Dithiothreitol Activation of the Insulin Receptor/Kinase Does Not Involve Subunit Dissociation of the Native α₂β₂ Insulin Receptor Subunit Complex

The subunit composition of the dithiothreitol-(DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing (0.1% SDS) or nondenaturing (0.1% Triton X-100) conditions. Pretreatment of ³²P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α₂β₂ insulin receptor complex (M_r 400 000) into the monomeric 95 000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS—polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α₂β₂ heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M_r 400 000 complex after the removal of DTT by gel filtration chromatography. Surprisingly, these apparently reoxidized insulin receptors were also observed to be functional with respect to insulin binding, albeit with a 50% decrease in affinity for insulin and insulin stimulation of the β subunit autophosphorylation. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT followed by incubation with excess N-ethylmaleimide prior to gel filtration chromatography in 0.1% Triton X-100. Under these conditions the insulin receptors migrated as the M_r 400 000 α₂β₂ complex. However, when this insulin receptor complex was subjected to nonreducing SDS—polyacrylamide gel electrophoresis, subsequent to gel filtration, only the M_r 95 000 β subunit was detected. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α₂β₂ insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α₂β₂ insulin receptor subunit complex.