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Characterization of an insulin receptor mutant lacking the subunit processing site
J F Williams1, D A McClain, T J Dull
1Department of Medicine, University of California, San Diego 92092.
The Journal of Biological Chemistry
|May 25, 1990
Summary
A modified human insulin proreceptor, lacking a specific cleavage site, was created. This mutant receptor shows reduced insulin binding and impaired signaling, indicating cleavage is vital for normal insulin receptor function.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The insulin receptor (IR) is crucial for glucose homeostasis.
- IR processing involves cleavage into alpha and beta subunits.
- The role of this cleavage in receptor function is not fully understood.
Purpose of the Study:
- To investigate the function of the human insulin proreceptor.
- To determine the necessity of the proreceptor cleavage site for insulin receptor activity.
Main Methods:
- Site-directed mutagenesis to delete the Arg-Lys-Arg-Arg cleavage site in the human insulin proreceptor cDNA.
- Transfection into Chinese hamster ovary cells.
- Immunoprecipitation, Western blotting, pulse-chase analysis, and photolabeling with a modified insulin analog.
Main Results:
- The mutant proreceptor was synthesized, glycosylated, and expressed on the cell surface but remained uncleaved.
- The mutant receptor exhibited significantly reduced insulin binding affinity.
- Internalization, down-regulation, and autophosphorylation of the mutant receptor upon insulin binding were defective.
Conclusions:
- Cleavage of the insulin proreceptor is essential for high-affinity insulin binding.
- Proreceptor cleavage is required for normal signal transduction, including beta subunit tyrosine kinase activation.
- This study highlights the critical role of post-translational processing in insulin receptor function.