title: Evidence for the formation of a heterotrimeric complex of leukaemia inhibitory factor with its receptor subunits in solution.
abstract: Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high-affinity, functional, receptor complex. Human LIF binds with unusually high affinity to a naturally occurring mouse soluble LIF receptor alpha-chain, and this property was used to purify a stable complex of human LIF and mouse LIF receptor alpha-chain from pregnant-mouse serum. Recombinant soluble human gp130 was expressed, with a FLAG(R) epitope (DYKDDDDK) at the N-terminus, in the methylotropic yeast Pichia pastoris and purified using affinity chromatography. The formation of a trimeric complex in solution was established by native gel electrophoresis, gel-filtration chromatography, sedimentation equilibrium analysis, surface plasmon resonance spectroscopy and chemical cross-linking. The stoichiometry of this solution complex was 1:1:1, in contrast with that of the complex of interleukin-6, the interleukin-6-specific low-affinity receptor subunit and gp130, which is 2:2:2.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research and The Cooperative Research Centre for Cellular Growth Factors, P.O. Royal Melbourne Hospital, Victoria 3050, Australia.
authors.forename: J G
authors.initials: JG
authors.lastname: Zhang
authors.forename: C M
authors.initials: CM
authors.lastname: Owczarek
authors.forename: L D
authors.initials: LD
authors.lastname: Ward
authors.forename: G J
authors.initials: GJ
authors.lastname: Howlett
authors.forename: L J
authors.initials: LJ
authors.lastname: Fabri
authors.forename: B A
authors.initials: BA
authors.lastname: Roberts
authors.forename: N A
authors.initials: NA
authors.lastname: Nicola
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D050822:Cytokine Receptor gp130; D006131:Growth Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015398:Signal Transduction; D012996:Solutions
chemical_list: D015703:Antigens, CD; D006131:Growth Inhibitors; C494199:IL6ST protein, human; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D050822:Cytokine Receptor gp130