Carcinogenesis Advance Access originally published online on March 19, 2007
Carcinogenesis 2007 28(7):1408-1417; doi:10.1093/carcin/bgm058
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EGFRvIII escapes down-regulation due to impaired internalization and sorting to lysosomes
Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark
1 Department of Radiation Biology, Section 6321, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
2 Department of Molecular Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark
* To whom correspondence should be addressed. Tel: +45 35 45 63 03; Fax: +45 35 45 63 01; Email: skovgaard{at}rh.dk
EGFRvIII is a mutant variant of the epidermal growth factor receptor (EGFR) found exclusively in various cancer types. EGFRvIII lacks a large part of the extracellular domain and is unable to bind ligands; however, the receptor is constitutively phosphorylated and able to activate downstream signaling pathways. Failure to attenuate signaling by receptor down-regulation could be one of the major mechanisms by which EGFRvIII becomes oncogenic. Using a cell system expressing either EGFR or EGFRvIII with no expression of other EGFR family members and with endogenous levels of key degradation proteins, we have investigated the down-regulation of EGFRvIII and compared it to that of EGFR. We show that, in contrast to EGFR, EGFRvIII is inefficiently degraded. EGFRvIII is internalized, but the internalization rate of the mutated receptor is significantly less than that of unstimulated EGFR. Moreover, internalized EGFRvIII is recycled rather than delivered to lysosomes. EGFRvIII binds the ubiquitin ligase c-Cbl via Grb2, whereas binding via phosphorylated tyrosine residue 1045 seems to be limited. Despite c-Cbl binding, the receptor fails to become effectively ubiquitinylated. Thus, our results suggest that the long lifetime of EGFRvIII is caused by inefficient internalization and impaired sorting to lysosomes due to lack of effective ubiquitinylation.
Abbreviations: BSA, bovine serum albumin; CHX, cycloheximide; DMEM, Dulbecco’s modified Eagle’s medium; d.n., dominant negative; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; RT, room temperature; TfR, transferrin receptor
These authors contributed equally to this work. Received October 10, 2006; revised March 2, 2007; accepted March 9, 2007.
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