Disassembly of Son-of-sevenless proteins from Grb2 during p21ras desensitization by insulin.
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.
Cherniack
A D
AD
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
Klarlund
J K
JK
Conway
B R
BR
Czech
M P
MP
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000187:drug effects / Q000378:metabolism*; D011972:Receptor, Insulin / Q000502:physiology; D015398:Signal Transduction; D020664:Son of Sevenless Proteins
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D020664:Son of Sevenless Proteins; D006160:Guanosine Triphosphate; D066246:ErbB Receptors; D011972:Receptor, Insulin; D016283:Proto-Oncogene Proteins p21(ras)