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Regulation of Growth Factor Receptor Degradation by ADP-Ribosylation Factor Domain Protein (ARD) 1

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Regulation of Growth Factor Receptor Degradation by ADP-Ribosylation Factor Domain Protein (ARD) 1 Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1 Victor Meza-Carmen1, Gustavo Pacheco-Rodriguez, Gi Soo Kang, Jiro Kato, Chiara Donati2, Chun-Yi Zhang3, Alessandro Vichi, D. Michael Payne, Souheil El-Chemaly4, Mario Stylianou, Joel Moss, and Martha Vaughan5 Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Martha Vaughan, March 9, 2011 (sent for review October 10, 2010) ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa pro- lular sites specified by targeting sequences, posttranslational tein containing a functional ADP-ribosylation factor (GTP hydro- modifications, and/or associated molecules (6, 7). Mammalian lase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase ARFs (ca. 20 kDa) are grouped as class I (ARFs 1–3), class II domains. ARD1 activation by the guanine nucleotide-exchange (ARFs 4 and 5), and class III (ARF6), based on similarities of factor cytohesin-1 was known. GTPase and E3 ligase activities of gene structure, protein sequences, and phylogenetic relationships ARD1 suggest roles in protein transport and turnover. To explore (8). The ARFs cycle between active GTP- and inactive GDP- this hypothesis, we used mouse embryo fibroblasts (MEFs) from bound forms dependent on the actions of GEFs, which accelerate ARD1-/- mice stably transfected with plasmids for inducible expres- the replacement of ARF-bound GDP with GTP, and GTPase- sion of wild-type ARD1 protein (KO-WT), or ARD1 protein with activating proteins (GAPs) that enhance intrinsic ARF GTPase inactivating mutations in E3 ligase domain (KO-E3), or containing hydrolysis of GTP (9). Activated ARF–GTP can interact stably persistently active GTP-bound (KO-GTP), or inactive GDP-bound with specific effector molecules, such as vesicle coat protein sub- (KO-GDP) GTPase domains. Inhibition of proteasomal proteases units of COPI coatomer complex (10), and Golgi-associated, in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted γ-adaptin ear-containing, ARF-binding adapter proteins of cla- in accumulation of these ARD1 proteins, whereas KO-E3 accumu- thrin-coated vesicles (6). lated without inhibitors. All data were consistent with the conclu- ADP-ribosylation factor domain protein 1 (ARD1), an atypical sion that ARD1 regulates its own steady-state levels in cells by member of the ARF family, is a ca. 64-kDa molecule with a BIOCHEMISTRY autoubiquitination. Based on reported growth factor receptor- C-terminal (amino acids 391–565) approximate 18-kDa ARF cytohesin interactions, EGF receptor (EGFR) was investigated in domain that is ca. 60% identical to class I ARFs (11). As ARF induced MEFs. Amounts of cell-surface and total EGFR were higher proteins lack detectable GTPase activity and require a GAP to in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels terminate activation, the intrinsic GTPase activity of ARD1 was in both mutants greater (p ¼ 0.001) after proteasomal inhibition. soon recognized (12). Direct functional interaction of recombi- Significant differences among MEF lines in content of TGF-β recep- nant 18-kDa ARF domain and ca. 46-kDa remainder (GAP do- tor III were similar to those in EGFR, albeit not as large. Differences main) of ARD1 was also shown (12). The 46-kDa N-terminal in amounts of insulin receptor mirrored those in EGFR, but did not region of ARD1 (1–390), termed “GAP domain,” although it in- reach statistical significance. Overall, the capacity of ARD1 GTPase cludes sequences with additional diverse functions (Fig. 1A), can to cycle between active and inactive forms and its autoubiquitina- activate ARF-domain GTPase activity to generate ARD1–GDP tion both appear to be necessary for the appropriate turnover of (12, 13). Three structural motifs (RING, B box, and coiled-coil or EGFR and perhaps additional growth factor receptors. RBCC) in this region gave ARD1 the designation TRIM23 in the tripartite motif (TRIM) family (14). RBCC sequences participate tyrosine receptors ∣ ubiquitination in protein–protein interactions, and the ARD1 RING exhibits E3 ubiquitin ligase activity demonstrated as apparent autoubiqui- esicular trafficking, which is responsible for intracellular tination in assays with recombinant ARD1 or fragments, E1, and Vtransport of proteins and/or lipids between and among orga- an appropriate E2, plus ubiquitin and ATP (15). A recent report nelles, is of major importance in regulating cell function and fate that the protein NF-κB essential modulator (NEMO) is a sub- (1). Extracellular stimuli, either via direct ligand interaction with strate for the E3 activity of ARD1 (16) did not relate that activity specific cell-surface receptors (e.g., growth factor) or in response to its ARF domain. to mechanical or environmental signals (e.g., pH, oxygen), also To explore the biological function(s) of ARD1, we prepared use vesicular trafficking to produce needed alterations in cellular from ARD1-null mice mouse embryo fibroblast (MEF) lines stably metabolism, growth, and/or motion (2). For example, after ligand transfected with plasmids for mifepristone (Mfp)-inducible expres- binding, plasma membrane receptors, such as the epidermal sion of ARD1, wild type, or with specific mutations that prevent growth factor receptor (EGFR) are internalized in clathrin- or caveolin-coated vesicles (3) and can be recycled, after ligand dissociation, to the plasma membrane via multivesicular bodies. Author contributions: V.M.-C., G.P.-R., J.M., and M.V. designed research; V.M.-C., G.P.-R., G.S.K., J.K., C.D., C.-Y.Z., A.V., D.M.P., and S.E.-C. performed research; V.M.-C., G.P.-R., M.S., Receptor degradation after covalent modification (e.g., phos- J.M., and M.V. analyzed data; and V.M.-C., G.P.-R., J.M., and M.V. wrote the paper. phorylation and/or ubiquitination) can occur through lysosomal The authors declare no conflict of interest. or proteasomal pathways (4). 1Present address: Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana Vesicle formation is initiated by guanine nucleotide-exchange de San Nicolás de Hidalgo, Morelia, Michoacán, 58030, Mexico. factor (GEF)-catalyzed activation of ADP-ribosylation factor 2Present address: Dipartimento di Scienze Biochimiche, Università di Firenze, 50134 (ARF) that results in its specific association with membrane sites Florence, Italy. – where ARF GTP recruits coat proteins and cargo, resulting in 3Present address: Epitomics, Inc., 863 Mitten Road, Suite 103, Burlingame, CA membrane deformation and initiation of budding. After move- 94010-1303. ment of a completed vesicle to its target and tethering/docking, 4Present address: Division of Pulmonary and Critical Care Medicine, Brigham and Women’s each transport step ends with membrane fusion (5). Biogenesis of Hospital, 75 Francis Street, Boston, MA 02115. several types of vesicles uses specific guanine nucleotide-binding 5To whom correspondence should be addressed. E-mail: [email protected]. GTPases as molecular switches for initiation and termination of This article contains supporting information online at www.pnas.org/lookup/suppl/ diverse pathways that deliver molecules to appropriate intracel- doi:10.1073/pnas.1103867108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1103867108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 26, 2021 ARD1 and mutants (see Fig. 1A) of the ARF domain that were persistently inactive or active. Cells constitutively overexpressing ARD1 protein failed to proliferate, which seems likely, in retro- spect, to have been a consequence (at least in part) of elevated levels of its E3 ligase activity. Endogenous ARD1 was finally detected by two different antibodies in HepG2 cells grown over- night with, but not without, proteasome inhibitors (Fig. 1B, arrow- head). This behavior mirrors reports of other RING-type E3 ubiquitin ligases capable of autoubiquitination that causes their instability in cells (17, 18) and detection only after inhibition of proteasomal proteases. Results were similar with fibroblasts grown from WT MEFs, although KO MEFs always, unfortu- nately, showed extensive nonspecific staining with our antipeptide antibodies (Fig. 1B). Our ARD1 KO mouse (C57/BL6) was initially generated for alternative exploration of the biological role of ARD1. Mice lack- ing the ARD1 gene appeared grossly normal and thus far present no obvious pathological phenotype or histopathology of major organs including brain, spleen, heart, kidneys, lung, and axial lymph nodes, or problems with breeding. Fatty liver without iden- tified cause appeared in some older KO mice. ARD1-null MEF lines that inducibly express WT or mutant ARD1 proteins under control of the CMV promoter were, therefore, prepared. Induced Expression of ARD1 in KO MEFs. RT-PCR confirmed appar- ent absence of ARD1 mRNA in KO MEFs (Fig. S1). We used ARD1-/- MEFs for Mfp-inducible expression of murine WT ARD1 or its mutant forms, which do not cycle between GTP- and GDP-bound forms. Mutation T418N interferes with GTP bind- ing, locking ARD1 in its inactive GDP-bound form, whereas K458I abolishes GTPase activity, producing persistently active ARD1–GTP (19). The Mfp-induced double E3 mutant with ala- nine replacing C34 and H54, amino acids critical for E3 ubiquitin Fig. 1. ARD1, a multifunctional protein. (A) Functional domains of 574 ligase activity, was also generated (15). We report data from eight amino acid human ARD1 with C-terminal (402–574) ARF sequence. ARD1 stably transfected MEF lines referred to, respectively, as KO-WT, contains an E3 ubiquitin ligase motif (positions 31–75) with critical C34 and KO-GDP, and KO-GTP (two lines of each), and one line each of H53, which were replaced with alanine in E3-inactive mutants. GAP function KO-E3 and KO-GFP (empty vector). No ARD1 protein was de- was assigned to amino acid 101–190, with amino acid 190–333 additionally tected in any of these MEFs before induction (Fig. 1C), but after required for domain interactions that result in GTP hydrolysis.
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