title: Engagement of the small GTPase Rab31 protein and its effector, early endosome antigen 1, is important for trafficking of the ligand-bound epidermal growth factor receptor from the early to the late endosome.
abstract: Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.
authors.affiliation: From the Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System, 1E Kent Ridge Road, Singapore 119228 and.
authors.forename: Christelle En Lin
authors.initials: CE
authors.lastname: Chua
authors.forename: Bor Luen
authors.initials: BL
authors.lastname: Tang
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport; D034622:RNA Interference; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: C509137:GAPVD1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; C484190:RAB31 protein, human; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
keywords: Endocytosis; Endosomes; Epidermal Growth Factor Receptor (EGFR); Rab; Trafficking