Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils.
We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism.
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Y
Y
Hanazono
T
T
Hosoi
T
T
Kuwaki
S
S
Matsuki
K
K
Miyazono
K
K
Miyagawa
F
F
Takaku
D000328:Adult; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000097:blood*; D011994:Recombinant Proteins / Q000378:metabolism; D013388:Succinimides
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D013388:Succinimides; D016179:Granulocyte Colony-Stimulating Factor; C019358:disuccinimidyl suberate