Molecular Characterization and Regulation of GTP-binding Proteins

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To understand better the roles of ARD1 in cells, we prepared from ARD1-null mice mouse embryo fibroblast (MEF) lines stably transfected with plasmids for mifepristone (Mfp)-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 auto-ubiquitination. Based on reported growth factor receptor-cytohesin interactions, epidermal growth factor 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 TGFβ 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 auto-ubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.