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 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated 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 of 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 separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.
D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D015638:Cytochalasin D / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry; D016184:Receptors, Colony-Stimulating Factor / Q000737:chemistry*; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism