INSULIN-LIKE GROWTH FACTOR-I (IGF-I) DEPENDENT PHOSPHORYLATION OF THE IGF-I RECEPTOR IN MG-63 CELLS

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63. by acting through the IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of the IGF-I receptor in this cell line. Serum starved MG-63 cells were metabolically labeled with [P-32]orthophosphoric acid and the cells were treated with IGF-I. Phosphotyrosine containing proteins were immunoprecipitated from the cell lysates with antiphosphotyrosine-Agarose and eluted with phenyl phosphate. Further immmunoprecipitation with IGF-I receptor monoclonal antibodies (alphaIR-3, 18E9) and analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography demonstrated IGF-I dependent autophosphorylation of the IGF-I receptor. Phosphoamino acid analysis of the IGF-I receptor beta subunit and the observation that antiphosphotyrosine-Agarose did not immunoprecipitate [S-35]methionine-labeled receptor from unstimulated cells, demonstrated that in the absence of IGF-I, the receptor was not phosphorylated on tyrosine residues. Western blotting of cell lysates with a monoclonal phosphotyrosine antibody did not identify the IGF-I receptor or pp185 but demonstrated IGF-I dependent phosphorylation on tyrosine residues in three other proteins, p110, p70 and p40.