CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction.
Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alpha-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross-reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure-function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix. Selected variants contained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha-independent receptor actions of CNTF. These CNTF analogs thus represent receptor-specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine.
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Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Rome.
Gloaguen
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Poiana
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Laufer
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002642:Chick Embryo; D020934:Ciliary Neurotrophic Factor; D003601:Cytotoxicity Tests, Immunologic; D004306:Dose-Response Relationship, Immunologic; D005786:Gene Expression Regulation / Q000235:genetics; D014644:Genetic Variation; D006242:Haptoglobins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics* / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000276:immunology; D017354:Point Mutation; D018121:Receptors, Cytokine / Q000276:immunology*; D014407:Tumor Cells, Cultured
D020934:Ciliary Neurotrophic Factor; D006242:Haptoglobins; D008024:Ligands; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D018121:Receptors, Cytokine