Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential

Glycoprotein 130 (gp130) is a type I transmembrane,protein and serves as th e common signal-transducing receptor subunit of the interleukin-6-type cyto kines. Whereas the membrane-distal half of the gp130 extracellular part con fers ligand binding and has been subject to intense investigation, the stru ctural and functional features of its membrane-proximal half are poorly und erstood. On the basis of predictions of tertiary structure, the membrane-pr oximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of-the polypeptides predicted to comprise each of these three domains. The recombinant proteins were ref olded from solubilized inclusion bodies in vitro, purified to homogeneity a nd characterized by means of size-exclusion chromatography and CD spectrosc opy. For the first time the prediction of three individual membrane-proxima l protein domains for gp130 has been verified experimentally. The three dom ains do not show intermediate-affinity or high-affinity interactions betwee n each other. Mapping of a neutralizing gp130 monoclonal antibody against D 4 suggested a particular functional role of this domain for gp130 activatio n, because above that an intrinsic tendency for low affinity oligomerizatio n was demonstrated for D4.