Purification and characterization of the receptor for murine granulocyte colony-stimulating factor.
A receptor for mouse granulocyte colony-stimulating factor (G-CSF) has been found on the cell surface of mouse myeloid leukemia cell line NFS-60. Chemical cross-linking of the receptor with radioiodinated G-CSF, followed by gel electrophoresis in the presence of sodium dodecyl sulfate, has revealed that the G-CSF receptor in the NFS-60 cells is a single polypeptide of Mr approximately 100,000-130,000. The receptor in the membrane fraction of NFS-60 cells were solubilized in an active form with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid. The solubilized receptor was purified approximately 100,000-fold to near homogeneity using a G-CSF affinity gel and gel filtration on a Superose 12 column, as measured by the selective precipitation of the 125I-G-CSF-receptor complex by polyethylene glycol. The purified G-CSF receptor has two classes of binding characteristics, one with an equilibrium dissociation constant (Kd) of 120-360 pM which is comparable with the Kd value for the cell-surface receptor, and the other with a higher Kd value of 2.6-4.2 nM. Analyses of the purified receptor by ligand blotting and sucrose density gradient centrifugation indicated that the low-affinity receptor is the monomer of the Mr 100,000-130,000 protein, whereas the high-affinity receptor consists of oligomers of the protein.
Osaka Bioscience Institute, Japan.
R
R
Fukunaga
E
E
Ishizaka-Ikeda
S
S
Nagata
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003115:Colony-Stimulating Factors / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016179:Granulocyte Colony-Stimulating Factor; D007700:Kinetics; D007942:Leukemia, Experimental; D051379:Mice; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003115:Colony-Stimulating Factors; D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor