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Members of the Arf family of GTP-binding (G) proteins, including the Arf-like (Arl) proteins and Sar1, regulate membrane traffic and organelle structure through the membrane recruitment of cargo-sorting coat proteins, modulation of membrane lipid composition, and interaction with regulators of other G proteins. New roles of Arf and Arl proteins are emerging, including novel functions at the Golgi complex and in cilia formation. Their function is under tight spatial control, mediated by guanine nucleotide-exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyse GTP exchange and hydrolysis, respectively. Important advances are being gained in our understanding of how these GEFs and GAPs are tightly regulated, and the functional networks that can be formed not only by the GEFs and GAPs themselves, but also inactive forms of the Arf proteins.
The Arf family of low molecular weight GTP-binding (G) proteins controls membrane traffic and organelle structure, and is regulated through a cycle of GTP-binding to activate and GTP hydrolysis to inactivate the G protein1, 2. Arfs have several important functions, including the recruitment of coat proteins that promote sorting of cargo into vesicles, the recruitment and activation of enzymes such as the phosphatidylinositol kinases that alter membrane lipid composition, and interaction with cytoskeletal factors (Table 1). There are 6 mammalian Arf proteins that can be divided into three classes based on sequence homology: Class I (Arfs1-3), Class II (Arfs 4-5) and Class III (Arf6) (Figure 1). The Class II Arfs arose late in animal cell evolution, possibly in metazoans, but Class I Arfs are highly conserved and are present in all eukaryotes examined to date. Hence in D. melanogaster and C. elegans, each class has a single Arf orthologue, but yeast lack Class II Arfs. Plants have Class I Arfs, and potentially also Class III Arfs (see Box 1).
In addition, there are over 20 Arf-like (Arl) proteins, which seem to have broader roles than Arf proteins. Some Arl proteins, including Arl1, Arl2 and Arf-related protein 1 (Arfrp1), are ancient with members in plants, yeast and metazoans, whereas others such as Arl11 arose later in evolution and are present only in vertebrates2. Sar1, present in all eukaryotes examined to date, is also considered to be a member of the Arf family due to the presence of an amino terminal amphipathic helix and its functional similarity to Arf1 in recruiting a coat complex during vesicle budding.
The study of Arf protein function was aided greatly by the early discovery of the regulators of Arf GTP binding and GTP hydrolysis. The Arf guanine nucleotide exchange factors (GEFs) all contain a conserved Sec7 domain that catalyses GDP release from, and GTP binding to, their substrate Arf. The GTPase activating proteins (GAPs) catalyse the hydrolysis of GTP-bound Arf and are critically important since Arfs have negligible intrinsic GTP hydrolysis activity. Arf GAPs all contain a conserved Zinc-finger GAP catalytic domain. The conserved, signature catalytic domains in the GEFs and GAPs are what facilitated the identification of these Arf regulators in all organisms from yeast to man. On the other hand, the Arf substrate specificity for these GEFs and GAPs remains unclear, and whether they also work on Arl proteins has yet to be determined.
In the past, G protein activity was viewed as a linear signaling pathway, with the GDP-bound form being inactive and the GTP-bound form initiating effector functions until it returned to the inactive, GDP-bound state. The GEFs and GAPs were thought of as ‘activators’ and ‘inactivators’, respectively, that controlled this on/off switch (Figure 1C). However, work on Arf and Arl proteins over the past decade has revealed that their signaling is more complex and that GEFs and GAPs can initiate their own physiological responses. We see evidence of Arf proteins acting in pairs or in series in the ER-Golgi system and at the PM. In this review, we emphasize how Arf proteins function as a network in which the Arf regulators participate in, and integrate Arf activities with other G protein signaling networks, in addition to initiating their own distinct signalling pathways. We highlight new Arf and Arl activities, discuss how GEFs and GAPs can act as scaffolds both as effectors and in initiating signalling responses, and describe how they participate in development and disease. The reader is referred to two excellent prior reviews that deal comprehensively with Arf1 and Arf6 function 1 and more broadly with Arf/Arl, GEF and GAP proteins 2.
