Ligand Binding Characteristics of the Carboxyl-terminal Domain of the Cytokine Receptor Homologous Region of the Granulocyte Colony-stimulating Factor Receptor

• Published in: The Journal of biological chemistry 1995-11, Vol.270 (46), p.27845-27851
• Main Authors: Anaguchi, H, Hiraoka, O, Yamasaki, K, Naito, S, Ota, Y
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters; Base Sequence; Carrier Proteins - biosynthesis; Cloning, Molecular; Disulfides - analysis; Escherichia coli; Escherichia coli Proteins; Factor Xa - metabolism; Kinetics; Magnetic Resonance Spectroscopy; Maltose-Binding Proteins; Mice; Models, Structural; Molecular Sequence Data; Monosaccharide Transport Proteins; Oligodeoxyribonucleotides; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Mapping; Plasmids; Protein Structure, Secondary; Receptors, Cytokine - chemistry; Receptors, Granulocyte Colony-Stimulating Factor - biosynthesis; Receptors, Granulocyte Colony-Stimulating Factor - chemistry; Receptors, Granulocyte Colony-Stimulating Factor - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.46.27845

The carboxyl-terminal domain (BC domain, roughly 100 amino acid residues) of the cytokine receptor homologous region in the receptor for murine granulocyte colony-stimulating factor was secreted as a maltose binding protein fusion into the Escherichia coli periplasm. The murine BC domain was prepared from the fusion protein by restriction protease factor Xa digestion and was purified to homogeneity. The purified BC domain specifically and stoichiometrically bound granulocyte colony-stimulating factor. This result indicates that the BC domain is also critical for ligand binding, as shown for the amino-terminal domain of the cytokine receptor homologous region (Hiraoka, O., Anaguchi, H., Yamasaki, K., Fukunaga, R., Nagata, S., and Ota, Y.(1994) J. Biol. Chem. 269, 22412-22419). The tertiary folding and the β-sheet structure of the BC domain were confirmed by NMR spectroscopy. The disulfide bond pattern suggested from peptide mapping was Cys -Cys and Cys -Cys . Disruption of the disulfide bonds suggested that both bonds are critical for maintaining the folding of the BC domain, although a BC domain lacking the second bond still retained ligand binding activity. Mutational analysis of the WS X WS sequence conserved in the cytokine receptor family suggested that this motif is critical for protein folding rather than for ligand binding.