Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity.
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)
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
D046911:Macromolecular Substances; D010748:Phosphopeptides; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin