Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation.
Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.
Ramos
Fresnida J
FJ
Dept. of Pharmacology, UTHSCSA, 7703 Floyd Curl Dr., San Antonio, TX 78229, USA.
Langlais
Paul R
PR
Hu
Derong
D
Dong
Lily Q
LQ
Liu
Feng
F
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D051383:GRB10 Adaptor Protein / Q000378:metabolism* / Q000502:physiology*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology; D034622:RNA Interference; D020871:RNA Stability; D011972:Receptor, Insulin / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D007328:Insulin; D007976:Leupeptins; D025801:Ubiquitin; D051383:GRB10 Adaptor Protein; D011972:Receptor, Insulin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde