Separation and characterization of the activated pool of colony-stimulating factor 1 receptor forming distinct multimeric complexes with signalling molecules in macrophages

Colony-stimulating factor I (CSF-I) triggers the activation of intracellula r proteins in macrophages through selective assembly of signalling complexe s, The separation of multimeric complexes of the CSF-1 receptor (CSF-IR) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-l-stimulated cel ls that neither possessed detectable tyrosine kinase activity nor formed co mplexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes, A small poo l of CSF-IR formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of ty rosine-phosphorylated proteins in CSF-l-stimulated cells. The complex showe d a considerable amount of CSF-1R complex-associated kinase activity. A det ectable level of the complex was also present in untreated cells. PI-3 kina se in the multimeric complex displayed low lipid kinase activity despite th e association with several proteins. The major pool of activated CSF-IR for med transient multimeric complexes with distinctly different tyrosine-phosp horylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2, A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities o f PI-3 kinase in these complexes support the notion that the activity of PI -3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separat e CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of th e distinct pools of the CSF-1R were dependent on the integrity of the micro tubular network.