Autophosphorylation of the insulin receptor in vitro. Designation of phosphorylation sites and correlation with receptor kinase activation.
Chemical degradation and antipeptide antibodies were used to study alterations in the structure and function of the human placental insulin receptor following autophosphorylation in vitro. Antibodies elicited to residues 1143-1162 (P2) of the human insulin proreceptor immunoprecipitated the native, phosphorylated receptor but not the unphosphorylated receptor. Since this antibody recognizes both forms of the receptor on immunoblots, it was concluded that the accessibility of the P2 domain to the antibody is increased by in vitro autophosphorylation. Chemical cleavage at either tryptophan or methionine residues followed by immunoprecipitation with antipeptide antibodies was used to map the in vitro autophosphorylation sites of the beta subunit of the insulin receptor. Two phosphorylated fragments were resolved. One, recognized by antibody elicited to amino acid residues 1328-1343 (P5), is derived from the carboxyl terminus of the beta subunit and includes tyrosine 1316. The other, recognized by antibody to P2, is located in a domain that includes tyrosine 1150. The rate of phosphorylation of this latter site correlates with the rate of activation of the insulin receptor kinase during in vitro autophosphorylation. The results support the following conclusions: autophosphorylation alters the conformation of the beta subunit of the insulin receptor; autophosphorylation in vitro leads to phosphorylation of tyrosine residues near the carboxyl terminus of the protein and in the P2 domain that includes tyrosine 1150; activation of the insulin receptor kinase correlates with autophosphorylation of the domain containing tyrosine 1150.
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000276:immunology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013997:Time Factors
D000906:Antibodies; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000266:Adenylyl Imidodiphosphate; D011494:Protein Kinases; D011972:Receptor, Insulin; D003488:Cyanogen Bromide