Evidence for exogenous substrate phosphorylation and dimer formation by the activated 190 kDa insulin proreceptor in vitro.
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 alpha-casein, as similarly observed for the mature receptor. Structurally, the phosphorylated proreceptor was identified as a 360 kDa homodimer under non-reducing condition.
Arakaki
R F
RF
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Comi
R J
RJ
Gorden
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D004220:Disulfides; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008214:Lymphocytes / Q000032:analysis; D046911:Macromolecular Substances; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014909:Wheat Germ Agglutinins
D004220:Disulfides; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D010455:Peptides; D011498:Protein Precursors; D014909:Wheat Germ Agglutinins; C027606:L-glutamic acid-L-tyrosine copolymer; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin