TI  - Two distinct and independent sites on <prot>IL-6</prot> trigger   <prot>gp 130</prot>   dimer formation
      and signalling.
PG  - 1942-51
AB  - The helical cytokine  <prot>interleukin (IL) 6</prot>  and its specific binding subunit
       <prot>IL-6R alpha</prot>  form a 1:1 complex which, by promoting homodimerization of the
      signalling subunit   <prot>gp130</prot>   on the surface of target cells, triggers
      intracellular responses. We expressed differently tagged forms of <prot>gp130</prot>
      and used them in solution-phase binding assays to show that the soluble
      extracellular domains of   <prot>gp130</prot>   undergo dimerization in the absence of
      membranes. In vitro receptor assembly reactions were also performed in the
      presence of two sets of <prot>IL-6</prot> 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 <prot>IL-6R alpha</prot> of these variants is normal
      but their biological activity is poor or absent. We demonstrate here that
      both the site 2 and site 3 <prot>IL-6</prot> variants complexed with <prot>IL-6R alpha</prot> bind a
      single <prot>gp130</prot> molecule but are unable to dimerize it, whereas the combined
      site 2/3 variants lose the ability to interact with <prot>gp130</prot>. 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   <prot>gp130</prot>   dimer is formed through direct binding at two
      independent and differently oriented sites on <prot>IL-6</prot>. Immunoprecipitation
      experiments further reveal that the fully assembled receptor complex is
      composed of two  <prot>IL-6</prot> , two  <prot>IL-6R alpha</prot>  and two  <prot>gp130</prot>  molecules. We propose
      here a model representing the <prot>IL-6</prot> receptor complex as hexameric, which
      might be common to other helical cytokines.
AD  - Istituto di Ricerche di Biologia Molecolare (IRBM), Pomezia, Rome, Italy.
