Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity
The authors have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the ..beta..-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22/sup 0/C with low concentrations of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the ..beta..-subunit was carried out before and after digestion, and the (/sup 32/P)phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the ..beta..-subunit (..cap alpha..Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the (/sup 32/P)phosphate originally found in the ..beta..-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. To determined the structural requirements for kinase activity, the insulin receptor was subjected to tryptic digestion for 30 s-30 min, such that the receptor was composed exclusively of 85- and 70-kDa fragments of the ..beta..-subunit. The 85-kDa fragment exhibited autophosphorylation at pY1, pY1a, and pY4. Both the 85- and 70-kDa fragments phosphorylated tyrosine residues in a synthetic decapeptide that has the sequence of the C-terminal domain of the ..beta..-subunit of human insulin rare in the receptor.
- Research Organization:
- Harvard Medical School, Boston, MA
- OSTI ID:
- 5514760
- Journal Information:
- Biochemistry; (United States), Vol. 26:8
- Country of Publication:
- United States
- Language:
- English
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62 RADIOLOGY AND NUCLEAR MEDICINE
RECEPTORS
AUTORADIOGRAPHY
MOLECULAR STRUCTURE
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
INSULIN
LIQUID COLUMN CHROMATOGRAPHY
PHOSPHORUS 32
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ENZYMES
HORMONES
HYDROLYSIS
ISOTOPES
LIGHT NUCLEI
LYSIS
MEMBRANE PROTEINS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
RADIOISOTOPES
SEPARATION PROCESSES
SOLVOLYSIS
TRANSFERASES
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