Recombinant human CNTF receptor alpha: production, binding stoichiometry, and characterization of its activity as a diffusible factor.
The primary ligand-binding protein (CNTFR alpha) of the multicomponent receptor for ciliary neurotrophic factor was produced in Escherichia coli. Using novel applications of size-exclusion chromatography and a protein gel-shift assay, we obtained quantitative separation of correctly refolded protein, as well as analytical monitoring of the refolding process and ligand binding. By these and other methods, we determined a 1:1 stoichiometry for the receptor-ligand complex. To investigate the proposed activity and mechanism of soluble CNTFR alpha as a diffusible factor, we studied the response of TF-1 cells which lack CNTFR alpha to various CNTF ligands and the stimulation of this response by sCNTFR alpha. The results show that sCNTFR alpha combines with CNTF and mediates cell survival with the same relative ligand specificity and relative affinity as the cell-surface form. Thus, soluble receptor can reconstitute on a cell surface active complexes that are analogous to the native complexes. Moreover, both the relative ligand potency in the absence of CNTFR alpha and the kinetics of the response to sCNTFR alpha indicate that the other components of the receptor complex contribute little, but measurably, to the specific potency of CNTF.
Panayotatos
N
N
REGENERON Pharmaceuticals Inc., Tarrytown, New York 10591-6707.
Everdeen
D
D
Liten
A
A
Somogyi
R
R
Acheson
A
A
D000818:Animals; D002470:Cell Survival / Q000502:physiology; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D009419:Nerve Tissue Proteins / Q000502:physiology; D010957:Plasmids; D017510:Protein Folding; D051381:Rats; D020801:Receptor, Ciliary Neurotrophic Factor; D017978:Receptors, Growth Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020934:Ciliary Neurotrophic Factor; D009419:Nerve Tissue Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins