IDENTIFICATION OF CILIARY NEUROTROPHIC FACTOR (CNTF) RESIDUES ESSENTIAL FOR LEUKEMIA INHIBITORY FACTOR-RECEPTOR BINDING AND GENERATION OF CNTF RECEPTOR ANTAGONISTS

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subuni t (CNTEPR alpha) and the signal transducers gp130 and leukemia inhibit ory factor receptor-beta (LIFR). The D1 structural motif, located at t he beginning of the D-helix of human CNTF, contains two amino acid res idues, F152 and K155, which are conserved among all cytokines that sig nal through LIFR, The functional importance of these residues was asse ssed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LI FR without affecting binding to CNTFR alpha or gp130, The resulting va riants behaved as partial agonists with varying degrees of residual bi oactivity in different cell-based assays. Simultaneous alanine substit ution of both F152 and K155 totally abolished biological activity. Com bining these mutations with amino acid substitutions in the D-helix, w hich enhance binding affinity for the CNTFR alpha, gave rise to a pote nt competitive CNTF receptor antagonist, This protein constitutes a ne w tool for studies of CNTF function in normal physiology and disease.