Arf6 Activation Research Articles

Inhibition of formyl peptide-stimulated phospholipase D activation by Fal-002-2 via blockade of the Arf6, RhoA and protein kinase C signaling pathways in rat neutrophils

Three recently developed selective phospholipase D (PLD) inhibitors N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide (VU0155056), (S)-N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide (VU0155069), and N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]decan-8-yl)ethyl)quinoline-3-carboxamide (VU0285655-1) inhibited O2 (•-) generation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel 2-phenyl-4-quinolone compound 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), which inhibited O2 (•-) generation, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC50 16.0 ± 5.0μM). Fal-002-2 attenuated the interaction of PLD1 with ADP-ribosylation factor (Arf) 6, Ras homology (Rho) A and protein kinase C (PKC) isoforms (α, βI, and βII), and also inhibited the membrane recruitment of Arf6 and RhoA in fMLP-stimulated neutrophils, but not in GTPγS-stimulated cell-free system. The cellular levels of GTP-bound Arf6 and GTP-bound RhoA were reduced by Fal-002-2. Fal-002-2 also attenuated the membrane recruitment of Rho-associated protein kinase 1, phosphorylation of myosin light chain 2 at Thr18/Ser19 and PLD1 at Thr147, and the interaction of Arf6 with both arfaptin 1 and phosphatidylinositol 4-phosphate 5-kinase 1A. The association between RhoA and Vav, the interaction of Vav with both Lyn and Lck, the membrane recruitment of Vav, and the phosphorylation of Vav at Tyr174, but not Src family at Tyr416, were all attenuated by Fal-002-2 in fMLP-stimulated neutrophils. These results indicate that Fal-002-2 is not a direct PLD inhibitor, but the inhibition of fMLP-stimulated PLD activity by Fal-002-2, which partly accounts for its suppression of O2 (•-) generation, is attributable to the blockade of both Arf6 and RhoA activation and attenuation of the interaction of Arf6, RhoA and PKC isoforms with PLD1 in rat neutrophils.

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

SummaryPrecise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.

The scaffolding protein GRASP/Tamalin directly binds to Dock180 as well as to cytohesins facilitating GTPase crosstalk in epithelial cell migration

BackgroundThe transition of epithelial cells from their normal non-motile state to a motile one requires the coordinated action of a number of small GTPases. We have previously shown that epithelial cell migration is stimulated by the coordinated activation of Arf and Rac GTPases. This crosstalk depends upon the assembly of a multi-protein complex that contains the Arf-activating protein cytohesin 2/ARNO and the Rac activating protein Dock180. Two scaffolding proteins that bind directly to cytohesin 2 organize this complex.ResultsWe now have found that Rac activation in response to hepatocyte growth factor (HGF) requires cytohesin 2 and Dock180. GRASP/Tamalin is one of the scaffolds that builds the complex containing cytohesin 2 and Dock180. We determine here that the Ala/Pro rich region of GRASP directly interacts with the SH3 domain of Dock180. By binding to both cytohesin 2/ARNO and Dock180, GRASP bridges the guanine nucleotide exchange factors (GEFs) that activate Arf and Rac, thereby promoting Arf-to-Rac signaling. Furthermore, we find that knockdown of GRASP impairs hepatocyte growth factor (HGF)-stimulated Rac activation and HGF-stimulated epithelial migration.ConclusionsGRASP binds directly both cytohesin 2 and Dock180 to coordinate their activities, and by doing so promotes crosstalk between Arf and Rac.

Abstract A66: NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling

Abstract The adhesion scaffolding protein NEDD9 was identified as potential pro-metastatic gene in several cancers. The molecular mechanisms of NEDD9-driven metastasis are still unknown. In this study, we show that expression of NEDD9 positively correlates with the invasive stage of breast cancer. We show that NEDD9 localizes to invadopodia and endosomes. Notably, NEDD9 depleted cells have increased levels of inactive surface receptors due to increase in fast recycling of Rab4 and Rab5 positive vesicles. Mechanistically, we found that NEDD9 binds to and scaffolds the Arf6 specific GAP - ASAP3, decreasing Arf6 activity. Thus, depletion of NEDD9 leads to activation of Arf6. Inhibition of Arf6 or re-expression of NEDD9 in shNEDD9 cells was sufficient to restore recycling rates, decrease the number of Rab4 and 5 positive vesicles and the invasive properties of tumor cells. Thus, in this work, we uncover the mechanistic basis of NEDD9-driven invasion and identify a new role for NEDD9 in Arf6-dependant endocytosis. Citation Format: Elena N. Pugacheva, Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Mark Culp, Robert Wysolmerski, Scott Weed, Alexey Ivanov. NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A66.

