Molecular modeling-guided mutagenesis of the extracellular part of gp130 leads to the identification of contact sites in the interleukin-6 (IL-6)center dot IL-6 receptor center dot gp130 complex

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 sis 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 B'C' loop (Val(252)) and in the F'G' loop (Gly(306), Lys(307)) of domain 3 and in the hinge region (Tyr(21S)) 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.