Rho-GTPase-activating Protein Interacting with Cdc-42-interacting Protein 4 Homolog 2 (Rich2): A NEW Ras-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (Rac1) GTPase-ACTIVATING PROTEIN THAT CONTROLS DENDRITIC SPINE MORPHOGENESIS*

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.     

Scaffold Protein X11α Interacts with Kalirin-7 in Dendrites and Recruits It to Golgi Outposts

Pyramidal neurons in the mammalian forebrain receive their synaptic inputs through their dendritic trees, and dendritic spines are the sites of most excitatory synapses. Dendritic spine structure is important for brain development and plasticity. Kalirin-7 is a guanine nucleotide-exchange factor for the small GTPase Rac1 and is a critical regulator of dendritic spine remodeling. The subcellular localization of kalirin-7 is thought to be important for regulating its function in neurons. A yeast two-hybrid screen has identified the adaptor protein X11α as an interacting partner of kalirin-7. Here, we show that kalirin-7 and X11α form a complex in the brain, and this interaction is mediated by the C terminus of kalirin-7. Kalirin-7 and X11α co-localize at excitatory synapses in cultured cortical neurons. Using time-lapse imaging of fluorescence recovery after photobleaching, we show that X11α is present in a mobile fraction of the postsynaptic density. X11α also localizes to Golgi outposts in dendrites, and its overexpression induces the removal of kalirin-7 from spines and accumulation of kalirin-7 in Golgi outposts. In addition, neurons overexpressing X11α displayed thinner spines. These data support a novel mechanism of regulation of kalirin-7 localization and function in dendrites, providing insight into signaling pathways underlying neuronal plasticity. Dissecting the molecular mechanisms of synaptic structural plasticity will improve our understanding of neuropsychiatric and neurodegenerative disorders, as kalirin-7 has been associated with schizophrenia and Alzheimer disease.     

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

Plexin‐B1 is a GTPase activating protein for M‐Ras,remodelling dendrite morphology

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin‐B1, induces axonal growth cone collapse by functioning as an R‐Ras GTPase activating protein (GAP). Here, we report that Plexin‐B1 shows GAP activity for M‐Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M‐Ras was upregulated during dendritic development. Knockdown of endogenous M‐Ras—but not R‐Ras—reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M‐Ras, M‐Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M‐Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M‐Ras(Q71L). M‐Ras(Q71L) stimulated extracellular signal‐regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down‐regulation of ERK was required for a Sema4D‐induced reduction of dendrite growth. Thus, we conclude that Plexin‐B1 is a dual functional GAP for R‐Ras and M‐Ras, remodelling axon and dendrite morphology, respectively.     

Convergent CaMK and RacGEF signals control dendritic structure and function

Structural plasticity of excitatory synapses is a vital component of neuronal development, synaptic plasticity and behavior, and its malfunction underlies many neurodevelopmental and psychiatric disorders. However, the molecular mechanisms that control dendritic spine morphogenesis have only recently emerged. We summarize recent work that has revealed an important connection between calcium/calmodulin-dependent kinases (CaMKs) and guanine-nucleotide-exchange factors (GEFs) that activate the small GTPase Rac (RacGEFs) in controlling dendritic spine morphogenesis. These two groups of molecules function in neurons as a unique signaling cassette that transduces calcium influx into small GTPase activity and, thence, actin reorganization and spine morphogenesis. Through this pathway, CaMKs and RacGEFs amplify calcium signals and translate them into spatially and temporally regulated structural remodeling of dendritic spines.     

ApoE receptor 2 regulates synapse and dendritic spine formation

Background

Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation—processes critical for learning and memory.

Methodology/Principal Findings

In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.

Conclusions/Significance

These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.     

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.     

Spine Formation Pattern of Adult-Born Neurons Is Differentially Modulated by the Induction Timing and Location of Hippocampal Plasticity

In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn’t respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP–induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.     

