Insulin and IGF-I stimulate phosphorylation of their respective receptors in intact neuronal and glial cells in primary culture

Previous studies have shown that insulin and IGF-I bind to their respective receptors and stimulate autophosphorylation of the receptor β subunits in detergent extracts of neuronal and glial cells. In the present study, intact neuronal and glial cells in primary culture have been utilized to characterize insulin- and IGF-I-stimulated phosphorylation of their receptors. Following [³²P]orthophosphate labeling and stimulation by insulin or IGF-I, the cells were solubilized and the phosphorylated receptors were partially purified on wheat germ agglutinin-agarose columns, and immunoprecipitated using anti-phosphotyrosine or anti-insulin receptor antibodies. Insulin stimulated the phosphorylation of its receptor β subunit (95 kD phosphoprotein) in a dose-dependent manner, within at least 20 seconds in both neuronal and glial cells. Additionally, a 102-kD phosphoprotein was observed in insulin-stimulated neuronal cells. Maximal stimulation of receptor phosphorylation occurred at 1 minute for the glial cells, and 10 minutes for the neuronal cells. IGF-I stimulated the phosphorylation of two phosphoproteins in intact neuronal and glial cells; a 95-kD protein and a 102-kD protein, in a dose-dependent manner. These observations demonstrate that both insulin and IGF-I stimulate the phosphorylation of the β subunits of their respective receptors in brain cells in a similar fashion to their effects on receptors from nonneural tissues.