title: GAPex-5 mediates ubiquitination, trafficking, and degradation of epidermal growth factor receptor.
abstract: Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Xiong
authors.initials: X
authors.lastname: Su
authors.forename: Chen
authors.initials: C
authors.lastname: Kong
authors.forename: Philip D
authors.initials: PD
authors.lastname: Stahl
mesh_terms: D011992:Endosomes / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005799:Genes, Dominant; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D021381:Protein Transport; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D018631:ras Proteins / Q000378:metabolism
chemical_list: C509137:GAPVD1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; D034741:RNA, Small Interfering; D025801:Ubiquitin; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors; D020696:rab5 GTP-Binding Proteins; D018631:ras Proteins