Construction and characterization of ciliary neurotrophic factor (CNTF) antagonists: microenvironmental difference in the CNTF receptor between rat and chicken cells for recognizing the D1 cap region.
Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.
Inoue
M
M
Sumitomo Pharmaceuticals Research Center, Konohana-ku, Osaka, Japan.
Karita
H
H
Nakayama
C
C
Noguchi
H
H
D000818:Animals; D002470:Cell Survival / Q000502:physiology; D002645:Chickens; D020934:Ciliary Neurotrophic Factor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D006131:Growth Inhibitors; D006801:Humans; D015850:Interleukin-6; D007457:Iodine Radioisotopes; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D016296:Mutagenesis / Q000502:physiology; D009414:Nerve Growth Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D009447:Neuroblastoma; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D020801:Receptor, Ciliary Neurotrophic Factor; D018121:Receptors, Cytokine / Q000737:chemistry / Q000235:genetics; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry / Q000235:genetics*; D053606:Receptors, OSM-LIF; D014407:Tumor Cells, Cultured
D020934:Ciliary Neurotrophic Factor; D003432:Cross-Linking Reagents; D006131:Growth Inhibitors; D015850:Interleukin-6; D007457:Iodine Radioisotopes; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; C507842:Lifr protein, rat; D008222:Lymphokines; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D018121:Receptors, Cytokine; D017475:Receptors, Nerve Growth Factor; D053606:Receptors, OSM-LIF