Identification of Residues Critical to the Activity of Human Granulocyte Colony-Stimulating Factor

Alanine scanning mutagenesis of human granulocyte colony-stimulating factor (G-CSF) was used to identify residues critical for the cell-proliferative activity of the protein. Fifty-eight residues, most of them on the protein surface, were independently mutated to alanine. Most of the variants retain...

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Bibliographic Details
Published in:Biochemistry (Easton) 1996-07, Vol.35 (28), p.9034-9041
Main Authors: Reidhaar-Olson, John F, De Souza-Hart, Janet A, Selick, Harold E
Format: Article
Language:English
Subjects:
Alanine - chemistry
Alanine - metabolism
Animals
Binding Sites
Cell Division
Cloning, Molecular
Escherichia coli - genetics
Gene Expression
Genes, Synthetic
Granulocyte Colony-Stimulating Factor - chemistry
Granulocyte Colony-Stimulating Factor - genetics
Granulocyte Colony-Stimulating Factor - metabolism
Granulocyte Colony-Stimulating Factor - pharmacology
Humans
Hydrogen Bonding
Mice
Models, Molecular
Mutagenesis, Site-Directed
Protein Binding
Receptors, Granulocyte Colony-Stimulating Factor - chemistry
Receptors, Granulocyte Colony-Stimulating Factor - metabolism
Tumor Cells, Cultured
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Summary:Alanine scanning mutagenesis of human granulocyte colony-stimulating factor (G-CSF) was used to identify residues critical for the cell-proliferative activity of the protein. Fifty-eight residues, most of them on the protein surface, were independently mutated to alanine. Most of the variants retained full biological activity; however, 15 mutants were significantly impaired in their ability to stimulate bone marrow cell proliferation in vitro. Four of these variants contain mutations at buried residues and two have substitutions at side chains involved in intramolecular hydrogen bonds. The remaining nine down mutations identify two regions on the surface of the molecule important for biological activity. Consistent with these observations, measurements of binding to NFS-60 cells indicate that the residues most important for receptor binding are Lys40 and Phe144 in site 1 and Glu19 in site 2. In addition to these residues, Val48 and Leu49 in site 1 and Leu15, Asp112, and Leu124 in site 2 are also important for biological activity. These results suggest the presence of two binding sites on the cytokine surface required for dimerization of the G-CSF receptor.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi952705x