The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor. Mol Cell Biol 2003 May;23(9):3363-72
Date
04/17/2003Pubmed ID
12697834Pubmed Central ID
PMC153198DOI
10.1128/MCB.23.9.3363-3372.2003Scopus ID
2-s2.0-0345381737 (requires institutional sign-in at Scopus site) 214 CitationsAbstract
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.
Author List
Vecchione A, Marchese A, Henry P, Rotin D, Morrione AAuthor
Adriano Marchese PhD Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsCalcium-Binding Proteins
Cell Membrane
Cells, Cultured
Chloroquine
Cysteine Endopeptidases
Cysteine Proteinase Inhibitors
Endosomal Sorting Complexes Required for Transport
Fibroblasts
GRB10 Adaptor Protein
Gene Expression
Insulin-Like Growth Factor I
Leupeptins
Ligands
Ligases
Lysosomes
Macromolecular Substances
Mice
Multienzyme Complexes
Mutation
Nedd4 Ubiquitin Protein Ligases
Precipitin Tests
Proteasome Endopeptidase Complex
Proteins
Receptor, IGF Type 1
Recombinant Proteins
Transport Vesicles
Ubiquitin
Ubiquitin-Protein Ligases
src Homology Domains