Evidence for exogenous substrate phosphorylation and dimer formation by the activated 190 kDa insulin proreceptor in vitro

• Published in: Biochemical and biophysical research communications Vol. 160; no. 1; pp. 296 - 302
• Main Authors: Arakaki, Richard F., Comi, Richard J., Gorden, Phillip
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 14.04.1989; Elsevier
• Subjects: Adenosine Triphosphate - metabolism; Biological and medical sciences; Cell Line; Cell receptors; Cell structures and functions; Chromatography; Disulfides; Fundamental and applied biological sciences. Psychology; Immunosorbent Techniques; Insulin - pharmacology; lymphocytes; Lymphocytes - analysis; Macromolecular Substances; Molecular and cellular biology; Peptides - metabolism; Phosphorylation; Protein Precursors - isolation & purification; Protein Precursors - metabolism; Protein Processing, Post-Translational; Protein-Tyrosine Kinases - metabolism; Receptor, Insulin - drug effects; Receptor, Insulin - isolation & purification; Receptor, Insulin - metabolism; Wheat Germ Agglutinins; Cell culture; Pancreatic hormone; Phosphorylation; Gel electrophoresis; Enzyme; Activation; Glycoproteins; In vitro; Insulin; Precursor; Protein hormone; Enzymatic method; Immunoprecipitation reaction; Affinity chromatography; Protein kinase; Dimerization; Lymphocyte; Hormonal receptor
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(89)91655-0

The insulin receptor is synthesized as a single chain, 190 kDa glycoprotein precursor, which undergoes proteolytic cleavage, carbohydrate processing, and fatty acylation to generate the mature receptor on the plasma membrane. The relationship of these post-translational modifications to the acquisition of receptor function, i.e. ligand binding and phosphokinase activity, is not fully understood. Therefore, the 190 kDa proreceptor and mature receptor kinase activities were separately examined in vitro, and their phosphorylation properties compared. The solubilized receptor precursor from IM-9 lymphocytes was purified by sequential lectin chromatography and, following site specific anti-receptor antibody immunoprecipitation, phosphokinase studies performed. The isolated proreceptor was activated by insulin and phosphorylated exogenous substrate α-casein, as similarly observed for the mature receptor. Structurally, the phosphorylated proreceptor was identified as a 360 kDa homodimer under non-reducing condition.