Abstract
The insulin receptor is a transmembrane protein dimer composed of two alphabeta monomers held together by inter-alpha-chain disulfide bonds. In a previous report we described a monomeric insulin receptor obtained by replacing Cys-524, -682, -683, and -685 with serine. The membrane-bound monomeric insulin receptors could be cross-linked to dimers in the presence of insulin, indicating that although covalent interactions had been abolished, noncovalent dimerization could still occur in the membrane. To eliminate noncovalent dimerization, we replaced all or some of Cys-524, -682, -683, and -685 with arginine or aspartic acid with the expectation that the electrostatic repulsion at these contact sites would prevent noncovalent dimerization. The results indicate that mutant insulin receptors that are able to form covalent dimers are expressed at the wild type level; mutants that can form noncovalent dimers are expressed at half the level of the wild type receptor, whereas insulin receptor mutants that cannot dimerize are expressed at less than 10% of the wild type level. To elucidate the mechanism of the decrease in expression of the mutant insulin receptors, we examined their subcellular localization and biosynthesis. The results suggest that the extent of expression of these mutant receptors is related to their ability to form covalent or noncovalent dimers at the proreceptor stage.
Highlights
The insulin receptor subunits are linked by disulfide bonds [7]
The membrane-bound monomeric insulin receptors could be cross-linked to dimers in the presence of insulin, indicating that covalent interactions had been abolished, noncovalent dimerization could still occur in the membrane
The results indicate that mutant insulin receptors that are able to form covalent dimers are expressed at the wild type level; mutants that can form noncovalent dimers are expressed at half the level of the wild type receptor, whereas insulin receptor mutants that cannot dimerize are expressed at less than 10% of the wild type level
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM), LipofectAMINE, fetal bovine serum, and phosphate-buffered saline (PBS) were purchased from Invitrogen. [125I]Insulin (50 pM) was incubated with the cells for 12 h at 4 °C in binding buffer containing 100 mM HEPES, pH 7.4, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 10 mM glucose, and 1% bovine serum albumin. The cells were washed 3 times with ice-cold binding buffer, solubilized in 0.1% SDS, and counted in a ␥ counter. The cells were scraped off the plates with a rubber policeman after a 5-min incubation in PBS supplemented with 5 mM Na2EDTA, pelleted by centrifugation at 1000 ϫ g for 3 min, and resuspended in 2 ml of ice-cold HEPES buffer (10 mM, pH 7.4) containing 50 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 2.5 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml chymostatin, and 1 g/ml pepstatin.
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