Arfs localize to membranes throughout the cell, including the plasma membrane, and the membranes of the secretory and the endosomal/lyosomal pathways. A distinguishing feature of Arf family G proteins is the presence of an amino-terminal amphipathic helix that is critical for membrane binding (Fig. 1A). In addition, all Arf proteins are myristoylated at the amino terminus and this cotranslational modification is required for membrane recruitment and biological activity. The myristoyl group and associated N-terminal amphipathic helix are inserted into the membrane upon GTP binding 3. Thus, in addition to changes in the effector binding regions upon exchange of GDP for GTP, Arf proteins undergo a second change in conformation that brings them into very close contact with the membrane (Figure 1)4. This distinguishes them from other small G proteins of the Ras superfamily, including the Ras, Rho and Rab Families, which have a long C-terminal linker to which their lipid membrane anchor is attached 2. Arf effectors are thus constrained to a position close to the membrane surface, in contrast to those of Rab and Rho, which can be located at a distance from the membrane 2. Some Arl proteins are myristoylated but most appear to lack this modification. In Arl8B, loss of hydrophobic residues in the amphipathic helix abolishes lysosomal localization 5. Interestingly, Arfrp1 (Arl3 in S. cerevisiae) and Arl8A and B, are acetylated rather than myristoylated at their amino-terminus 2. In Sar1, the amino-terminal amphipathic helix binds directly to membranes and induces membrane curvature 6,
Unlike Rab and Rho G proteins, no guanosine diphosphate dissociation inhibitor (GDI) proteins have been identified for Arfs or Arls. Arf1 and Arf3 appear to be released from membranes upon GTP hydrolysis in cells. Arf6, however, remains bound to membranes in its GDP-bound conformation, and there is evidence that Arf4 and Arf5 remain bound to ER-Golgi intermediate compartment (ERGIC) membranes in their GDP-bound form 7, 8. This raises the possibility that Arf proteins bound to membranes in their GDP-bound form might interact with membrane-localized partners and mediate signalling. Evidence for this idea is emerging for Arf6 (see below), and suggests that distinct signalling pathways might be coordinated through the nucleotide state of these constitutively membrane-bound Arf family proteins. Sar1 and some of the Arls, such as Arl1, Arl4 and Arl8, are cytosolic when GDP-bound, similarly to Arf1 2, 9 and it remains to be determined whether this is true for other Arl proteins.
In humans, there are 15 Arf GEFs, divided into six subfamilies, as well as the Sar1 GEF mSec12 (Table 2). No specific Arl GEFs have yet been identified, although the Arf1 GEF Syt1p in yeast apparently also has activity towards Arl1 10. The 31 identified mammalian Arf GAPs fall into nine major subgroups based on their domain structure (Table 2). Two Arl GAPs have been identified (see below). GEFs and GAPs are recruited to very specific sites within cells to not only catalyse GTP exchange and hydrolysis, respectively, but also to assemble protein complexes at these sites independently of their catalytic activity (Figure 1C). In this way, versatile signalling networks can be assembled that can respond dynamically to extra- and intracellular signals.
Following activation on membranes, GTP-bound Arfs recruit coat proteins, lipid-modifying enzymes, tethers and other effector molecules that influence membrane trafficking and organelle structure (Table 1)1, 2. For example, Arf1 recruits the cytosolic coatomer complex I (COPI) to Golgi membranes, allowing sorting of cargo proteins into COPI-coated vesicles 11. Arf proteins at the trans-Golgi network (TGN) also recruit the heterotetrameric clathrin adaptor proteins (AP), AP1, AP3 and AP4 and the three monomeric Golgi-localized γ-ear-containing, ADP-ribosylation factor-binding proteins (GGAs 1-3) 12. These various coat proteins specifically bind to and incorporate cargo proteins into forming vesicles for sorting and transport to their correct destination. Arfs can also recruit and activate enzymes that alter membrane lipid composition. Phospholipase D (PLD), which hydrolyses phosphatidylcholine to generate phosphatidic acid, is activated by all Arf proteins and also by Arl113. All Arf proteins can both recruit and activate phosphatidylinositol 4-phosphate, 5-kinase (PIP5K), an enzyme that phosphorylates phosphatidylinositol 4-phosphate (PI4P) at the 5-position to generate phosphatidylinositol 4,5-bisphosphate (PI4,5P2) 1. For example, Arf6, at the cell periphery directly affects activity of PIP5K at the PM and thus regulates PI4,5P2 levels there 1. At the Golgi, Arf1 recruits and stimulates the activity of phosphatidyl insoitol 4-kinase, forming PI4P, an important membrane lipid for Golgi function 14. Arf1 also binds to PI4P-specific pleckstrin homology domains contained in a family of oxysterol-binding proteins, believed to function in lipid homeostasis at the Golgi 14.
