Relationship between the subunit structure of insulin receptor and its competence to bind insulin and undergo phosphorylation.
Insulin receptor partially purified from human placenta by chromatography on immobilized wheat germ agglutinin was subjected to affinity cross linking to determine the relationship between the subunit structure of the multiple forms of the insulin receptor and their competence to bind insulin and undergo autophosphorylation. It was demonstrated that, whereas the 340-kDa intact receptor undergoes autophosphorylation, the 290- and 320-kDa insulin binding forms of the receptor do not. Phosphorylation at tyrosyl residues in the intact receptor was verified using a new facile method for determination of phosphorylated amino acids. The competence of the phosphorylated 340-kDa protein to bind insulin was demonstrated using a double-probe labeling protocol wherein receptor phosphorylated with [gamma-32P]ATP was cross-linked with disuccinimidyl suberate (DSS) in the presence of N epsilon B29-biotinylinsulin. The observation that succinylavidin, by virtue of its interaction with biotinyl residues, decreased the electrophoretic mobility of receptor radiochemically labeled with 32P indicated that the phosphorylated 340-kDa protein was competent to bind insulin. This result is compelling evidence that the 340-kDa phosphorylated species is insulin receptor itself, rather than a closely associated contaminant. Treatment of the receptor with the crosslinking agent DSS produced (after reduction and denaturation) alpha-dimer, beta-dimer, and a smaller amount of tetramer. This observation is consistent with a symmetrical, tetrameric, alpha 2 beta 2 structure for insulin receptor from human placenta, and excludes previously proposed alternative structures containing one alpha and one beta chain.
D000255:Adenosine Triphosphate / Q000378:metabolism; D003432:Cross-Linking Reagents; D005260:Female; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D011972:Receptor, Insulin / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D003432:Cross-Linking Reagents; D007328:Insulin; D046911:Macromolecular Substances; D013388:Succinimides; C025360:insulin, biotinyl-; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin; C019358:disuccinimidyl suberate