Solubilization and assay of a colony-stimulating factor receptor from murine macrophages.
The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).
D000818:Animals; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000378:metabolism; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D016184:Receptors, Colony-Stimulating Factor; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism
D003115:Colony-Stimulating Factors; D011956:Receptors, Cell Surface; D016184:Receptors, Colony-Stimulating Factor