The five Arf proteins in humans, Arf1, 3, 4, 5, and 6, are ubiquitously expressed. Studies to date have focused mainly on Arf1 at the Golgi and Arf6 at the PM, but Arfs 3, 4 and 5 are also present on Golgi membranes (Figure 2A). Surprisingly, depletion experiments using RNA interference show that no single Arf, including Arf1, is required for Golgi function; instead, Arfs function in pairs at particular steps in Golgi transport 15. For example, Arf1 and Arf4 act redundantly during transport in the early secretory pathway 15. Consistent with this observation, Arf4 localizes to the ERGIC and cis Golgi 8, and together with Arf1 at the cis Golgi, it organizes trafficking between these compartments 16.
Arf1 and Arf3 are identical except for 7 amino-acid differences in their amino and carboxy regions and previously they were thought to function and localize identically. However, a Golgi-targeting sequence contained within the α-3 helix of Arf1 and Arf3 targets a chimera between Arf6 and Arf1 to the early Golgi 17. Furthermore, Arf3 localizes specifically to the TGN (Figure 2A) and this localization depends on 4 Arf3-specific amino acids contained in the N-terminal amphipathic helix, which are conserved among Arf3 homologues 18. Arf3, but not Arf1, becomes cytosolic at 20°C, the temperature at which exit from the TGN is blocked18. Thus, Arf3 might have an additional critical role during exit from the Golgi.
Several important functions for Class II Arfs at the TGN have now been defined (Figure 2A). In an elegant series of studies, Arf4 was found to specifically recognize the VxPx cytosolic targeting motif in retinal rhodopsin to facilitate its transport into the rod outer segment, a specialized cilium (Figure 2B)19. This ciliary targeting complex includes, in addition to Arf4, Rab11, FIP3 (a shared Arf and Rab11 effector) and ASAP1, an Arf GAP 20. Exactly how this complex facilitates the packaging of rhodopsin into post-Golgi carriers has yet to be determined but, interestingly, rhodopsin itself initiates the complex formation by recruiting Arf4. The rhodopsin-binding site on Arf4 is in the α-3 helix 19, the same region that in Arf1 binds the SNARE protein membrin to mediate targeting to the early Golgi17; thus, this region might generally allow Arf protein binding to membrane receptors. Arf4 and Arf5 can also directly bind to the calcium-dependent activator protein for secretion (CAPS), which regulates exocytosis of dense core vesicles from nerve terminals 21. It is the GDP-bound form of the Arf that binds to the PH domains of CAPS proteins, and knockdown of CAPS, Arf4 or Arf5 causes retention of chromagrannin, a marker for dense core vesicles, in the Golgi, suggesting that Arf4 and 5 together with CAPS regulates the release of dense core vesicles from the Golgi (Figure 2A). How these roles of Arf4 and 5 at the TGN in specialized cells can be reconciled with findings of Arf4 localization to, and Arf4 and 5 functioning at, the early Golgi in other cells 8, 15, 16 is not known.
