The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor.
The adapter protein Grb10 belongs to a superfamily of related proteins, including Grb7, -10, and -14 and Caenorhabditis elegans Mig10. Grb10 is an interacting partner of the insulin-like growth factor I receptor (IGF-IR) and the insulin receptor (IR). Previous work showed an inhibitory effect of mouse Grb10 (mGrb10alpha) on IGF-I-mediated mitogenesis (A. Morrione et al., J. Biol. Chem. 272:26382-26387, 1997). With mGrb10alpha as bait in a yeast two-hybrid screen, mouse Nedd4 (mNedd4-1), a ubiquitin protein ligase, was previously isolated as an interacting protein of Grb10 (A. Morrione et al., J. Biol. Chem. 274:24094-24099, 1999). However, Grb10 is not ubiquitinated by Nedd4 in cells. Here we show that in mouse embryo fibroblasts overexpressing Grb10 and the IGF-IR (p6/Grb10), there is a strong ligand-dependent increase in ubiquitination of the IGF-IR compared with that in parental cells (p6). This increased ubiquitination is associated with a shorter half-life and increased internalization of the IGF-IR. The IGF-IR is stabilized following treatment with both MG132 and chloroquine, indicating that both the proteasome and lysosomal pathways mediate degradation of the receptor. Ubiquitination of the IGF-IR likely occurs at the plasma membrane, prior to the formation of endocytic vesicles, as it is insensitive to dansylcadaverine, an inhibitor of early endosome formation in IGF-IR endocytosis. Grb10 coimmunoprecipitates with the IGF-IR and endogenous Nedd4 in p6/Grb10 cells, suggesting the presence of a Grb10/Nedd4/IGF-IR complex. Ubiquitination of the IGF-IR in p6/Grb10 cells is severely impaired by overexpression of a catalytically inactive Nedd4 mutant (Nedd4-CS), which also stabilizes the receptor. Likewise, overexpression of a Grb10 mutant lacking the Src homology 2 (SH2) domain impaired ubiquitination of the IGF-IR in parental p6 and p6/Grb10 cells, indicating that Grb10 binding to Nedd4 is critical for ubiquitination of the receptor. These results suggest a role for the Grb10/Nedd4 complex in regulating ubiquitination and stability of the IGF-IR, and they suggest that Grb10 serves as an adapter to form a bridge between Nedd4 and the IGF-IR. This is the first demonstration of regulation of stability of a tyrosine kinase receptor by the Nedd4 (HECT) family of E3 ligases.
Vecchione
Andrea
A
Department of Urology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Marchese
Adriano
A
Henry
Pauline
P
Rotin
Daniela
D
Morrione
Andrea
A
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D056827:Endosomal Sorting Complexes Required for Transport; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051383:GRB10 Adaptor Protein; D015870:Gene Expression; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D056827:Endosomal Sorting Complexes Required for Transport; C495959:Grb10 protein, mouse; D007976:Leupeptins; D008024:Ligands; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011506:Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D051383:GRB10 Adaptor Protein; D007334:Insulin-Like Growth Factor I; D002738:Chloroquine; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D017526:Receptor, IGF Type 1; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde