Abstract
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.
Highlights
From the Research Division,Joslin Diabetes Center, Department of Medicine, Brigham and Women’s Hospitaland Harvard Medical School, Boston, Massachusetts02215
C(IRyEs664477aanndd ICRySBsSe2s,z68~36.68863,~r6essspheacbvteieevnelyre).pClaocmedpawrietdhwSietrh the wild-type receptor (IRwT), both mutant receptors displayed altered sensitivities to dithiothreitol with respect to insulin binding and reduction of oligomeric forms
Chinese hamster ovarycells expressing IRS647did not exhibit basal or insulin-stimdistribution of Cys residues involved in the covalent attachment of receptor subunits, the exact Cys residues involved ineitherclass I or I1 interactionsareunknown
Summary
Were removed, and the cells were treated with 1 ml PBS, 0.1%BSA containing 25 mM NEM for 10 min at room temperature, followedby two washes in ice-cold PBS. '251-Insulinbinding (10 PM)was carried. Insulin-stimulated Receptor Autophosphorylation in Intact CellsAutophosphorylation in intact cells was done as previously described the Cys mutants, intact cells were incubated for 10 min at room temperature in PBS (pH 7.4) containing 0.1% BSA in the absence or presence of increasing concentrations of DTT (0-10mM).Any free sulfhydryl groups that were generated by the reduction with DTT were blocked by alkylation with [21]. Following removal of any excess NEM, '251-insulin phate-free medium (Gibco) containing 0.5 mCi/ml [32P]orthophos- binding was carried out overnight at 4 "C. phate (Du Pont-New England Nuclear), followedby stimulation with DTT treatment of cells expressing wild-type insulin recep-. Pellets were washed three times with 1 ml of buffer containing 50 mM HEPES contrast, insulin binding to both IRS682*683a,6n8d5IRS647decreased following treatment with the lowest concentration of DTT used (0.5 mM). CHO cells transfected with pSVEneo; or ZW k h -
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