EVIDENCE FOR THE FORMATION OF A HETEROTRIMERIC COMPLEX OF LEUKEMIA INHIBITORY FACTOR WITH ITS RECEPTOR SUBUNITS IN SOLUTION

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF recep tor alpha-chain and gp130) in order to form a high-affinity, functiona l, 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 LI F 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 usin g affinity chromatography. The formation of a trimeric complex in solu tion was established by native gel electrophoresis, gel-filtration chr omatography, sedimentation equilibrium analysis, surface plasmon reson ance spectroscopy and chemical crosslinking. The stoichiometry of this solution complex was 1:1:1, in contrast with that of the complex of i nterleukin-6, the interleukin-6-specific low-affinity receptor subunit and gp130, which is 2:2:2.