ARAP2 Signals through Arf6 and Rac1 to Control Focal Adhesion Morphology*[S

Focal adhesions (FAs) are dynamic structures that connect the actin cytoskeleton with the extracellular matrix. At least six ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), including ARAP2 (an Arf6 GAP), are implicated in regulation of FAs but the mechanisms for most are not well defined. Although Rac1 has been reported to function downstream of Arf6 to control membrane ruffling and cell migration, this pathway has not been directly examined as a regulator of FAs. Here we test the hypothesis that ARAP2 promotes the growth of FAs by converting Arf6·GTP to Arf6·GDP thereby preventing the activation of the Rho family GTP-binding protein Rac1. Reduced expression of ARAP2 decreased the number and size of FAs in cells and increased cellular Arf6·GTP and Rac1·GTP levels. Overexpression of ARAP2 had the opposite effects. The effects of ARAP2 on FAs and Rac1 were dependent on a functional ArfGAP domain. Constitutively active Arf6 affected FAs in the same way as did reduced ARAP2 expression and dominant negative mutants of Arf6 and Rac1 reversed the effect of reduced ARAP2 expression. However, neither dominant negative Arf6 nor Rac1 had the same effect as ARAP2 overexpression. We conclude that changes in Arf6 and Rac1 activities are necessary but not sufficient for ARAP2 to promote the growth of FAs and we speculate that ARAP2 has additional functions that are effector in nature to promote or stabilize FAs.

Mimicking p14ARF Phosphorylation Influences Its Ability to Restrain Cell Proliferation

The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered loci in human cancer. It is generally accepted that ARF is involved in oncogenic checkpoint pathways by sensitizing incipient cancer cells to undergo growth arrest or apoptosis through both p53-dependent and independent pathways. While intensive studies have been focused on ARF activation at the transcriptional level, only recently mechanisms governing ARF turnover have been identified. Here, we show for the first time that p14ARF is a PKC target. Prediction analysis showed many potential phosphorylation sites in PKC consensus sequences within ARF protein, and, among them, the threonine at position 8 was the most conserved. Substitution of this threonine influences both ARF stability and localization. Furthermore, a phosphomimetic ARF mutation reduces the ability to arrest cell growth although the ability to bind MDM2 and stabilize p53 result unaffected. Thus we propose that phosphorylation of ARF in both immortalized and tumor cell lines could be a mechanism to escape ARF surveillance following proliferative and oncogenic stress.

Arf activation at the Golgi is modulated by feed-forward stimulation of the exchange factor GBF1

ADP-ribosylation factors (Arfs) play central roles in the regulation of vesicular trafficking through the Golgi. Arfs are activated at the Golgi membrane by guanine-nucleotide-exchange factors (GEFs) that are recruited from cytosol. Here, we describe a novel mechanism for the regulation of recruitment and activity of the ArfGEF Golgi-specific BFA resistance factor 1 (GBF1). Conditions that alter the cellular Arf-GDP:Arf-GTP ratio result in GBF1 recruitment. This recruitment of GBF1 occurs selectively on cis-Golgi membranes in direct response to increased Arf-GDP. GBF1 recruitment requires Arf-GDP myristoylation-dependent interactions suggesting regulation of a membrane-bound factor. Once recruited, GBF1 causes increased Arf-GTP production at the Golgi, consistent with a feed-forward self-limiting mechanism of Arf activation. This mechanism is proposed to maintain steady-state levels of Arf-GTP at the cis-Golgi during cycles of Arf-dependent trafficking events.

Targeting of the Arf-GEF GBF1 to lipid droplets and Golgi membranes

Lipid droplet metabolism and secretory pathway trafficking both require activation of the Arf1 small G protein. The spatiotemporal regulation of Arf1 activation is mediated by guanine nucleotide exchange factors (GEFs) of the GBF and BIG families, but the mechanisms of their localization to multiple sites within cells are poorly understood. Here we show that GBF1 has a lipid-binding domain (HDS1) immediately downstream of the catalytic Sec7 domain, which mediates association with both lipid droplets and Golgi membranes in cells, and with bilayer liposomes and artificial droplets in vitro. An amphipathic helix within HDS1 is necessary and sufficient for lipid binding, both in vitro and in cells. The HDS1 domain of GBF1 is stably associated with lipid droplets in cells, and the catalytic Sec7 domain inhibits this potent lipid-droplet-binding capacity. Additional sequences upstream of the Sec7 domain-HDS1 tandem are required for localization to Golgi membranes. This mechanism provides insight into crosstalk between lipid droplet function and secretory trafficking.

