Construction and characterization of a monomeric insulin receptor.
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.
Wu
James Jianping
JJ
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Guidotti
Guido
G
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003545:Cysteine; D017931:DNA Primers; D019281:Dimerization; D007328:Insulin / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011972:Receptor, Insulin; D003545:Cysteine