract

Cytokines of the interleukin-6 (IL-6)-type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL-6, interleukin-11, ciliary neurotrophic factor, neuropoietin, cardiotrophin-1, and cardiotrophin-1-like-cytokine, interact with specific ligand binding receptor proteins. High- and low-affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine-receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc-fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL-6 and interleukin-b receptor (IL-6R) by means of fluorescence-correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell-free condition the binding affinities and dynamics of IL-6 and IL-6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL-6/IL-6R complex and only at higher ligand concentrations does it bind a second IL-6/IL-6R complex. This view contrasts with the current perception of IL-6 receptor activation and reveals an alternative receptor activation mechanism.

Figure 1.

Purification and characterization of the recombinant gp130-Fc fusion protein. (A) Analysis of the different purification steps by SDS PAGE. M, molecular weight marker. Three different sgp130Fc concentrations were used. (B) Size-exclusion chromatography elution profile of the recombinant gp130-Fc fusion protein monitored at 280 nm. The void volume is denoted by a, and fraction b corresponds to the expected size of the sgp130Fc. The absorption is given in arbitrary units (au). (C) CD spectrum of the gp130-Fc fusion protein in the far UV.

Figure 2.

Fluorescence labeling of Hyper-IL-6. H-IL-6 was labeled using the affinity labeling dye EVOblue50. The diffusion times of the pure labeling dye and two labeled H-IL-6 fractions are shown.

Figure 3.

Fluorescence correlation spectroscopy (FCS). (A) Titration of the labeled Hyper-IL-6 with increasing concentrations of sgp130Fc, FCS Analysis 1-component fit, measurement time 5 × 30 sec, determined K_D = 0.06 nM. (B) Competition titration with varying concentrations of unlabeled Hyper-IL-6 (1 nM sgp130Fc, 0.5 nM labeled Hyper-IL-6), FCS Analysis 1-component fit, measurement time 5 × 30 sec, determined IC₅₀ = 0.86 nM.

Figure 4.

Fluorescence intensity multiple distribution analysis (FIMDA). (A) Titration of labeled H-IL6 (open squares, 1 nM H-Il-6; closed triangles, 10 nM Hyper-IL-6) with increasing concentrations of sgp130Fc, FIMDA 1 component analysis (only the molecular brightness is depicted), measurement time 5 × 30 sec, brightness maximum for low concentration of labeled Hyper-IL-6 (open squares, 1 nM H-Il-6) at 0.5 nM sgp130Fc, brightness maximum for high concentration of labeled Hyper-IL-6 (closed triangles, 10 nM H-Il-6) at 5 nM sgp130Fc. (B) Titration of labeled Hyper-IL-6 with increasing concentrations of sgp130Fc, FIMDA 3 component analysis, Hyper-IL-6* free: brightness, 25.3 kHz; diffusion time, 654 μsec. (sgp130Fc)(Hyper-IL-6*)² complex: brightness, 48.1 kHz; diffusion time, 1200 μsec. (sgp130Fc)(Hyper-IL-6*) complex: brightness, 24.3 kHz; diffusion time, 1144 μsec.

Figure 5.

Two possibilities of Interleukin-6 receptor assembly. In both reaction pathways the first step is the formation of an IL-6/IL-6R complex. The second step is the association with one gp130 molecule and subsequent dimerization of this complex to form the final hexameric complex (A); the association with two gp130 molecules leading to the tetrameric complex, which then can bind another IL-6/IL-6R complex to form the hexameric complex (B).