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Volume 272, Number 38, Issue of September 19, 1997 pp. 23748-23757
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Modeling-guided Mutagenesis of the Extracellular Part of gp130 Leads to the Identification of Contact Sites in the Interleukin-6 (IL-6)·IL-6 receptor·gp130 Complex

(Received for publication, March 25, 1997, and in revised form, May 9, 1997)

Ursula Horsten , Gerhard Müller-Newen , Claudia Gerhartz , Axel Wollmer , John Wijdenes ^§ , Peter C. Heinrich and Joachim Grötzinger

From the  Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany and ^§ Diaclone, 1 Bd Fleming BP 1985, F-25020 Besançon Cedex, France

The transmembrane protein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal-transducing subunit. In the case of interleukin-6 (IL-6), the signal-transducing complex is composed of the ligand IL-6, the IL-6 receptor (IL-6R, gp80, CD126), and at least two gp130 (CD130) molecules. The extracellular part of the signal transducer gp130 consists of six fibronectin type III-like domains. It has recently been shown that the three membrane distal domains bind to the IL-6·IL-6R complex. A structural model of the IL-6·IL-6R·gp130 complex enabled us to propose amino acid residues in these domains of gp130 interacting with IL-6 bound to its receptor. The proposed amino acid residues located in the BC loop (Val²⁵²) and in the FG loop (Gly³⁰⁶, Lys³⁰⁷) of domain 3 and in the hinge region (Tyr²¹⁸) connecting domains 2 and 3 of gp130 were mutated to disturb ternary complex formation. Binding of wild type and mutants of the extracellular region of gp130 was studied by use of a co-precipitation assay and Scatchard analysis. All mutants showed decreased binding to the IL-6·IL-6R complex. Biological function of the membrane-bound gp130 mutants was studied by STAT (signal transducer and activator of transcription) activation in COS-7 cells and by proliferation of stably transfected Ba/F3 cells. Reduced binding of the mutants was accompanied by decreased biological activity. The combined approach of molecular modeling and site-directed mutagenesis has led to the identification of amino acid residues in gp130 required for complex formation with IL-6 and its receptor.

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