title: Polyubiquitination of insulin-like growth factor I receptor (IGF-IR) activation loop promotes antibody-induced receptor internalization and down-regulation.
abstract: Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.
authors: Department of Research Pathology, Genentech, South San Francisco, California 94080.
authors: Yifan
authors: Y
authors: Mao
authors: Department of Research Pathology, Genentech, South San Francisco, California 94080.
authors: Yonglei
authors: Y
authors: Shang
authors: Department of Protein Chemistry, Genentech, South San Francisco, California 94080.
authors: Victoria C
authors: VC
authors: Pham
authors: Department of Protein Chemistry, Genentech, South San Francisco, California 94080.
authors: James A
authors: JA
authors: Ernst
authors: Department of Protein Chemistry, Genentech, South San Francisco, California 94080.
authors: Jennie R
authors: JR
authors: Lill
authors: Department of Molecular Biology, Genentech, South San Francisco, California 94080. Electronic address: sscales@gene.com.
authors: Suzie J
authors: SJ
authors: Scales
authors: Department of Research Pathology, Genentech, South San Francisco, California 94080. Electronic address: jzha@crownbio.com.
authors: Jiping
authors: J
authors: Zha
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000494:pharmacology*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007152:Immunologic Capping / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1