Comparison of insulin receptor tyrosine phosphorylation under in vitro and in situ conditions: assessment of specific protein tyrosine phosphorylation without the use of 32P-phosphate-labeled substrates.
An assay which permitted a comparison of insulin receptor tyrosine phosphorylation induced under in vivo and in situ conditions was developed. It was demonstrated that the level of tyrosine phosphorylation of the receptor induced by insulin under in situ conditions exceeded by 2.3-fold the level of tyrosine phosphorylation induced under in vitro conditions. In addition, chronically treated, down-regulated cells demonstrated a significant decrease in the level of insulin receptor tyrosine phosphorylation compared to cells acutely treated with insulin. The procedure utilized an antibody specific for the insulin receptor which allowed separation of the receptor from other cellular proteins which are potential substrates for tyrosine phosphorylation. A second iodinated antibody specifically directed against phosphotyrosine residues allowed quantitation of the phosphorylated residues. Since the use of 32P-labeled substrates for phosphorylation was not required, this procedure allowed a comparison of protein phosphorylation induced under in vitro and in situ conditions. This procedure should permit investigations into the roles of other cellular factors, such as phosphatases and serine kinases, in modulating protein tyrosine phosphorylation.
Knutson
V P
VP
Department of Pharmacology, University of Texas Medical School, Houston 77225.
Buck
R A
RA
D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D007457:Iodine Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine; D011863:Radioimmunoassay; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D007328:Insulin; D007457:Iodine Radioisotopes; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin