Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro
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 one major and two 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, two 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 our experimental conditions, no significant change in the amount of (TSP)phosphoserine or (TSP)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-TSP)ATP and MnS , very little phosphorylation occurred in the absence of insulin.
- Research Organization:
- Harvard Medical School, Boston, MA
- OSTI ID:
- 6085870
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 16; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELEMENTS
HORMONES
HYDROXY ACIDS
IN VITRO
IN VIVO
INSULIN
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
METALS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
RADIOISOTOPES
RATS
RECEPTORS
RODENTS
SEPARATION PROCESSES
SERINE
THREONINE
TRACER TECHNIQUES
TRANSITION ELEMENTS
TUMOR CELLS
VANADIUM
VERTEBRATES