Recent discoveries show that Arf1 regulates lipid transfer proteins within the Golgi and promotes the formation of lipid droplets, neutral lipid storage organelles enclosed by a phospholipid monolayer, at the ERGIC (Figure 2A). At the Golgi, Arf1 recruits the lipid transfer proteins CERT and FAPP2 14 through interaction with their PH domains, which can also bind PI4P. CERT mediates the non-vesicular transport of ceramide from the ER to the Golgi and FAPP2 mediates the transfer of glucosylceramide from the cytosolic side of the early Golgi to the trans-Golgi 22. Exactly how the directionality of this transfer occurs and the role that Arf1 has is not yet clear. The finding that Arf1 associates with GBF1 and COPI during lipid droplet formation was unexpected. These proteins were identified in an RNAi screen of lipid droplet formation in Drosophila 23 and also appeared in proteomic analyses of lipid droplets along with other trafficking proteins, which led to the idea that lipid droplets interface with multiple membrane trafficking pathways 24. In particular, the delivery of two proteins, adipose triglyceride lipase (ATGL) and adipophilin (ADRP), to the surface of lipid droplets requires Arf1, GBF1 and COPI and possibly the COPII machinery in mammalian cells 25; similar results were obtained in Drosophila S2 cells 26. Another Arf family member, Arfrp1, is highly expressed in adipocytes, and mice that lack Arfrp1 in adipose tissue show severe defects in lipid storage and enhanced lipolysis 27. Finally, in some cell types Arf1 at the plasma membrane affects endocytosis of proteins anchored to the membrane by a glycosylphosphatidyl inositol (GPI) linkage28. This may also require the Arf GEF GBF1 29 and could be related to the other lipid-regulating functions of Arf1.
A great deal of work on Arf6 function has been summarized in a previous review 1, so here we focus on more recent advances. In mammals, Arf6 is not required for early embryonic development, but the knock-out mice die at mid-gestation or shortly after birth with impaired liver development 30. This phenotype suggests that the critical physiological roles of Arf6 take place after birth and is consistent with reported effects of Arf6 on cell adhesion, cell migration, wound healing and metastasis.
Arf6 is present at the plasma membrane and influences both the cortical actin cytoskeleton and endosomal membrane recycling 1 (Figure 3). At the plasma membrane, Arf6 changes the membrane lipid composition through activation of PI4P 5-kinase (PIP5K) and PLD, resulting in the generation of PI4,5P2 and PA. These lipids are important for sorting proteins within the membrane, for the formation of clathrin-coated pits during endocytosis, and for the recruitment and activation of Rho family G proteins such as Rac to alter actin polymerization. There is some evidence that Arf6 can interact with adaptor protein 2 (AP2) 31 and with AP2 and clathrin during G-protein coupled receptor cell signalling 32. A recent study has found that Arf6 enters cells in clathrin-coated vesicles to facilitate the rapid recycling of transferrin receptor back to the plasma membrane through interaction with the microtubule motor adaptor protein JIP4 after clathrin uncoating 33. In some cells, Arf6 associates with endosomal membranes derived from clathrin-independent forms of endocytosis and mediates recycling of this membrane back to the plasma membrane34. Recycling endosomes return membrane proteins that are important for cell adhesion and migration back to the plasma membrane34, 35. Arf6 regulation of such endosomal membrane trafficking is required for the polarized delivery of Cdc42, Rac and the Par6 complex to the leading edge of migrating cells 36, which can alter both adhesion to the extracellular matrix through focal adhesions and actin-based protrusions. Hence, regardless of the mode of endocytosis, Arf6 is important for recycling from multiple endocytic pathways.
The crucial functions of Arf6 in membrane lipid modification, establishment of cell polarity and promotion of endocytic recycling are conserved in yeast and Drosophila 1. Arf3p, the yeast Arf6 homologue, contributes to PI4,5P2 levels at the plasma membrane 37 and also affects polarization events such as bud site selection 38 in S. cerevisiae and new end take off growth in S. pombe 39. ArfB, the Arf6 homologue in the filamentous fungi Aspergillus nidulans, localizes to both the plasma membrane and endomembranes, and regulates endocytosis and polarity establishment during hyphal growth 40. In Drosophila, deletion of the Arf6 homologue blocks the rapid endocytic recycling required for cytokinesis in spermatocytes, resulting in male sterililty, but no other phenotypes were reported41 Interestingly, Arf6 interacts with JPI4 to control a motor switch mechanism regulating endosomal trafficking in cytokinesis 42. The crystal structure of Arf6 in complex with JIP4 shows that residues adjacent to the switch regions are structural determinants for the specific binding of JIP4 to Arf6 43.
