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Phosphorylation state and biological function of a mutant human insulin receptor Val996
R Yamamoto-Honda1, O Koshio, K Tobe
1Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan.
Abstract:
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.
Insights
A mutated human insulin receptor (Val996) lacking tyrosine kinase activity still undergoes seryl and threonyl phosphorylation but fails to mediate post-receptor insulin effects like glucose uptake.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The human insulin receptor (hIR) is a transmembrane receptor tyrosine kinase.
- Insulin binding initiates intracellular signaling cascades crucial for glucose homeostasis and cell growth.
- The tyrosine kinase domain is essential for signal transduction, but the role of specific mutations is under investigation.
Purpose of the Study:
- To investigate the functional consequences of a specific mutation (Glycine 996 to Valine) in the human insulin receptor.
- To determine if the Val996 mutant receptor, deficient in tyrosine kinase activity, can mediate insulin's post-receptor effects.
- To compare the signaling capabilities of the Val996 mutant with the wild-type receptor.
Main Methods:
- Creation of Chinese hamster ovary (CHO) cell transfectants expressing the wild-type and Val996 mutant human insulin receptors.
- Assessment of receptor processing, insulin binding, and tyrosine kinase activity.
- Analysis of cellular substrate phosphorylation (pp 185, ribosomal protein S6), glucose incorporation into glycogen, and thymidine incorporation into DNA.
- Evaluation of insulin-mediated receptor internalization and downregulation.
Main Results:
- The Val996 mutation did not affect receptor processing or insulin binding but abolished tyrosine kinase activity.
- The Val996 mutant exhibited seryl and threonyl phosphorylation, unlike a previously studied tyrosine kinase-deficient mutant.
- Cells expressing the Val996 mutant showed no increased insulin sensitivity in substrate phosphorylation, glucose uptake, or DNA synthesis compared to untransfected cells.
- Insulin internalization and receptor downregulation were impaired in cells expressing the Val996 mutant.
Conclusions:
- The tyrosine kinase activity of the human insulin receptor is essential for mediating insulin's post-receptor effects, including cellular signaling and receptor trafficking.
- Seryl and threonyl phosphorylation of the Val996 mutant receptor do not substitute for the lack of tyrosine kinase activity in downstream signaling.
- The Val996 mutant receptor provides a tool to dissect the specific roles of tyrosine kinase activity in insulin action.