title: Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.
abstract: ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.
authors.affiliation: Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Meza-Carmen
authors.forename: Gustavo
authors.initials: G
authors.lastname: Pacheco-Rodriguez
authors.forename: Gi Soo
authors.initials: GS
authors.lastname: Kang
authors.forename: Jiro
authors.initials: J
authors.lastname: Kato
authors.forename: Chiara
authors.initials: C
authors.lastname: Donati
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Zhang
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vichi
authors.forename: D Michael
authors.initials: DM
authors.lastname: Payne
authors.forename: Souheil
authors.initials: S
authors.lastname: El-Chemaly
authors.forename: Mario
authors.initials: M
authors.lastname: Stylianou
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Martha
authors.initials: M
authors.lastname: Vaughan
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000235:genetics; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D020823:ADP-Ribosylation Factor 1