Transfection of cDNA with G----T point mutation at the cleavage site of insulin receptors to COS 7 cells.
To study whether the G----T point mutation of insulin proreceptors at the cleavage site which changed -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser- caused unprocessed insulin receptors with decreased insulin binding affinity, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. After site-directed mutagenesis, an expression vector pGEM3SV was used to make a plasmid which contained full-length HIRcDNA behind SV40 early promoter. Transfection of normal HIR cDNA produced normal insulin receptors on the plasma membranes in COS 7 cells. However, transfection of cDNA with the mutation resulted in the presence of 210K proreceptors in the plasma membranes with decreased insulin binding ability (35% of normal). These results suggest that the mutation, not the defect of converting enzyme, was the cause for unprocessed insulin proreceptors in the patients with insulin resistance.
Kobayashi
M
M
Third Department of Medicine, Shiga University of Medical Science, Ohtsu, Japan.
Sugibayashi
M
M
Sasaoka
T
T
Egawa
K
K
Shigeta
Y
Y
Tamaki
M
M
Nakamura
E
E
Teraoka
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007333:Insulin Resistance / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004247:DNA; D011972:Receptor, Insulin