The RhoG/ELMO1/Dock180 signaling module is required for spine morphogenesis in hippocampal neurons

Dendritic spines are actin-rich structures, the formation and plasticity of which are regulated by the Rho GTPases in response to synaptic input. Although several guanine nucleotide exchange factors (GEFs) have been implicated in spine development and plasticity in hippocampal neurons, it is not known how many different Rho GEFs contribute to spine morphogenesis or how they coordinate the initiation, establishment, and maintenance of spines. In this study, we screened 70 rat Rho GEFs in cultured hippocampal neurons by RNA interference and identified a number of candidates that affected spine morphogenesis. Of these, Dock180, which plays a pivotal role in a variety of cellular processes including cell migration and phagocytosis, was further investigated. We show that depletion of Dock180 inhibits spine morphogenesis, whereas overexpression of Dock180 promotes spine morphogenesis. ELMO1, a protein necessary for in vivo functions of Dock180, functions in a complex with Dock180 in spine morphogenesis through activating the Rac GTPase. Moreover, RhoG, which functions upstream of the ELMO1/Dock180 complex, is also important for spine formation. Together, our findings uncover a role for the RhoG/ELMO1/Dock180 signaling module in spine morphogenesis in hippocampal neurons.     

Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling

ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.     

Essential Role of Neuron-Enriched Diacylglycerol Kinase (DGK), DGKβ in Neurite Spine Formation,Contributing to Cognitive Function

Background

Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKβ is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated.

Methodology/Principal Findings

We, therefore, developed DGKβ KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKβ. In addition, overexpression of DGKβ in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKβ, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKβ but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed.

Conclusions/Significance

These results demonstrate that membrane-localized DGKβ regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory.     

Cellular Source-Specific Effects of Apolipoprotein (Apo) E4 on Dendrite Arborization and Dendritic Spine Development

Apolipoprotein (apo) E4 is the leading genetic risk factor for Alzheimer’s disease (AD), and it has a gene dose-dependent effect on the risk and age of onset of AD. Although apoE4 is primarily produced by astrocytes in the brain, neurons can also produce apoE4 under stress conditions. ApoE4 is known to inhibit neurite outgrowth and spine development in vitro and in vivo, but the potential influence of apoE4’s cellular source on dendritic arborization and spine development has not yet been investigated. In this study, we report impairments in dendritic arborization and a loss of spines, especially thin (learning) and mushroom (memory) spines, in the hippocampus and entorhinal cortex of 19–21-month-old female neuron-specific-enolase (NSE)-apoE4 and apoE4-knockin (KI) mice compared to their respective apoE3-expressing counterparts. In general, NSE-apoE4 mice had more severe and widespread deficits in dendritic arborization as well as spine density and morphology than apoE4-KI mice. The loss of dendritic spines, especially mushroom spines, occurred in NSE-apoE4 mice as early as 7–8 months of age. In contrast, glial fibrillary acidic protein (GFAP)-apoE4 mice, which express apoE4 solely in astrocytes, did not have impairments in their dendrite arborization or spine density and morphology compared to GFAP-apoE3 mice at both ages. These results indicate that the effects of apoE4 on dendrite arborization, spine density, and spine morphology depend critically on its cellular source, with neuronal apoE4 having more detrimental effects than astrocytic apoE4.     

The F-BAR Protein Rapostlin Regulates Dendritic Spine Formation in Hippocampal Neurons

Pombe Cdc15 homology proteins, characterized by Fer/CIP4 homology Bin-Amphiphysin-Rvs/extended Fer/CIP4 homology (F-BAR/EFC) domains with membrane invaginating property, play critical roles in a variety of membrane reorganization processes. Among them, Rapostlin/formin-binding protein 17 (FBP17) has attracted increasing attention as a critical coordinator of endocytosis. Here we found that Rapostlin was expressed in the developing rat brain, including the hippocampus, in late developmental stages when accelerated dendritic spine formation and maturation occur. In primary cultured rat hippocampal neurons, knockdown of Rapostlin by shRNA or overexpression of Rapostlin-QQ, an F-BAR domain mutant of Rapostlin that has no ability to induce membrane invagination, led to a significant decrease in spine density. Expression of shRNA-resistant wild-type Rapostlin effectively restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the protein kinase C-related kinase homology region 1 (HR1) or Src homology 3 (SH3) domain did not. In addition, knockdown of Rapostlin or overexpression of Rapostlin-QQ reduced the uptake of transferrin in hippocampal neurons. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin, also reduced spine density and the transferrin uptake. These results suggest that Rapostlin and Rnd2 cooperatively regulate spine density. Indeed, Rnd2 enhanced the Rapostlin-induced tubular membrane invagination. We conclude that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine formation through the regulation of membrane dynamics.     

ACAPs are arf6 GTPase-activating proteins that function in the cell periphery

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.     

ASAP3 is a focal adhesion-associated Arf GAP that functions in cell migration and invasion

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.     

KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX

Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.     

Arf GTPase-activating proteins and their potential role in cell migration and invasion

Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.Key words: Arf GAP, Arf, effector, ADP-ribosylation factor, GTPase-activating protein, focal adhesions, podosomes, invadopodia, cell migrationCell migration involves adhesive structures in which the cell membrane is integrated with the actin cytoskeleton.¹ Cells acquire a spatial asymmetry to enable them to turn intracellular generated forces into a net cell body translocation. With the asymmetry, there is a clear distinction between the cell front and rear. Active membrane processes, including lamellipodia and filopodia, take place primarily around the cell front. Extension of both filopodia and lamellipodia is coupled with local actin polymerization, which generates protrusive force. In some cells, focal complexes form at the leading edge of lamellipodia and filopodia. Focal complexes are specialized surfaces of the plasma membrane that mediate attachment to the substratum, providing traction and allowing the cell edge to protrude. Focal complexes mature with cell migration to form another specialized surface in the plasma membrane, focal adhesions (FAs). FAs localize to the termini of stress fiber bundles and serve in longer-term anchorage at the rear of the cell.² A contractile force is generated at the rear of the cell by the myosin motors to move the cell forward and cell-substratum (extracellular matrix) attachments are released to retract the cell rear. In some cells, podosomes are adhesive structures that mediate cell migration and sometimes invasion.The structures involved in cell migration that are affected by Arf GAPs are FAs, podosomes and invadopodia. FAs contain multiple proteins, including integrins, which are transmembrane proteins.³ The extracellular part of integrins binds to the extracellular matrix. The cytoplasmic domains of integrins associate with multiple signaling proteins as well as proteins that are part of the actin cytoskeleton, thereby coordinating signaling events involved in cell migration and linking the extracellular matrix to the cytoskeleton. Cytoplasmic proteins critical to the function of FAs and that are often used as markers of FAs include vinculin, paxillin and focal adhesion kinase. At least five distinct Arf GAPs have been found to associate with FAs, including GIT1, GIT2, ASAP1, ASAP3 and ARAP2.⁴Podosomes and invadopodia are related structures induced by action of Src (reviewed in ref. ⁵). They contain some proteins in common with FAs, but do have some differences that likely reflect different function and/or regulation. For example, podosomes contain ASAP1 but not ASAP3.⁶ Podosomes and invadopodia have not been examined for the presence of other Arf GAPs. Like FAs, podosomes and invadopodia mediate adhesion to extracellular surfaces. In addition, they are points of degradation of the extracellular matrix and may transfer tension along the extracellular matrix to enable the cell to move. Consistent with the function in motility, podosomes and invadopodia are dynamic structures, turning over in minutes. Podosomes are found in normal physiology of cells including smooth muscle cells, osteoclasts and macrophages and in Src-transformed fibroblasts. Invadopodia are observed in transformed cells, such as cells derived from breast cancers.Two families of GTP-binding proteins within the Ras superfamily, Rho and Arf, are involved in both actin and membrane remodeling. RhoA regulates stress fibers (bundles of actin filaments that traverse the cell and are linked to the extracellular matrix through FAs) and the assembly of FAs.⁷ Rac1 regulates membrane ruffling and lamellipodia formation.⁸ Cdc42 regulates filopodia formation.⁹ Activation of Cdc42 has been shown to lead to the sequential activation of Rac1 and then RhoA in growth factor stimulated fibroblasts.Arf proteins regulate membrane traffic and the actin cytoskeleton.