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 [32P]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 immunoprecipitation with IGF-I receptor monoclonal antibodies (alpha IR-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 [35S]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.
Lopaczynski
W
W
Endocrinology Section, National Cancer Institute, Bethesda, MD 20892.
Harris
S
S
Nissley
P
P
D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D012516:Osteosarcoma; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine; D017526:Receptor, IGF Type 1 / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1