Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.
The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.
Paonessa
G
G
Istituto di Ricerche di Biologia Molecolare (IRBM), Pomezia, Rome, Italy.
Graziani
R
R
De Serio
A
A
Savino
R
R
Ciapponi
L
L
Lahm
A
A
Salvati
A L
AL
Toniatti
C
C
Ciliberto
G
G
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D001665:Binding Sites; D002460:Cell Line; D050822:Cytokine Receptor gp130; D014644:Genetic Variation; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019947:Receptors, Interleukin-6; D015398:Signal Transduction
D015703:Antigens, CD; C494199:IL6ST protein, human; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D050822:Cytokine Receptor gp130