¹⁰ There are six mammalian Arf proteins, divided into three classes based on their amino-acid sequence. Arf12 and 3 are class I, Arf4 and Arf5 are class II and Arf6 is the single member of the class III group. Arf1 and Arf6 have been the most extensively studied. Most work has focused on Arf1 function in the Golgi apparatus and endocytic compartments although Arf1 has been found to affect paxillin recruitment to FAs and trafficking of epidermal growth factor receptor from the plasma membrane. Arf6 affects the endocytic pathway and the peripheral actin cytoskeleton.The function of Rho and Arf family proteins depends on a cycle of binding and hydrolyzing GTP. However, Rho and Arf family proteins have slow intrinsic nucleotide exchange. Rho family proteins have slow intrinsic GTPase activity and Arf family proteins have no detectable intrinsic GTPase activity. The cycle of GTP binding and hydrolysis is driven by accessory proteins called guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho family proteins are also regulated by guanine nucleotide dissociation inhibitors, which prevent spontaneous activation in the cytoplasm.Arf GAPs are enzymes that catalyze the hydrolysis of GTP bound to Arf proteins, thereby converting Arf•GTP to Arf•GDP. Thirty-one genes in human encode proteins with Arf GAP domains (Fig. 1). The Arf GAP family is divided into ten subgroups based on domain structure and phylogenetic analysis.¹¹ Six subgroups contain the Arf GAP domain at the N-terminus of the protein. Four groups contain a tandem of a PH, Arf GAP and Ankyrin repeat domains. The Arf GAP nomenclature is mostly based on the protein domain structure. For instance, the ASAP first identified, ASAP1, contains Arf GAP, SH3, Ank repeat and PH domains; ARAPs contain Arf GAP, Rho GAP, Ank repeat and PH domains; ACAPs contain Arf GAP, coiled-coil (later identified as BAR domain), Ank repeat and PH domains; and AGAPs contain Arf GAP, GTP-binding protein-like, Ank repeat and PH domains.Open in a separate windowFigure 1Domain structure of the Arf GAP family. The schematic representation of the ten groups of proteins containing the Arf GAP domain is not drawn to scale. Abbreviations used are: ALPS, ArfGAP1 lipid-packing sensor domain; Ank, Ankyrin repeats; Arf GAP, Arf GTPase activating domain; BAR, Bin/Amphiphysin/Rvs domain; CALM, CALM binding domain; CB, clathrin box; CC, coiled-coiled domain; FG repeats, multiple copies of the XXFG motif; GLD, GTP-binding protein-like domain; PBS, paxillin binding site; PH, pleckstrin homology domain; Pro (PxxP)₃, cluster of three proline-rich (PxxP) motifs; Pro (D/ELPPKP)₈, eigth tandem Prolin-rich (D/ELPPKP) motifs; RA, Ras association motif; Rho GAP, Rho GTPase activating domain; SAM, sterile α-motif; SH3, Src homology 3 domain; SHD, Spa homology domain. *ASAP2 and ASAP3 lack the Pro (D/ELPPKP)₈ motifs. ASAP3 has no SH3 domain. ^&AGAP2 has a splice variant with three N-terminal PxxP motifs, called PIKE-L. @ARAP2 has an inactive Rho GAP domain.The subcellular localization and function of a number of Arf GAPs have been identified. Arf GAP1, Arf GAP2 and Arf GAP3 are found in the Golgi apparatus where they control membrane traffic by regulating Arf1•GTP levels.^12,13 Arf GAP1 has also been proposed to directly contribute to the formation of transport intermediates.¹⁴ SMAPs and AGAP1 and AGAP2 are associated with endosomes and regulate endocytic trafficking.^14,15 ASAPs, ARAPs and Gits are associated with FAs. ASAPs, ARAPs and ACAPs are found in actin-rich membrane ruffles. ASAP1 is also found in invadopodia and podosomes.⁴ We propose that common to all Arf GAPs is that they laterally organize membranes, which maintain surfaces of specialized functions such as FAs and podosomes/invadopodia. Some Arf GAPs function primarily as Arf effectors with the turnover rate of the specialized membrane surface being determined by the catalytic rate of the GAP. Other Arf GAPs function as Arf regulators that integrate several signals.ASAP1 is an example of an Arf GAP that may function as an Arf effector to regulate podosomes and invadopodia. ASAP1 is encoded by a gene on the short arm of chromosome 8. The gene is amplified in aggressive forms of uveal melanoma and cell migration rates correlate with ASAP1 expression levels in uveal melanoma¹⁶ and other cell types. ASAP1 function depends on cycling among four cellular locations, cytosol, FAs, lamellipodia and podosomes/invadopodia. ASAP1 is necessary for the formation of podosomes/invadopodia.^17,18The structural features of ASAP1 that are required to support podosome formation have been examined.^17,18 ASAP1 contains, from the N-terminus, BAR, PH, Arf GAP, Ank repeat, proline rich and SH3 domains (Fig. 2A).¹⁹ There are two major isoforms, ASAP1a and ASAP1b that differ in the proline rich domain. ASAP1a contains three SH3 binding motifs within the proline rich region including an atypical SH3 binding motif with 6 consecutive prolines. The atypical SH3 binding motif is absent in ASAP1b (Fig. 2A). ASAP1 also has a highly conserved tyrosine between the Ank repeat and proline rich domains that is a site of phosphorylation by the oncogene Src.