title: Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity.
abstract: We 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 degrees C with low concentrations (5-10 micrograms/mL, pH 7.4) of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the beta-subunit was carried out before and after digestion, and the [32P]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. Mild trypsin digestion reduced the apparent molecular mass of the beta-subunit from 95 to 85 kDa, and then to 70 kDa. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the beta-subunit (alpha Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the [32P]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. Treatment of the intact receptor with staphylococcal V8 protease also converted the beta-subunit to an 85-kDa fragment that did not bind to alpha Pep-1, contained about 50% of the initial radioactivity, and lacked pY2 and pY3. Elastase rapidly degraded the receptor to inactive fragments between 37 and 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D012697:Serine Endopeptidases; D014357:Trypsin / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D010748:Phosphopeptides; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin