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1 Evotec Technologies GmbH, D-40225 Düsseldorf, Germany
2 Biochemisches Institut, Christian Albrechts Universität zu Kiel, D-24098 Kiel, Germany
(RECEIVED September 13, 2004; FINAL REVISION November 17, 2004; ACCEPTED November 18, 2004)
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.
Keywords: gp130; cytokine; confocal fluorescence correlation spectroscopy; interleukin-6
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041117105.
Reprint requests to: Joachim Grötzinger, Biochemisches Institut, Christian Albrechts Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany; e-mail: jgroetzinger{at}biochem.uni-kiel.de; fax: +49-431-880-5007.
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