Characterization of the insulin receptor kinase from human erythrocytes.
The insulin receptor from human erythrocytes was studied for receptor-associated tyrosine kinase activity. Receptor phosphorylation was performed by incubation of the receptor with [gamma-32P]ATP and Mn2+ in the presence or absence of insulin, and the phosphorylated receptor was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In Triton X-100-solubilized, and partially purified, receptor preparations, insulin stimulated the phosphorylation of a 95,000 dalton protein in a dose-dependent fashion. Immunoprecipitation with antiinsulin receptor antibodies indicates that this 95,000 dalton protein corresponds to the beta-subunit of the insulin receptor. Phosphoaminoacid analysis revealed that 32P incorporation occurred predominantly on tyrosine residues of the beta-subunit. In addition, the insulin receptor kinase catalyzed the phosphorylation of Histone H2b. Dose response curves for insulin-stimulated phosphorylation of the beta-subunit and Histone H2b were sigmoidal. Both half-maximal effects were observed at 3 X 10(-9) M insulin with maximal effects at 10(-6) M. When insulin binding was examined under the same conditions as phosphorylation, the dose-response curves for receptor occupancy and the kinase activation were nearly superimposable, indicating few or no spare receptors for this response to insulin. Insulin-stimulated receptor phosphorylation was diminished by treatment with N-ethylmaleimide, indicating that the receptor possesses sulfhydryl groups, which are important for its enzyme activity. This study demonstrates that insulin receptor kinase from human erythrocytes shares characteristics which are similar to those found in other classical insulin target cells and tissues.
D000227:Adenine Nucleotides / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D011972:Receptor, Insulin / Q000378:metabolism
D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D006657:Histones; D007328:Insulin; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D005033:Ethylmaleimide