Cysteine 647 in the insulin receptor is required for normal covalent interaction between alpha- and beta-subunits and signal transduction.
We have investigated the structural and functional properties of two mutant insulin receptors in which Cys647 and Cys682,683,685 have been replaced with Ser (IRS647 and IRS682,683,685, respectively). Compared with the wild-type receptor (IRWT), both mutant receptors displayed altered sensitivities to dithiothreitol with respect to insulin binding and reduction of oligomeric forms. Subunit composition of the oligomeric forms of the receptors as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors indicated that Cys682,683,685 are required for normal heterotetrameric structure and that Cys647 plays a major role in the normal covalent association of the alpha- and beta-subunits. Under nonreducing conditions, the affinity-labeled IRS647 migrated, almost exclusively, as a 230-kDa species which appeared to represent an alpha 2 form of the receptor. Furthermore, Chinese hamster ovary cells expressing IRS647 did not exhibit basal or insulin-stimulated autophosphorylation, suggesting that Cys647 is also required for signal transduction.
Cheatham
B
B
Joslin Diabetes Center, Department of Medicine, Boston, Massachusetts 02215.
Kahn
C R
CR
D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000378:metabolism; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007328:Insulin; D007457:Iodine Radioisotopes; D012694:Serine; D004247:DNA; D011972:Receptor, Insulin; D003545:Cysteine; D004229:Dithiothreitol