¹⁸Open in a separate windowFigure 2ASAP1 function in podosome and invadopodia formation. (A) Domain structure of ASAP1 splice variants. ASAP1a contains three proline-rich motifs, P1, P2 and P3. P1 and P3 contain a typical (PxxP) motif. P2 contains six prolines. ASAP1b contains only P1 and P3. (B) Model of ASAP1 functioning as an Arf effector to regulate podosome and invadopodia formation. ASAP1 integrates signals from Src, PIP₂ and Arf•GTP. For abbreviations of the domain structure of ASAP1 see Figure 1. Other abbreviations: PIP₂, phosphoinositides 4,5-biphosphate; Arf1, ADP-ribosylation factor 1.The BAR domain is a bundle of 3 α-helices that homodimerizes to form a boomerang-shaped structure.^20,21 BAR domains sense or induce membrane curvature.²⁰ ASAP1 has been found to induce curvature dependent on its BAR domain.²² BAR domains are also protein binding sites.²¹ The BAR domain of ASAP1 binds to FIP3, a Rab11 and Arf6 binding proteins.²³ Arf6-dependent targeting of ASAP1 is likely mediated by FIP3.²³ Deletion of or introduction of point mutations into the BAR domain render ASAP1 inactive in supporting podosome formation. The relative role of membrane tubulation and protein binding in mediating the effect of the BAR domain on podosome formation has not been explored.The SH3 domain of ASAP1 binds to focal adhesion kinase²⁴ and pyk2.²⁵ Either deletion of or introduction of point mutations into the SH3 domain abrogates the ability of ASAP1 to support podosome formation.¹⁸ The molecular basis for the function of the SH3 domain in podosome formation is not known. The proline rich domain binds to Src¹⁹ and CrkL.²⁶ Whether it also binds to cortactin has not been resolved. Reports also conflict regarding the importance of the proline rich domain for podosomes/invadopodia formation.^17,18Three signals impinge on ASAP1 to drive podosome formation (Fig. 2B). A conserved tyrosine between the Ank and proline rich motifs is phosphorylated by Src.^18,25 Mutation of the tyrosine to phenylalanine results in a protein that functions as a dominant negative blocking podosome formation. ASAP1 with the tyrosine changed to glutamate can support podosome formation, but the mutant ASAP1 is not sufficient to drive podosome formation.¹⁸ Based on these results, phosphorylation of the conserved tyrosine is necessary but not sufficient to support podosome formation. Phosphatidylinositol 4,5-bisphosphate (PIP₂) binds to the PH domain, which stimulates GAP activity in vitro.²⁷ ASAP1 with mutations in the PH domain that abrogate binding, does not support podosome formation (Jian, Bharti and Randazzo PA, unpublished observations). Point mutations in the PH domain affect both the K_m and the k_cat for GAP activity. The effect of mutating the PH domain on the ability of ASAP1 to support podosome formation may be consequent to changes in binding Arf1•GTP; it is not likely the result of loss of GAP activity. ASAP1 with a point mutation in the GAP domain that prevents GAP activity but not Arf1•GTP binding is able to support podosome formation whereas a point mutant of ASAP1 that cannot bind Arf1•GTP does not (Jian, Bharti and Randazzo PA, unpublished observations).¹⁸ These data support the idea that ASAP1 integrates three signals, (1) PIP₂, (2) Src and (3) Arf1•GTP. In response to the signals, ASAP1 functions as a scaffold and directly alters the lipid bilayer to create a domain within the plasma membrane that becomes a podosome. In this model, ASAP1 is functioning as an Arf effector and the GAP activity may regulate the turnover of podosomes.ASAP3, another ASAP-type protein, is found in FAs.⁶ Reducing ASAP3 expression also reduces cell migration and invasion of Arf GAPs in cell migration mammary carcinoma cells through matrigel. Although ASAP3 does not affect the ability to form FAs, it does affect stress fiber formation and may affect focal adhesion maturation (Ha, Chen and Randazzo PA, unpublished observations).⁶ The molecular mechanisms underlying the effects of ASAP3 on the cytoskeleton are being examined including the possibility that, like ASAP1, ASAP3 integrates several signals and functions as an Arf effector.ARAPs are examples of Arf GAP family proteins that function as Arf regulators. In common with ASAPs, they integrate a number of signaling pathways and affect the actin cytoskeleton. Three genes encode ARAPs in humans.