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Disassembly of Son-of-sevenless proteins from Grb2 during p21ras desensitization by insulin
A D Cherniack1, J K Klarlund, B R Conway
1Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
Abstract:
Insulin receptor signaling acutely stimulates GTP loading of p21ras, apparently by mobilizing complexes of Grb2 and the guanine nucleotide exchangers Son-of-sevenless (Sos) 1 and 2 to associate with tyrosine-phosphorylated proteins in the plasma membrane. Here we show that in 32P-labeled 3T3-L1 adipocytes the elevated cellular concentrations of [32P]GTP-bound p21ras in response to insulin return to near basal levels after 20-30 min of hormone stimulation, while insulin receptors remain activated. Lysates of such desensitized cells were quantitatively immunoprecipitated with an antiserum recognizing both Sos1 and Sos2 proteins or a specific anti-Sos2 antiserum. Immunoblot analysis of these precipitates revealed that insulin causes a marked hyperphosphorylation of Sos1 and a 50% decrease in Grb2 associated with Sos proteins under these conditions. Similarly, anti-Grb2 immunoprecipitates of such lysates revealed the presence of decreased Sos1 protein due to insulin action. The disassembly of Grb2 from Sos proteins slightly precedes the time course of p21ras deactivation in response to insulin. These data are consistent with the hypothesis that the dissociation of Grb2 from Sos proteins caused by insulin in 3T3-L1 cells mediates p21ras deactivation and desensitization.
Insights
Insulin signaling initially activates p21ras GTP loading. However, prolonged stimulation leads to Grb2-Sos protein dissociation, causing p21ras deactivation and insulin desensitization in 3T3-L1 adipocytes.
Area of Science:
- Cellular signaling pathways
- Molecular mechanisms of insulin action
- Ras protein regulation
Background:
- Insulin receptor signaling activates p21ras via Grb2-Sos complexes.
- Desensitization of insulin signaling occurs despite sustained receptor activation.
Purpose of the Study:
- Investigate the molecular mechanisms behind insulin-induced p21ras deactivation and signaling desensitization.
- Determine the role of Grb2-Sos protein interactions in insulin response.
Main Methods:
- Utilized 32P-labeled 3T3-L1 adipocytes to study insulin signaling.
- Performed quantitative immunoprecipitation with anti-Sos and anti-Grb2 antisera.
- Analyzed protein phosphorylation and complex formation via immunoblotting.
Main Results:
- Insulin stimulation led to transient p21ras activation followed by deactivation.
- Hyperphosphorylation of Sos1 and decreased Grb2 association with Sos proteins were observed.
- Dissociation of Grb2 from Sos proteins preceded p21ras deactivation.
Conclusions:
- Insulin-induced Grb2-Sos dissociation mediates p21ras deactivation.
- This dissociation is a key event in insulin signaling desensitization in 3T3-L1 cells.