The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation
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Abstract:
We have previously demonstrated that the human insulin receptor, mutated in the ATP-binding domain of the beta-subunit, is kinase-defective and fails to mediate multiple post-receptor actions of insulin in stably transfected Chinese hamster ovary cells (Chou, C.-K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, O. M. (1987) J. Biol. Chem. 262, 1842-1847). This study addresses the role of protein-tyrosine kinase activity in insulin-mediated receptor down-regulation. Although the mutant insulin proreceptor was properly processed and able to bind insulin like the wild-type human receptor, it differed from the latter in the following respects: 1) it failed to mediate internalization of surface-bound radiolabeled ligand; 2) it did not undergo short- or long-term down-regulation in response to 1 microM insulin; 3) it did not exhibit ligand-promoted receptor turnover; and 4) it was not phosphorylated on either tyrosine or serine residues in response to insulin. Although the cells transfected with the mutant receptor failed to respond to insulin-mediated insulin receptor down-regulation, they were able to down-regulate their insulin-like growth factor I receptors in response to insulin-like growth factor I or high concentrations of insulin and were sensitive to monoclonal antibody-induced down-regulation of their insulin receptors. Antibody-mediated receptor internalization alone, however, was unable to mimic at least one action of insulin, thymidine incorporation into DNA, and did not lead to any phosphorylation of the receptor. It is concluded that either the protein-tyrosine kinase activity of the insulin receptor or its phosphorylation state is essential for ligand-mediated receptor down-regulation.
Insights
The insulin receptor's protein-tyrosine kinase activity is essential for insulin-mediated receptor down-regulation. A mutated receptor, lacking this kinase activity, failed to internalize or down-regulate in response to insulin.
Area of Science:
- Cell biology
- Molecular endocrinology
- Signal transduction
Background:
- The human insulin receptor (IR) plays a crucial role in insulin signaling.
- Previous studies showed a mutated IR in the ATP-binding domain is kinase-defective.
- The role of IR's protein-tyrosine kinase (PTK) activity in receptor down-regulation remained unclear.
Purpose of the Study:
- To investigate the role of protein-tyrosine kinase activity in insulin-mediated insulin receptor down-regulation.
- To determine if kinase activity is required for insulin receptor internalization and degradation.
- To assess the impact of impaired kinase activity on other insulin-mediated cellular responses.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cells stably transfected with either wild-type or a kinase-defective mutant human insulin receptor.
- Assessed insulin binding, ligand internalization, receptor phosphorylation (tyrosine and serine), and receptor down-regulation in response to insulin.
- Investigated insulin-like growth factor I (IGF-I) receptor regulation and antibody-induced insulin receptor down-regulation.
Main Results:
- The kinase-defective mutant insulin receptor bound insulin but failed to mediate ligand internalization, receptor down-regulation, or receptor phosphorylation (tyrosine/serine) upon insulin stimulation.
- Cells expressing the mutant receptor did not undergo insulin-induced receptor down-regulation.
- Cells with the mutant receptor could still down-regulate IGF-I receptors and insulin receptors via antibody stimulation, but antibody-mediated internalization alone did not trigger downstream signaling like thymidine incorporation.
Conclusions:
- Protein-tyrosine kinase activity of the insulin receptor is essential for ligand-mediated receptor down-regulation.
- Phosphorylation state of the insulin receptor is critical for its proper down-regulation following insulin binding.
- Kinase-independent pathways may regulate other aspects of receptor function or signaling.