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.
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 B'C' loop (Val252) and in the F'G' 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.
Horsten
U
U
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany.
Müller-Newen
G
G
Gerhartz
C
C
Wollmer
A
A
Wijdenes
J
J
Heinrich
P C
PC
Grötzinger
J
J
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019556:COS Cells; D002455:Cell Division; D050822:Cytokine Receptor gp130; D005455:Fluorescent Antibody Technique; D015850:Interleukin-6 / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D013329:Structure-Activity Relationship; D014162:Transfection
D015703:Antigens, CD; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D050822:Cytokine Receptor gp130