Studies on the interleukin-6-type cytokine signal transducer gp130 reveal a novel mechanism of receptor activation by monoclonal antibodies.
The transmembrane glycoprotein gp130 belongs to the family of hematopoietic cytokine receptors. It represents the common signal transducing receptor component of the so called interleukin-6-type cytokines. For several cytokine receptors including gp130 it has been shown that receptor activation cannot only be achieved by the natural ligand but also by single monoclonal antibodies raised against the receptor ectodomain. These findings have been interpreted in a way that dimerization of cytokine receptors is sufficient for receptor activation. Here we show that the recently described gp130-activating antibody B-S12 actually consists of two different monoclonal antibodies. By subcloning of B-S12 the monoclonal antibodies B-S12-A5 and B-S12-G7 were obtained. The individual antibodies are biologically inactive, in combination they exert B-S12-like activity on hepatoma cells. On Ba/F3 cells stably transfected with gp130 a combination of B-S12-G7 with another monoclonal gp130 antibody, B-P8, is required to stimulate proliferation. Using gp130 deletion mutants we show that all three antibodies map to domains 2 and 3 of gp130 which constitute the cytokine binding module. The individual antibodies inhibit activation of the signal transducer by interleukin-6 and interfere with binding of interleukin-6 to gp130. Interestingly, the combination of B-S12-G7 and a Fab fragment of B-P8 retains biological activity. We conclude from our data that (i) the monoclonal antibodies activate gp130 by mimicking the natural ligand and (ii) enforcement of gp130 dimerization is not sufficient for receptor activation but additional conformational requirements have to be fulfilled.
Müller-Newen
G
G
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, D-52074 Aachen, Germany. mueller-newen@RWTH-Aachen.de
Küster
A
A
Wijdenes
J
J
Schaper
F
F
Heinrich
P C
PC
D000209:Acute-Phase Proteins / Q000096:biosynthesis; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D050822:Cytokine Receptor gp130; D017931:DNA Primers; D015850:Interleukin-6 / Q000276:immunology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009500:Neutralization Tests; D019947:Receptors, Interleukin-6 / Q000378:metabolism; D015398:Signal Transduction
D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D017931:DNA Primers; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D019947:Receptors, Interleukin-6; D050822:Cytokine Receptor gp130