Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.
Plasma membrane insulin receptors, affinity labeled by covalent crosslinking to receptor-bound 125I-labeled insulin, are shown to appear as a heterogeneous population of three major disulfide-linked complexes (Mr 350,000, 320,000, and 290,000) upon electrophoresis in highly porous dodecyl sulfate/polyacrylamide gels in the absence of reductant. This pattern is consistent in all rat and human tissues that were analyzed. Upon reduction of disulfide bonds, each of these receptor structures is dissociated in two successive steps. Low concentrations of dithiothreitol promote a first step of disulfide bond reduction in which the Mr 350,000 species splits into a Mr 210,000 form and the Mr 290,000 species splits into a Mr 160,000 form. In contrast, both the Mr 210,000 and Mr 160,000 receptor fragments are generated from the native Mr 320,000 species upon partial reduction, indicating an asymmetrical structure. The second step of receptor reduction occurs upon treatment of the native disulfide-linked receptor complexes with high concentrations of dithiothreitol. Under these conditions, the Mr 350,000 receptor yields a Mr 125,000 subunit, denoted as alpha, and a Mr 90,000 subunit, denoted as beta, whereas the Mr 290,000 receptor dissociates into the alpha subunit and a Mr 49,000 subunit, denoted as beta 1. The Mr 320,000 receptor band is found to consist of alpha, beta, and beta 1 subunits upon complete reduction. The partially reduced Mr 210,000 receptor fragment is composed of the alpha subunit disulfide-linked to the beta subunit, whereas the Mr 160,000 species consists of the alpha subunit disulfide-linked to the beta 1 subunit. Thus, the stoichiometry of the three ubiquitous native insulin receptor structures of Mr 350,000, 320,000, and 290,000 are (alpha) 2 (beta) 2, (alpha) 2 (beta) (beta 1), and (alpha) 2 (beta 1) 2, respectively.
D000818:Animals; D003432:Cross-Linking Reagents; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011972:Receptor, Insulin / Q000032:analysis*; D014018:Tissue Distribution
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D011972:Receptor, Insulin