The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

The function of Arf6 has been investigated largely by using the T27N and the Q67L mutants, which are thought to be blocked in GDP- and GTP-bound states, respectively. However, these mutants have been poorly characterized biochemically. Here, we found that Arf6(T27N) is not an appropriate marker of the inactive GDP-bound form because it has a high tendency to lose its nucleotide in vitro and to denature. As a consequence, most of the protein is aggregated in vivo and localizes to detergent-insoluble structures. However, a small proportion of Arf6(T27N) is able to form a stable complex with its exchange factor EFA6 at the plasma membrane, accounting for its dominant-negative phenotype. To define the cellular localization of Arf6-GDP, we designed a new mutant, Arf6(T44N). In vitro, this mutant has a 30-fold decreased affinity for GTP. In vivo, it is mostly GDP bound and, in contrast to the wild type, does not switch to the active conformation when expressed with EFA6. This GDP-locked mutant is found at the plasma membrane, where it localizes with EFA6 and Ezrin in actin- and phosphatidylinositol (4,5)-bisphosphate-enriched domains. From these results, we conclude that the Arf6 GDP-GTP cycle takes place at the plasma membrane.

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of ϳ0.5 M EFA6-PH for PI(4,5)P 2 . Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P 2 . These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P 2 , and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.The ADP-ribosylation factor (Arf) family, which includes six isoforms, plays a key role in the intracellular vesicular transport Likewise, the binding of GDP-bound Arfs to their guanine nucleotide exchange factors (GEFs) 2 is critical to localize the Arf proteins to their proper membrane (for review, see Ref. 4). The Arf specific GEF family, also referred to as the Sec7 family, comprises 15 members divided in two classes, the large and the small GEFs, which are responsible for the activation of 6 Arf isoforms. This larger number of GEFs suggests that each Arf is under the control of several ArfGEFs. Growing evidence indicates that the ArfGEFs are targeted to specific compartments where they will activate a cognate Arf. Little is known about how the large GEFs, GBF1, Big1, and Big2 are targeted to their working compartments, respectively cis-Golgi, endoplasmic reticulum-Golgi intermediate (5), and trans-Golgi network (6). In contrast, the small Arf-GEF family members (Arno/Cytohe-* This work was supported by grants from Association pour la Recherche contre le Cancer, Cancé ropô le PACA (Provence-Alpes-Cô te d'Azur), and Agence Nationale de la Recherche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indi...

In epithelial cells, the tight junction (TJ) functions as a permeability barrier and is involved in cellular differentiation and proliferation. Although many TJ proteins have been characterized, little is known about the sequence of events and temporal regulation of TJ assembly in response to adhesion cues. We report here that the deubiquitinating enzyme USP9x has a critical function in TJ biogenesis by controlling the levels of the exchange factor for Arf6 (EFA6), a protein shown to facilitate TJ formation, during a narrow temporal window preceding the establishment of cell polarity. At steady state, EFA6 is constitutively ubiquitinated and turned over by the proteasome. However, at newly forming contacts, USP9x-mediated deubiquitination protects EFA6 from proteasomal degradation, leading to a transient increase in EFA6 levels. Consistent with this model, USP9x and EFA6 transiently co-localize at primordial epithelial junctions. Furthermore, knockdown of either EFA6 or USP9x impairs TJ biogenesis and EFA6 overexpression rescues TJ biogenesis in USP9x-knockdown cells. As the loss of cell polarity is a critical event in the metastatic spread of cancer, these findings may help to understand the pathology of human carcinomas.

One of the earliest events in epithelial carcinogenesis is the dissolution of tight junctions and cell polarity signals that are essential for normal epithelial barrier function. Here, we report that EFA6B, a guanine nucleotide exchange factor for the Ras superfamily protein Arf6 that helps assemble and stabilize tight junction, is required to maintain apico-basal cell polarity and mesenchymal phenotypes in mammary epithelial cells. In organotypic three-dimensional cell cultures, endogenous levels of EFA6B were critical to determine epithelial-mesenchymal status. EFA6B downregulation correlated with a mesenchymal phenotype and ectopic expression of EFA6B hampered TGFb-induced epithelial-to-mesenchymal transition (EMT). Transcriptomic and immunohistochemical analyses of human breast tumors revealed that the reduced expression of EFA6B was associated with loss of tight junction components and with increased signatures of EMT, cancer stemness, and poor prognosis. Accordingly, tumors with low levels of EFA6B were enriched in the aggressive triple-negative and claudin-low breast cancer subtypes. Our results identify EFA6B as a novel antagonist in breast cancer and they point to its regulatory and signaling pathways as rational therapeutic targets in aggressive forms of this disease.

Summary b2-adrenergic receptor (b2AR), a member of the GPCR (G-protein coupled receptor) family, is internalized in a ligand-and b-arrestindependent manner into early endosomes, and subsequently recycled back to the plasma membrane. Here, we report that b-arrestin promotes the activation of the small G protein Arf6, which regulates the recycling and degradation of b2AR. We demonstrate in vitro that the C-terminal region of b-arrestin1 interacts directly and simultaneously with Arf6GDP and its specific exchange factor EFA6, to promote Arf6 activation. Similarly, the ligand-mediated activation of b2AR leads to the formation of Arf6GTP in vivo in a b-arrestindependent manner. Expression of either EFA6 or an activated Arf6 mutant caused accumulation of b2AR in the degradation pathway. This phenotype could be rescued by the expression of an activated mutant of Rab4, suggesting that Arf6 acts upstream of Rab4. We propose a model in which Arf6 plays an essential role in b2AR desensitization. The ligand-mediated stimulation of b2AR relocates barrestin to the plasma membrane, and triggers the activation of Arf6 by EFA6. The activation of Arf6 leads to accumulation of b2AR in the degradation pathway, and negatively controls Rab4-dependent fast recycling to prevent the re-sensitization of b2AR.

