CDB15:0000305 CNTF — CNTFR

ABSTRACT: A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described. The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag. Bound cytokine is revealed by alkaline phosphatase-conjugated avidin. Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively. Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution. Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis. The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF. The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems. The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants.

ABSTRACT: Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low affinity LIF receptor. For CNTF, addition of a third subunit, or alpha subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high-affinity LIF receptor, by adding a soluble form of the alpha CNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/alpha CNTF receptor bound to gp130 with an affinity of 3-5 x 10(-10)M, whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.