Abstract
A mutant insulin receptor was constructed by replacing cysteine residues Cys(524), Cys(682), Cys(683), and Cys(685) with serine. The mutant was expressed in COS7 and Chinese hamster ovary cells, did not form covalently linked dimers, and was present at the cell surface. There was half as much insulin binding activity at the cell surface in cells expressing the mutant compared with that in cells expressing the wild type receptor. The intracellular processing of the mutant receptor was affected, since its beta-subunit migrated more slowly than that of the wild type receptor on SDS-PAGE. The mutant was capable of insulin-dependent autophosphorylation and phosphorylation of insulin receptor substrate-1 in vivo and could be cross-linked into receptor dimers when membrane-bound. The amount of insulin-dependent autophosphorylation of the mutant receptor was half that of the wild type receptor. However, after solubilization the monomeric insulin receptor had minimal autophosphorylation activity, and, unlike the naturally occurring monomeric receptor tyrosine kinases, the solubilized monomeric insulin receptor did not dimerize in response to insulin binding as determined by sucrose density gradient centrifugation.
Highlights
In a previous work, we reported that the cysteine residues involved in the class I disulfides between the ␣ chains were Cys524 and Cys682 [9]
Construction of a Monomeric Insulin Receptor—Previously, we identified Cys524 and Cys682 as the residues involved in the class I disulfide bonds between the ␣ chains [9]
All of the other mutants with only three mutations of these residues are present as a mixture of dimers and monomers, as is the IRC524S,C682S mutant previously described
Summary
We reported that the cysteine residues involved in the class I disulfides between the ␣ chains were Cys524 and Cys682 [9]. Whereas the involvement of Cys524 in the class I disulfides had been suggested by other investigators (6 – 8), evidence that at least one of the cysteine residues of the triplet at positions 682, 683, and 685 formed an additional disulfide bond was presented subsequently by Sparrow et al. We became concerned that the dimerization might be caused by formation of disulfide bonds between Cys683 and Cys685 To investigate this possibility, we constructed another insulin receptor by replacing Cys683 and Cys685, in addition to Cys524 and Cys682, with serine. The new receptors did not form covalent dimers, were expressed on the cell surface, and underwent autophosphorylation in response to insulin while membrane-bound. In the monomeric state the insulin receptors had decreased insulin-dependent kinase activity
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