Identification of the alpha beta monomer of the adipocyte insulin receptor by insulin binding and autophosphorylation.
The insulin receptor consists of an insulin-binding subunit (alpha) of 135,000 daltons. More recently, it has been documented that the receptor undergoes insulin-stimulated autophosphorylation that predominantly labels a 95,000-dalton (beta) subunit. We solubilized rat adipocyte insulin receptors in Triton X-100 and partially purified the protein on a wheat germ agglutinin-Sepharose affinity column. Subsequently, we labeled the two subunits of the receptor independently by using 125I-labeled insulin for the 135,000-dalton alpha-subunit and 32P for the 95,000-dalton beta-subunit. Sucrose density gradient sedimentation and NaDodSO4/PAGE were used to characterize the native, oligomeric structure of the receptor. In 0.1% Triton X-100, the receptor sedimented as a single peak of s20,w = 10.2 S as detected by both 125I and 32P. NaDodSO4/PAGE under nonreducing conditions revealed a large species that appeared to be alpha 2 beta 2 and, to a lesser extent, alpha beta. Treatment of the solubilized, partially purified receptor with 10 mM dithiothreitol led to the partial conversion of the 10.2S species to a smaller one sedimenting at 6.6 S. The composition of this species was determined to be alpha beta by nonreducing NaDodSO4/PAGE. Our results suggest that detergent-solubilized insulin receptors can exist as dimers and monomers. The oligomeric structure of receptors functional in the cell membrane cannot be immediately deduced from these results due to the possibility of artifacts arising from membrane disruption and extraction procedures. However, the ability to label the two subunits of the receptor separately should facilitate a detailed study of its oligomeric structure both in solution and in the membrane.
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*
D007328:Insulin; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; C008147:insulin, iodo-; D011972:Receptor, Insulin