Phosphorylation state and biological function of a mutant human insulin receptor Val996.
Chinese hamster ovary (CHO) cell transfectants that expressed human insulin receptors whose glycine 996 was substituted by valine were studied. Receptor processing and insulin binding were unaffected by this mutation; however, this mutant insulin receptor had little or no tyrosine kinase activity. Nevertheless, the Val996 mutant exhibited seryl and threonyl phosphorylation in both the basal and insulin-stimulated state in intact cells. This is in contrast to the Lys----Ala1018 tyrosine kinase deficient mutant (Russell, D. S., Gherzi, R., Johnson, E. L., Chou, C-K., and Rosen, O. M. (1987) J. Biol. Chem. 262, 11833-11840). Cells expressing the normal human receptor were 10-fold more sensitive to insulin than the untransfected CHO cells with respect to phosphorylation of a cellular substrate (pp 185) on tyrosyl residues, glucose incorporation into glycogen, thymidine incorporation into DNA, and phosphorylation of ribosomal protein S6. Cells expressing the mutant receptor exhibited the same insulin sensitivity as the untransfected CHO cells. Insulin was rapidly internalized in cells expressing the normal human receptor and the number of receptors expressed on the cell surface was decreased in response to exposure to insulin. However, little insulin was internalized in cells expressing the mutant receptor, and the number of receptors on the cell surface was not significantly diminished in response to exposure to insulin. It is concluded that despite the occurrence of seryl and threonyl phosphorylations, post-receptor effects of insulin described above are not mediated by the tyrosine kinase-deficient receptor, Val996.
Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan.
R
R
Yamamoto-Honda
O
O
Koshio
K
K
Tobe
Y
Y
Shibasaki
K
K
Momomura
M
M
Odawara
T
T
Kadowaki
F
F
Takaku
Y
Y
Akanuma
M
M
Kasuga
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014633:Valine
D007328:Insulin; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin; D014633:Valine