Members of the Arf family of small G proteins are involved in membrane traffic and organelle structure. They control the recruitment of coat proteins, and modulate the structure of actin filaments and the lipid composition of membranes. The ADP-ribosylation factor 6 (Arf6) isoform and the exchange factor for Arf6 (EFA6) are known to regulate the endocytic pathway of many different receptors. To determine the molecular mechanism of the EFA6/Arf6 function in vesicular transport, we searched for new EFA6 partners. In a twohybrid screening using the catalytic Sec7 domain as a bait, we identified endophilin as a new partner of EFA6. Endophilin contains a Bin/Amphiphysin/Rvs (BAR) domain responsible for membrane bending, and an SH3 domain responsible for the recruitment of dynamin and synaptojanin, two proteins involved, respectively, in the fission and uncoating of clathrin-coated vesicles. By using purified proteins, we confirmed the direct interaction, and identified the N-BAR domain as the binding motif to EFA6A. We showed that endophilin stimulates the catalytic activity of EFA6A on Arf6. In addition, we observed that the Sec7 domain competes with flat but not with highly curved lipid membranes to bind the N-BAR. In cells, expression of EFA6A recruits endophilin to EFA6A-positive plasma membrane ruffles, whereas expression of endophilin rescues the EFA6A-mediated inhibition of transferrin internalization. Overall, our results support a model whereby EFA6 recruits endophilin on flat areas of the plasma membrane to control Arf6 activation and clathrin-mediated endocytosis.small GTP-binding proteins | membrane curvature | vesicular trafficking T he ADP ribosylation factor family, which includes six members, is known to regulate different stages of vesicular trafficking (reviewed in refs. 1, 2). The most abundant isoform, Arf1, controls the membrane trafficking at the level of the Golgi apparatus by regulating in a GTP-dependent manner the recruitment of the COPI coat complex (3-5) and the two clathrin adaptors AP-1 (6, 7) and GGAs (8) onto the Golgi membranes. By activating lipid-modifying enzymes, Arf1 is able to change the lipid composition of the donor compartment membrane, thus facilitating membrane dynamic (9-12). ADP-ribosylation factor 6 (Arf6), the most distant isoform, is thought to regulate plasma membrane and endosomal trafficking. Similarly to Arf1, Arf6 activates the PLD (13, 14) and the type I PI4P5K (15, 16) to produce, respectively, phosphatidic acid, which is a fusogenic lipid, and phosphatidylinositol 4-5 bisphosphate, which is known to regulate clathrin-dependent endocytosis. Arf6 and PIP 2 cooperate (at least in vitro) to recruit AP-2 onto lipid membranes, suggesting a role for Arf6 in the formation of clathrin-coated pits (17). In addition to this putative role during the initial steps of internalization, Arf6 has been shown to interact and recruit Nm23-H1, a protein believed to control the dynamin-dependent fission of endocytic vesicles (18). These different observations have clarified the mole...

We have previously reported that EFA6, exchange factor for Arf6, is implicated upon E-cadherin engagement in the process of epithelial cell polarization. We had found that EFA6 acts through stabilization of the apical actin ring onto which the tight junction is anchored. Mutagenesis experiments showed that both the catalytic domain of EFA6 and its C-terminal domain were required for full EFA6 function. Here we address the contribution of the specific substrate of EFA6, the small G protein Arf6. Unexpectedly, depletion of Arf6 by RNA interference or expression of the constitutively active fast-cycling mutant (Arf6T157N) revealed that Arf6 plays an opposing role to EFA6 by destabilizing the apical actin cytoskeleton and the associated tight junction. However, in complementation experiments, when the C-terminal domain of EFA6 is co-expressed with Arf6T157N, it reverts the effects of Arf6T157N expressed alone to faithfully mimic the phenotypes induced by EFA6. In addition, we find that the two signaling pathways downstream of EFA6, i.e. the one originating from the activated Arf6GTP and the other one from the EFA6 C-terminal domain, need to be tightly balanced to promote the proper reorganization of the actin cytoskeleton. Altogether, our results indicate that to regulate the tight junction, EFA6 activates Arf6 through its Sec7 catalytic domain as it modulates this activity through its C-terminal domain.The internal bodily cavities are covered by a monolayer of epithelial cells that provides the first line of protection to pathogenic agents by acting as a physical barrier and by supporting mucosal immunity. Epithelial tissues are submitted to major remodeling during which epithelial cells are turned into mesenchymal cells along a process named epithelial-mesenchymal transition. Conversely, mesenchymal cells differentiate into epithelial cells in a process named mesenchymal-epithelial transition. Both processes take place during normal development and pathologic conditions. These phenotypical transition events are not supported by one unique process but rather refer as to a large variety of cell plasticity phenomenon in response to various pathways. One major challenge in modern cell biology, especially oncology, is to decipher the molecular mechanisms underlying these biological events (1-3).Polarized epithelial cells present two distinct plasma membrane domains; they are the apical domain exposed to the lumen and a basolateral domain contacting the extracellular matrix and the neighboring cells. These two domains are separated by the most apical junctional complex named the tight junction (TJ) 3 that is a hallmark of epithelial cell polarity. The TJ serves several functions; a barrier function to control the paracellular transport, a fence function to prevent the intermixing of proteins and lipids of the outer leaflet of the apical and basolateral domains, delivery site for exocytotic vesicles, and finally a signaling platform for the regulation of cell polarity, proliferation, and differentiation (4). The tetras...