Arf6 has been implicated in both the assembly and disassembly of adherens junctions in polarized epithelial cells (Figure 3)1. During adherens junction formation, Par3 recruits a scaffolding protein FRMD4A that binds to Cytohesin GEFs, which leads to activation of Arf6 44. Treatment of fully polarized epithelial cells with hepatocyte growth factor leads to activation of Arf6, most likely through the Arf GEF GEP100/BRAG2 45, and activation of Rac, which causes disassembly of adherens junctions by stimulating endocytosis of E-cadherin1. Hence, depending on the signalling complex assembled, either formation or disassembly of adherens junctions, can be achieved through activation of Arf6. There is also some evidence that the Arf6 GEF EFA6 affects tight junction assembly46. Arf6 activation has also been reported at the onset of tubulogenesis, a developmental progression from polarized epithelia to tubular structures, and perturbation of the Arf6 GTP/GDP cycle inhibits tubule formation 47.
Arf proteins carry out their action through a regulated cycle of GTP binding and hydrolysis. This allows Arfs to engage and disengage with its effectors with spatial and temporal specificity, and in some cases may allow Arf-GDP to bind other classes of effector. Arf6-GDP binds several TBC (Tre-2/Bub/Cdc16) domain-containing proteins, which often have Rab GAP activity 48. Arf6-GDP binds both TBC1D24, a protein mutated in familial infantile myoclonic epilepsy 49, and the TRE17 oncogene 50. TRE17 binding to Arf6 increases its activation50; although TRE17 does not itself have GEF activity towards Arf6, it may facilitate interaction of Arf6 with another GEF. Arf6-GDP also binds to the Kalirin family of Rho GEFs through their spectrin-like repeat domain51, and recruits Kalirin to the membrane where it subsequently activates Rac and RhoG to regulate actin dynamics (Figure 3)51. Hence, Arf6-GDP and Arf6-GTP both interact with regulatory proteins of other small G proteins, allowing alternative signalling pathways to be activated depending on the nucleotide bound (see Figure 1C). This raises the intriguing possibility that other GDP-bound Arf or Arl proteins might also bind unique effector proteins.
Turning off Arf6 is important for its biological function. In some cells, expression of the constitutively active mutant of Arf6, Q67L, leads to the accumulation of early endosomes containing plasma membrane proteins that enter cells independently of clathrin; failure to inactivate Arf6 blocks further trafficking of this membrane to recycling or to other destinations52. Immediately upon platelet activation, Arf6-GTP levels fall and this inactivation precedes and is required for the subsequent activation of Rac 53. Arf6 is important for the disassembly of adherens junctions 1 and, more recently, active Arf6 was shown to disrupt the formation of epithelial cysts54. The Slit-Robo4 signalling pathway is important for maintaining barrier function in the vascular network and Robo4 interacts with paxillin to recruit Arf GAP proteins such as Git1 to inactivate Arf6 55; this Arf6 inactivation suppresses protrusive activity of the endothelial cells and neovascularization. Git2 and Arf6 inactivation are also important for maintaining the podosome, an actin-rich sealing zone in osteoclasts 56. Finally, an unusual way to turn off Arf6 appears to be through non-canonical ubiquitylation of Arf6, catalysed by Fbx8, an Fbox and Sec7 domain-containing protein 57. Fbx8 is diminished or lacking in several cancer cell lines, consistent with roles for Arf6 in cancer cell metastasis 58.
Similarly to Arf1, Arl1 and Arl2 arose early in evolution and share common effectors in plants, yeast and mammals. Arl1 recruits GRIP-domain golgins to the TGN 2. It also mediates TGN localization of arfaptins, which contain BAR domains that induce the formation of tubules and vesicles at the TGN 59. Whereas Arl1 functions in vesicle trafficking similarly to Arfs, Arl2 has a highly conserved function in regulating microtubule-based processes 2. Arl3 is closely related to Arl2, but is found only in cells with cilia, where it regulates microtubule-based processes at the cilial basal body 2, 60 (Figure 2B). ELMOD2 has been reported as a GAP for Arl2, but also has activity against Arf1 and Arf6, which is surprising given that it has no homology to Arf GAPs 61; the physiological relevance of this activity remains to be determined. RP2 acts as a GAP for Arl3 during intraflagellar transport and ciliogenesis.