Total Synthesis of AMF-26, an Antitumor Agent for Inhibition of the Golgi System, Targeting ADP-Ribosylation Factor 1

An effective method for the total synthesis of 1 (AMF-26), a potentially promising new anticancer drug that disrupts the Golgi system by inhibiting the ADP-ribosylation factor 1 (Arf1) activation, has been developed for the first time. The construction of the chiral linear precursor (a key to the synthesis) was achieved by the asymmetric aldol reaction followed by the computer-assisted predictive stereoselective intramolecular Diels-Alder reaction. The global antitumor activity of the totally synthetic 1 against a variety of human cancer cells was assessed using a panel of 39 human cancer cell lines (JFCR39), and it was shown that the synthetic 1 strongly inhibited the growth of several cancer cell lines at concentrations of less than 0.04 μM. Biological assays of novel derivatives, 26 and 31, which have different side-chains at the C-4 positions in the Δ¹,²-octalin backbone, disclosed the importance of the suitable structure of the side-chain containing conjugated multidouble bonds.

Grp1‐associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)‐dependent membrane trafficking pathway

GRASP interacts with Grp1 (general receptor for phosphoinositides 1; cytohesin 3), which catalyses nucleotide exchange on and activation of Arf6 (ADP-ribosylation factor-6). Arf6 is a low-molecular-mass GTPase that regulates key aspects of endocytic recycling pathways. Overexpressed GRASP accumulated in the juxtanuclear ERC (endocytic recycling compartment). GRASP co-localized with a constitutively inactive mutant of Arf6 in the ERC such that it was reversed by expression of wild-type Grp1. Co-expression of GRASP and Grp1 promoted membrane ruffling, a cellular hallmark of Arf6 activation. GRASP accumulation in ERC was found to block recycling of the MHC-I (major histocompatibility complex-I), which is trafficked by the Arf6-dependent pathway. In contrast, overexpression of GRASP had no effect on the recycling of transferrin receptors, which are trafficked by a clathrin-dependent pathway. The findings suggest that GRASP regulates the non-clathrin/Arf6-dependent, plasma membrane recycling and signalling pathways.

ARF1-regulated coatomer directs the steady-state localization of protein kinase C epsilon at the Golgi apparatus

Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β′-COP). This receptor also functions as a vesicle coat protein in the secretory pathway where it is regulated by the small GTP-binding protein ADP-ribosylation factor, ARF1. We inhibited ARF1 activation to test the requirement for RACK2/β′-COP in PKCε localization in NIH3T3 fibroblasts. We found that steady-state localization of PKCε at the Golgi complex requires ARF1-regulated RACK2/β′-COP function. By contrast, we did not observe any defects in phorbol ester-induced translocation when ARF1 was inhibited. We also found that PKCε bound to isolated membranes through two distinct mechanisms. One mechanism was dependent upon RACK2/β′-COP while a second was RACK2/β′-COP-independent and stimulated by phorbol esters. Finally, we show that RACK2/β′-COP affects the subcellular distribution of a constitutively active form of PKCε, in a manner similar to what we observed for wild-type PKCε. Together, our data support a role for RACK2/β′-COP in the steady-state localization of PKCε at the Golgi apparatus, which may be independent of its role during PKCε translocation to the cell surface.

Abstract 19123: Brag2, An Arf6 Gef, Regulates Integrin-dependent Adhesion in Endothelial Cells and is Involved in Angiogenesis

β1-integrins are essential for angiogenesis. However, the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis are unclear. Brag2 is a guanine nucleotide exchange factor for the small GTPase ARF6, which is involved in the traffic of receptors and in the regulation of actin cytoskeleton. Here, we demonstrate that siRNA-mediated silencing of Brag2 reduced basal and VEGF-induced activation of Arf6 and significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and 3-dimensional migration of EC. Transfection with Brag2 siRNA increased focal adhesions and enhanced adhesion of EC on β1-integrin ligands, such as fibronectin and collagen. In line with these results, silencing of Brag2 significantly increased the surface expression of the α5β1-integrin. Moreover, silencing of Brag2 significantly reduced the endocytosis of β1-integrins and specifically of the active (matrix bound) α5β1-integrin present in focal adhesions, suggesting that Brag2 may contribute to the disassembly of focal adhesions during migration via β1-integrin-endocytosis. Strikingly, in vivo silencing of the Brag2 orthologues in zebrafish embryos with morpholinos perturbed vascular development by leading to defects of the parachordal vessels, the dorsal longitudinal anastomotic vessel and the intersomitic vessels. Beyond developmental angiogenesis, in vivo silencing of Brag2 with intravitreal injection of shRNA significantly reduced pathological retinal neovascularization in the model of retinopathy of prematurity (32 % inhibition) and pathological choroidal neovascularization in a model of laser-induced choroidal neovascularization. These data reveal that the Arf6 GEF, Brag2 is essential for developmental and pathological angiogenesis probably by mediating EC migration and angiogenic sprouting through regulation of adhesion by promoting integrin internalization.

