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Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

, : Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro. Journal of Biological Chemistry 260(16): 9470-9478

Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the .beta.-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed 1 major and 2 minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10-7 M) increased the phosphorylation of the .beta.-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, 2 phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under the experimental conditions, no significant change in the amount of [32P]phosphoserine or [32P]phosphothreonine associated with the .beta.-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with [.gamma.-32P]ATP and Mn2+, very little phosphorylation occurred in the absence of insulin. In this preparation, insulin rapidly stimulated autophosphorylation of the receptor on tyrosine residues only and high performance liquid chromatography analysis of the .beta.-subunit digested with trypsin revealed 1 minor and 2 major phosphopeptides. The elution position of the minor peptide corresponded to that of the major phosphotyrosine-containing peptide obtained from the .beta.-subunit of the insulin-stimulated receptor labeled in vivo. The elution position of 1 of the major phosphopeptides that occurred during in vitro phosphorylation corresponded to the minor phosphotyrosine-containing peptide phosphorylated in vivo. The other major in vitro phosphotyrosine-containing peptide was not detected in vivo. Apparently, tyrosine phosphorylation of the insulin receptor occurs rapidly following insulin binding to intact cells; the level of tyrosine phosphorylation remains constant for up to 1 h; the specificity of the receptor kinase or accessbility of the phosphorylation sites are different in vivo and in vitro. Apparently, tyrosine phosphorylation is an early intracellular signal in insulin action, but that caution should be used when relating in vitro data to in vivo phosphorylation events.

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Accession: 005151596

PMID: 3848433

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