Disassembly of Son-of-sevenless Proteins from Grb2 during p21
Desensitization by Insulin (*)
- From the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
Abstract
Insulin receptor signaling acutely stimulates GTP loading of p21
, 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 
P-labeled 3T3-L1 adipocytes the elevated cellular concentrations of [
P]GTP-bound p21
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 p21
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 p21
deactivation and desensitization.
Footnotes
-
↵* This work was supported by Grant DK30648 from the National Institutes of Health (to M. P. C.) and postdoctoral fellowships from the Juvenile Diabetes Foundation International (to A. D. C. and B. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- DMEM
-
Dulbecco's modified Eagle's medium
- PAGE
-
polyacrylamide gel electrophoresis.
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↵2 J. K. Klarlund, A. D. Cherniack, and M. P. Czech, submitted for publication.
-
- Received November 18, 1994.
- Revision received December 7, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