Thrombosis and Inflammation Are Coupled by a Common Thioredoxin Reductase Pathway Downstream of the P2×7 Receptor.

AbstractAbstract 2218The purinergic P2X7 receptor contributes to thrombosis by promoting tissue factor (TF) activation and the release of prothrombotic microparticles (MP). On primed macrophages, P2X7 stimulation induces the procoagulant activity of cell surface TF and the release of MP that carry TF, integrin β1 and protein disulfide isomerase (PDI). The generation of TF+ MP is dependent on extracellular thiol-disulfide exchange, but intermediates leading to MP generation downstream of P2X7 signaling are incompletely defined. Tracking of cell surface TF by confocal microscopy shows that constitutive internalization of TF is prevented by P2X7 activation. In non-stimulated cells, inhibition of dynamin-dependent endocytosis retains TF on the cell surface, rapidly upregulates TF activity, and releases procoagulant MP carrying TF and integrin β1. Integrin recycling is dependent on the small GTPase ARF6 that is found incorporated into MP released when internalization is blocked. In contrast, activation of P2X7 releases ARF6 into the MP-depleted supernatant rather than associated with MP. Decreased ARF6 GTP loading in P2X7-stimulated cells and additional data in integrin α4 S988A mutant mice with reduced ARF6 activity support the conclusion that P2X7 interrupts constitutive ARF6 recycling to increase cell surface availability of TF-integrin complexes for incorporation into MP. Confocal imaging shows that P2X7 stimulation promotes filopodia formation and the peripheral transport of TF to the tips of filipodia. Proteomics identifies γ-actin as a thiol-regulated MP protein that is released by P2X7 signaling. We show that specific inhibition of thioredoxin reductase attenuates P2X7-induced actin remodeling and filopodia formation, the generation of highly procoagulant MP carrying γ-actin and PDI, and the increased availability of extracellular thioredoxin and free thiols. Importantly, inhibition of thioredoxin reductase also prevented the processing and release of the pro-inflammatory cytokine IL1β. Thus, the thioredoxin reductase system is a crucial intermediate downstream of P2X7 activation that may couple coagulation and inflammation in cardiovascular diseases. Disclosures:No relevant conflicts of interest to declare.

Arf6-GEF BRAG1 Regulates JNK-Mediated Synaptic Removal of GluA1-Containing AMPA Receptors: A New Mechanism for Nonsyndromic X-Linked Mental Disorder

Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disabilities. BRAG1 is a guanine nucleotide exchange factor for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif; however, the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors, depresses AMPA receptor (AMPA-R)-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic XLID.

ADP-ribosylation factors 1 and 6 regulate Wnt/β-catenin signaling via control of LRP6 phosphorylation

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

GEP100/Arf6 Is Required for Epidermal Growth Factor-Induced ERK/Rac1 Signaling and Cell Migration in Human Hepatoma HepG2 Cells

BackgroundEpidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined.Methodology/Principal FindingsWe found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration.Conclusions/SignificanceTaken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization.

Acute Activation of β2-Adrenergic Receptor Regulates Focal Adhesions through βArrestin2- and p115RhoGEF Protein-mediated Activation of RhoA

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.

Arf6 guanine-nucleotide exchange factor, cytohesin-2, interacts with actinin-1 to regulate neurite extension

Proper regulation of morphological changes in neuronal cells is essential for their differentiation. Complex signaling mechanisms mediate a variety of morphological changes such as formation of neurites. It is well established that a number of small GTPases control neurite behavior before the connection with the target tissue. However, their regulatory mechanisms remain to be fully understood. Here, we show that the Arf6 guanine-nucleotide exchange factor (GEF), cytohesin-2 (CYTH2), interacts with the cytoskeletal protein actinin-1 (ACTN1) and regulates neurite extension in N1E-115 cells used as the model. Knockdown of ACTN1, as well as that of CYTH2, in cells inhibits cellular Arf6 activity and neurite extension. The C-terminal polybasic region of CYTH2 participates in interacting directly with the EFh2 domain of ACTN1. Expression of CYTH2 mutant deficient of the EFh2 domain in cells also inhibits Arf6 activation and neurite extension. Furthermore, FRET analysis detects that the respective interactive region peptides, tagged with cell-permeable short peptides, greatly decrease Arf6 activation at growth cones in a time-dependent manner. Collectively, the signaling through CYTH2 and ACTN1 properly regulates neurite extension in N1E-115 cells, demonstrating the unexpected interaction of CYTH2 and ACTN1 in the regulation of cellular Arf6 activity involved in neurite extension.