Arl3, Arl6 and Arl13 affect intraflagellar transport and ciliogenesis (Figure 2 B and C). Cilia are vital for cell signalling and differentiation, and their impaired formation is responsible for many genetic disorders 62. Bardet-Biedl Syndrome is a complex genetic disease that can be caused by any one of 14 genes associated with ciliogenesis. Transport of membrane proteins into the cilium is driven by a complex of proteins, called the BBSome. BBSome subunits have ‘coat-like’ attributes and similar structural folds to those found in COPI and AP complexes, suggesting that the BBSome can sort specific cargo for transport (Figure 2C). Arl6 is a BBS subunit (BBS3) and is required in its GTP-bound form to recruit the BBSome onto the plasma membrane to drive cargo sorting into cilia 63. Structural and biochemical analysis has shown that one of the mutations in Arl6 (BBS3) that causes Bardet-Biedel Syndrome, T31R, leads to a nonfunctional Arl6 that cannot bind GTP 64. This supports the idea that Arl6 recruits the BBSome complex to membranes for formation of BBSome-coated vesicles. Arl13 is mutated in patients with Joubert Syndrome, a rare, complex cerebral disorder characterized by developmental delays and mental retardation. It is also involved in intraflagellar transport (Figure 3B) and, in C. elegans, Arl13 associates with the doublet segment of the cilium and its loss results in shortened cilia65, 66.
Retinitis pigmentosa is a retinal degeneration disease, and mutations in the retinitis pigmentosa 2 gene (RP2) are responsible for a large fraction of the most severe X-linked form. RP2 was identified as a GAP for Arl3, and mutations associated with retinitis pigmentosa compromise Arl3 GAP activity 67. Arl3 localizes to the photoreceptor segment connecting to the cilium (Figure 2B), and Arl3 (-/-) mice have abnormal kidney and photoreceptor development, indicating its importance in primary cilia 68. RP2 localizes to the basal body and centriole at the base of the photoreceptor cilium, but also to the adjacent Golgi and apical plasma membrane 69.
Furthermore, RP2 promotes vesicle trafficking from the Golgi to the base of the cilium in mammalian cells69, presumably acting together with Arf4, ASAP1 and FIP3. Intriguingly, Drosophila Arl3 (also called dead end) regulates actin polymerization and vesicular trafficking to the plasma membrane, which are important for tracheal morphogenesis 70. Hence Arl3 appears to link microtubule-based processes and vesicular trafficking during development.
Arl8 might also coordinate microtubule and vesicular trafficking. Arl8 localizes to late endosomes and lysosomes (Figure 3) in both humans and worms, and mediates transport of endocytic proteins between these two compartments 71. Arl8 also facilitates the axonal transport of presynaptic cargo proteins in vesicles, preventing their premature aggregation 9. Exactly how these two functions of Arl8 are related is not clear but they might both involve transport along microtubules 2.
A great deal of progress has been made in identifying Arf GEFs, and an unexpectedly broad range of roles have been revealed for these regulators, including both the coordination of membrane trafficking with lipid homeostasis and signalling at the plasma membrane (Table 2). Because GEFs ensure the precise temporal and spatial activation of Arfs, their own localization mechanisms are crucial for understanding their cellular roles. These mechanisms are turning out to be quite complex, even for the simplest of the Arf GEFs, members of the Cytohesin/Arno family. Membrane trafficking is crucial to numerous developmental and physiological processes, and the specific functions of different Arf GEFs in these pathways and their links to disease are now being revealed.
