title: The N-terminus of gp130 is critical for the formation of the high-affinity interleukin-6 receptor complex.
abstract: Interleukin-6 (IL-6) mediates its activity through binding to two cell-surface receptors. The high-affinity human IL-6 receptor complex consists of two transmembrane anchored subunits: a ligand-specific, low-affinity IL-6 receptor and the high-affinity converter and signal transducing, gp130. Previously, using recombinant forms of human IL-6 and the extracellular ('soluble') domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130), we have shown that the high-affinity IL-6R complex is hexameric, consisting of two molecules each of IL-6, sIL-6R and sgp130 (Ward et al., 1994, J. Biol. Chem. 269: 23286-23289). This paper investigates the role of the N-terminal region of gp130 in the formation of the high-affinity IL-6R complex. Using recombinant sgp130 produced with a FLAG octapeptide epitope (DYKDDDDK) at the N-terminus (sgp130-FLAG), we demonstrate, using biosensor analysis and size-exclusion chromatography, that modification of the N-terminus of sgp130 interferes with the in vitro in solution formation of the stable hexameric IL-6 receptor complex. Rather, sgp130-FLAG interacts with IL-6 and sIL-6R with a much lower affinity and forms a stable lower-order ternary complex. However, this lower-order complex is inconsistent with the solution molecular weight of a trimeric complex, as measured by size-exclusion chromatography. In contrast, N-terminal modification of the sgp130 with the FLAG epitope did not interfere with the binding of leukemia inhibitory factor or oncostatin-M (other cytokines that signal through gp130) to sgp130. These data support our model of the hexameric IL-6 receptor complex, which is biased towards the association of two IL-6.IL-6R.gp130 trimers, and postulates the critical involvement of the N-terminal Ig-like domain of gp130 in tethering the two trimers to form the stable hexamer (Simpson et al., 1997, Prot. Sci. 6: 929-955).
authors.affiliation: Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Melbourne, Australia.
authors.forename: R L
authors.initials: RL
authors.lastname: Moritz
authors.forename: L D
authors.initials: LD
authors.lastname: Ward
authors.forename: G F
authors.initials: GF
authors.lastname: Tu
authors.forename: L J
authors.initials: LJ
authors.lastname: Fabri
authors.forename: H
authors.initials: H
authors.lastname: Ji
authors.forename: K
authors.initials: K
authors.lastname: Yasukawa
authors.forename: R J
authors.initials: RJ
authors.lastname: Simpson
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D000970:Antineoplastic Agents; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods; D002850:Chromatography, Gel; D050822:Cytokine Receptor gp130; D016207:Cytokines; D000939:Epitopes; D005656:Fungal Proteins / Q000378:metabolism; D006131:Growth Inhibitors; D006801:Humans; D018836:Inflammation Mediators; D015850:Interleukin-6 / Q000378:metabolism*; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D053683:Oncostatin M; D010455:Peptides; D010843:Pichia; D011485:Protein Binding; D011487:Protein Conformation; D019947:Receptors, Interleukin-6 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis; D020349:Surface Plasmon Resonance
chemical_list: D015703:Antigens, CD; D000970:Antineoplastic Agents; D016207:Cytokines; D000939:Epitopes; D005656:Fungal Proteins; D006131:Growth Inhibitors; C494199:IL6ST protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D008562:Membrane Glycoproteins; C508351:OSM protein, human; D010455:Peptides; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D053683:Oncostatin M; D050822:Cytokine Receptor gp130