Solubilization and assay of a colony-stimulating factor receptor from murine macrophages

✍ Scribed by Yee-Guide Yeung; Paul T. Jubinsky; E. Richard Stanley

• Publisher: John Wiley and Sons
• Year: 1986
• Tongue: English
• Weight: 680 KB
• Volume: 31
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.240310403

✦ Synopsis

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 5774.2 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 -150%. The binding of '*'I-CSF-l to intact cells and membrane preparations was consistent with the existence of a single class of highaffinity receptor sites. In contrast, the equilibrium binding of 1251-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent &s 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 highaffiity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0°C and -70°C but dissociated at 37°C. Dissociation also occurred at 0°C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl) .