There is particular interest in understanding how Arf GEFs are recruited to membranes to regulate Arf activation. BIG1 and BIG2 localize to the TGN and endosomes, where they have both distinct and overlapping functions 72, 73. In contrast, GBF1 localizes predominantly to the cis-Golgi (Figure 2A)74, where it controls transport of membrane proteins through the secretory pathway 75. The activity of phosphodiesterase 3A is important for recruitment of BIG1 and BIG2 to the trans Golgi 76. By contrast, Rab1 77 and PI4P generated by PI4 kinase IIIα 78 are involved in recruitment of GBF1 to membranes. Other close connections between Golgi Arfs and PI4P have emerged recently. In yeast there is an interesting synergy observed between the Arf1 GEF Gea2p and PI4P produced by Pik1p (the yeast homologue of PI4 kinase IIIβ). Both are simultaneously required to activate the aminophospholipid translocase (flippase) Drs2p at the TGN during formation of AP1/clathrin vesicles 79.
PI 4 kinases are essential for viral replication, and notably produce the PI4P-enriched membrane environment that recruits the enteroviral RNA polymerases 80. GBF1 is required for the replication of numerous viruses, including enteroviruses, hepatitis C virus and coronaviruses 81-84. In enteroviral sytems, both GBF1 and PI4 kinase IIIβ are recruited coordinately to membranes by the viral 3A protein to promote formation of functional viral replication complexes 80 near ER exit sites (Figure 2A).
Yel1p is an EFA6-like GEF for the Arf6 orthologue Arf3 in yeast, and localizes to the plasma membrane of the emerging bud 85. Similarly to its mammalian orthologues, the PH domain of Yel1p is required for membrane targeting but, interestingly, multiple regions of the protein are important for precise spatial localization of this GEF 85.
GEP100/BRAG2, an Arf6 GEF, also has a PH domain critical for membrane targeting and in breast cancer cells is specifically recruited to the EGF receptor upon EGF stimulation, through direct interaction of its PH domain with the EGF receptor 86. This interaction requires phosphorylation on specific tyrosine residues and thus the recruitment of BRAG2 couples receptor activation to Arf6 activation 86. BRAG2 is overexpressed in many breast cancer cell lines and depletion of BRAG2 by siRNA blocks cell invasion in in vitro and animal tumor models 86. These observations add to others that have implicated Arf6 and its activation in a number of models of cancer cell invasion and metastasis 1, 58.
At the cell periphery, the Cytohesin/Arno GEFs function in plasma membrane-endosomal membrane trafficking pathways, and in signal transduction pathways important for cell proliferation, immune response and growth control 87, 88. Members of this GEF family can catalyse exchange on both Arf1 and Arf6 in vitro and in cells, although in vitro they are more efficient GEFs for Arf1 87. Recent insights have been gained into how cytohesin activation is spatially regulated, and how its autoinhibition is relieved (Figure 4). In addition to phosphoinositide binding at the membrane, the PH domains of Cytohesin family members interact with the GTP-bound forms of Arf6 89 and Arl4 90, 91, leading to cytohesin recruitment and further activation of Arf6 or Arf1 at the membrane. A crystal structure of the Sec7 domain in tandem with the PH domain of Cytohesin-3/Grp1 revealed that it adopts an autoinhibited conformation. The C-terminal helix that follows the PH domain and the linker between the Sec7 and PH domains block the catalytic site 92. Interaction of the PH domain with Arf6-GTP and phosphoinonsitides (either PIP2 or PIP3), as well as the interaction of the polybasic C-terminus of Cytohesin/Arno with acidic phospholipids, all contribute to relieving this autoinhibition (Figure 4)92. Reconstitution of the Cytohesin/Arno exchange assay on liposomes, in the presence of both activating Arf6-GTP and substrate Arf1, revealed that mutations in the PH domain of Cytohesin/Arno that abolished interaction with Arf6-GTP were completely inactive 93. Together these studies demonstrate how precise spatial regulation of Cytohesin/Arno activation is achieved. A specific phosphoinositide (PIP2 and/or PIP3), additional acidic phospholipids and an active Arf localized in the plasma membrane must all coincide to relieve autoinhibition, thus restricting the membrane domain at which these GEFs can become active.
