Studies on the Interleukin-6-type Cytokine Signal Transducer gp130 Reveal a Novel Mechanism of Receptor Activation by Monoclonal Antibodies

Abstract

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.Fast and reliable intercellular communication is a requirement for the development, maintenance, and function of multicellular organisms. A central mode of communication is the release of mediators by one cell that are recognized specifically by the cognate receptors on the plasma membrane of target cells. Receptors act like a kind of molecular switches that respond to ligand binding with the triggering of cytoplasmic signal transduction pathways. These signaling cascades often target to nuclear DNA leading to specific cellular responses like growth, differentiation, or even apoptosis. The molecular mechanisms of receptor activation by the ligand depends on the type of receptor involved. The commonly accepted activation mechanism for most receptors spanning the membrane only once is ligand-induced receptor dimerization (1, 2). Important receptors sharing this feature belong to the families of receptor tyrosine kinases and cytokine receptors.The type I transmembrane glycoprotein gp130 is a cytokine receptor that represents the common signal transducing receptor component of the IL-6

Determination of the Disulfide Structure andN-Glycosylation Sites of the Extracellular Domain of the Human Signal Transducer gp130

“…Thus, a conformational change in gp130 would be required to reposition this protective loop and expose Cys 469 to possible disulfide bond formation with another gp130 molecule. Mü ller-Newen and colleagues (73) have recently demonstrated, using a panel of monoclonal antibodies, that the enforcement of gp130 dimerization alone is not sufficient for receptor activation and that an additional conformation requirement is needed. A similar phenomenon has also been reported for the erythropoietin receptor (74) and is also applicable to other types of receptors from both eukaryotic and prokaryotic origins (75), where dimerization alone is not sufficient for intracellular signaling but a further conformational change within the receptor dimer induced by ligand binding may be essential to propagate the signal across the cell membrane.…”

Due to interleukin-6 type cytokine redundancy only glycoprotein 130 receptor blockade efficiently inhibits myeloma growth

“…14,28,29 These antibodies induce receptor dimerization, thereby mimicking the effect of the natural ligand. 30 The critical role for gp130 in early plasmacytoma development was recently demonstrated in a retroviral bone marrow transduction-trans-…”

Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer

“…One of the key cytokines is IL-6, which was originally identified as B-cell stimulatory factor-2 and was shown to affect hybridoma growth and also stimulate antibody productivity (Makishima et al 1992). In the proposed mechanism, IL-6 binds to IL-6R and gp130 (Bravo et al 1998;Hibi et al 1990;Muller-Newen et al 2000) to form hexameric complex composed of two molecules of each component (Paonessa et al 1995;Skiniotis et al 2005;Ward et al 1996). This in turn triggers homodimerization and activation of gp130, leading to activation of the Janus kinase family (JAK1, JAK2, TYK2) of signal transducers, which further activate transcription activators (STAT1, STAT3) and mitogen-activated protein kinases (MAP-KKK, MAPKK, MAPK) (Matsuda et al 1999).…”