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Molecular and Cellular Biology, May 2003, p. 3363-3372, Vol. 23, No. 9
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.9.3363-3372.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Grb10/Nedd4 Complex Regulates Ligand-Induced Ubiquitination and Stability of the Insulin-Like Growth Factor I Receptor

Andrea Vecchione,1,2 Adriano Marchese,3 Pauline Henry,4 Daniela Rotin,4 and Andrea Morrione1*

Departments of Urology,1 Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107,3 Parco Scientifico Biomedico di Roma San Raffaele, 00128 Rome, Italy,2 Program in Cell Biology and Biochemistry Department, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X84

Received 30 October 2002/ Returned for modification 11 December 2002/ Accepted 3 February 2003

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 (mGrb10{alpha}) on IGF-I-mediated mitogenesis (A. Morrione et al., J. Biol. Chem. 272:26382-26387, 1997). With mGrb10{alpha} 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.

* Corresponding author. Mailing address: Department of Urology and Kimmel Cancer Center, Thomas Jefferson University, BLSB Room 631, 233 South 10th St., Philadelphia, PA 19107. Phone: (215) 503-4519. Fax: (215) 923-0249. E-mail: Andrea.Morrione{at}mail.tju.edu.

Molecular and Cellular Biology, May 2003, p. 3363-3372, Vol. 23, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.9.3363-3372.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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