¹¹ Each of the ARAPs is comprised of a SAM, five PH, Arf GAP, Rho GAP, Ank repeat and Ras association domains. Two of the five PH domains have the consensus sequence for binding to the signaling lipid phosphoinositide 3,4,5-triphosphate (PIP₃); however, when examined for ARAP1, PIP₃ was not involved in membrane targeting (Campa F, Balla and Randazzo PA, unpublished observations).Examination of the role of ARAP2 in FA formation has provided information about the function of the GAP activity in the cellular function of an Arf GAP. ARAP2 selectively uses Arf6 as a substrate and, different from ARAP1 and ARAP3, has an inactive Rho GAP domain. The Rho GAP domain, however, retains the ability to selectively bind to RhoA•GTP. Also different from ARAP1 and ARAP3, ARAP2 associates with FAs. Cells with reduced expression of ARAP2, consequent to siRNA treatment, have fewer FAs and stress fibers and more focal complexes than control cells. The formation of FAs and stress fibers can be restored by expressing recombinant wild type ARAP2. A mutant of ARAP2 that lacks Arf GAP activity, while retaining the ability to bind to Arf6•GTP, cannot restore FA and stress fiber formation. Similarly, expression of a mutant of ARAP2 that is not able to bind RhoA•GTP cannot reverse the effect of reducing expression of endogenous ARAP2.²⁸ These results support the idea that ARAP2 functions as an Arf GAP that is an effector of RhoA.The model of ARAP2 functioning as a RhoA effector can explain the effects of ARAP2 on FAs (Fig. 3). Arf6•GTP is involved in the formation of Rac1•GTP.²⁹ Rac1•GTP drives lamellipodia and focal complex formation. The conversion of focal complexes to FAs is accompanied by an increase in RhoA•GTP and a decrease in Rac1•GTP. ARAP2 could function to mediate the reciprocal changes in RhoA and Rac1. RhoA•GTP formation leads to the activation of ARAP2. As a consequence of Arf6 GAP activity, Arf6•GTP is converted to Arf6•GDP. With reduced Arf6•GTP, Rac1•GTP concentration also decreases.Open in a separate windowFigure 3Model of ARAP2 as an Arf regulator that controls focal adhesion formation. In this model, ARAP2 functions as a RhoA effector. The inactive Rho GAP domain of ARAP2 binds to RhoA•GTP, which contributes to activation of Arf6 GAP activity. ARAP2 hydrolyzes its substrate Arf6•GTP into Arf6•GDP. Subsequent to Arf6•GTP hydrolysis, Rac1•GTP concentration decreases. For abbreviations of the domain structure of ARAP2 see Figure 1.The Arf GAP activity of other ARAPs may also be critical for cellular functions of the protein. Furthermore, the Rho GAP activity is slow for ARAP1 and ARAP3. It is possible that ARAP1 and ARAP3 can function as Rho effectors with an active Rho GAP domain analogously to ASAP1 functioning as an Arf effector. Further definition of the cellular function of ARAP1 and ARAP3 will provide opportunities to test this idea.We have provided two examples of Arf GAPs that affect cell adhesion and migration. In one case, the Arf GAP appears to function as an Arf effector. In the other case, the Arf GAP functions as a regulator of Arf. The difference in function was discerned using Arf GAP mutants. If functioning as an Arf effector, an Arf GAP mutant that can bind Arf•GTP but not induce hydrolysis can reverse the effect of reduced endogenous Arf GAP, whereas a mutant that cannot bind Arf•GTP cannot replace endogenous Arf GAP. When working as an Arf regulator, a mutant that can bind Arf•GTP but not induce GTP hydrolysis cannot replace endogenous Arf GAP. Whether functioning as an effector or regulator, the rate of GAP activity determines the turnover rate of a specialized membrane surface maintained by Arf.The Arf GAPs have specific sites of action within cells. Some contribute to malignancy, such as ASAP1, ASAP3, AGAP2 and SMAP1.³⁰ The molecular basis of cellular function of each Arf GAPs is distinct. Here, we describe one Arf GAP that functions as an Arf effector and another that functions as an Arf regulator. Each class of Arf GAP has distinct sets of protein binding partners. Furthermore, catalytic mechanism differs among the GAPs. Because of these differences, Arf GAPs may be useful therapeutic targets for cancer therapy.     

Modeling the dynamic behaviors of the COPI vesicle formation regulators,the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain–containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain–containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.