Characterization of unprocessed insulin proreceptors in COS 7 cells transfected with cDNA with Arg735----Ser735 point mutation at the cleavage site.
We previously reported on patients with severe insulin resistance due to unprocessed insulin proreceptors. A structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G----T point mutation appeared to be the cause for failure to process the proreceptors. To determine whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin receptors and to elucidate the structural and binding characteristics of the proreceptors, we transfected cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in cells transfected with either normal or mutated cDNA, and insulin binding was 40 and 14 times higher than that of nontransfected cells, respectively. The declining rate of insulin binding after reaching the maximum was delayed in cells transfected with mutated cDNA. Affinity cross-linking and surface-labeling studies showed a 135-kilodalton (kD), normal alpha-subunit in the cells transfected with normal cDNA and a 210-kD proreceptor in the mutant cells. The proreceptors were cleaved by trypsin to yield normal-sized alpha- and beta-subunits. The sensitivity to trypsin was similar to that demonstrated in patients' cells, and the most effective concentration for the cleavage was 0.025%. Autophosphorylation resulted in decreased 32P incorporation into proreceptors of cells transfected with mutated cDNA at both basal and insulin-stimulated states, without a change in insulin sensitivity. Competitive binding studies with insulin, proinsulin, and miniproinsulin showed that the proreceptors had a lower relative affinity for proinsulin, but this characteristic disappeared after trypsin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Sugibayashi
M
M
Third Department of Medicine, Shiga University of Medical Science, Japan.
Shigeta
Y
Y
Teraoka
H
H
Kobayashi
M
M
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics*; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D010766:Phosphorylation; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D007328:Insulin; D015345:Oligonucleotide Probes; D011498:Protein Precursors; C051512:insulin proreceptor; D004247:DNA; D011972:Receptor, Insulin; D014357:Trypsin