We do not know whether Arf6, Arf1 or both are the primary substrates for the cytohesins. However, Arf6-GTP is more efficient in relieving autoinhibition of cytohesins than Arf1-GTP, both in vitro and in cells 89, 92. The activation of cytohesins by a GTP-bound Arf family member raises the question of whether they can engage in a positive feedback loop whereby the substrate itself can stimulate further exchange. Indeed, such a loop has been demonstrated in vitro for Arf193.
There is also evidence that cytohesins might mediate a cascade of activation from Arf6 to Arf1. Cells expressing constitutively active Arf6Q67L have increased levels of Arf1-GTP89. Arf1 affects several processes at the plasma membrane, including recruitment of proteins to focal adhesions and during phagocytosis. In the forming phagocytic cup, Arf6-GTP is recruited earlier than Arf1-GTP, at a stage that requires rapid insertion of new membrane 94. Hence the Arf6-cytohesin-Arf1 cascade might ensure a high level of activated Arf protein here. Arf6 is less abundant than Arf1 in cells, and as both Arf1 and Arf6 can recruit effectors such as PI4P 5 kinase and PLD, processes requiring an acute activation of such effectors may rely on the more abundant Arf1 to provide an adequate supply. In support of this idea, both Arf1 and Arf6, through cytohesins, contribute to activation of PI4P 5 kinase and PLD in the insulin signalling pathway 95. In addition to Arf6-Cytohesin-Arf1 or possible Arl4-Cytohesin-Arf6 cascades, there is a conserved Arl cascade in which yeast Arl3-GTP (and its mammalian orthologue ARFRP1) recruits Arl1 to TGN membranes 2. In this case, it is not known whether an Arl GEF is involved. Hence Arf family cascades could be common, and could explain the Golgi Arfs that act in pairs.
Use of the specific Cytohesin inhibitor SecinH3 has revealed roles for this family of GEFs in the insulin and ErbB receptor tyrosine kinase signalling pathways 96-98. Cytohesins are positive activators of insulin signalling both in Drosophila and mammalian cells, and are important for cell growth and for insulin sensitivity in human liver cells 97, 98. They regulate insulin signalling by binding CNK1, a scaffolding molecule important for Ras, PI3kinase and Akt signalling 95. CNK1 recruits cytohesins in an insulin-dependent manner to the plasma membrane, where they generate a PIP2-enriched microdomain that is essential for PI3K/AKT activation. Other scaffolding proteins interact with the coiled-coil domain of Cytohesin/Arno, including GRASP and IPCEF, which mediate Dock180 interaction with Cytohesin/Arno 99. Interestingly, assembly of this scaffolding complex promotes Rac activation and cell migration, indicating that these scaffolds assemble a signalling complex that determines a specific downstream output upon Arf activation 99. Cytohesins also affect integrin signalling in the immune system, and Cytohesin-1 can activate β2integrins in dendritic cells 100; this raises the possibility that this may occur through a scaffolding role of cytohesins.
Levels of Arf6 and both the EFA6 and Cytohesin family GEFs markedly increase in the mammalian brain after birth, suggesting important roles in post-natal nervous system development 101. Experiments in isolated hippocampal neurons indicate that Arf6, EFA6 and the cytohesins might affect neurite and dendritic spine development 102, 103.
In humans, mutations in the Arf1 GEF BIG2 are linked to autosomal recessive periventricular heterotopia (ARPH), a disease in which the cerebral cortex is severely underdeveloped due to failure of neurons in the lateral ventricular proliferative zone to migrate to the cortex 104. This impaired migration arises from a defect in vesicular trafficking that alters the adhesive properties of these neurons 105. Disease alleles include an early frameshift mutation that deletes most of the BIG2 protein 104.
Members of the BRAG/IQSEC family of Arf GEFs are extremely abundant in neuronal post-synaptic densities, and can serve as GEFs for Arf6 87. BRAG1 and BRAG2 are vital for neuronal development. BRAG1/IQSEC2 is mutated in X-linked nonsyndromic intellectual disability, also referred to as mental retardation. Three point mutations isolated from patients map to the Sec7 domain and result in proteins that cannot activate Arf6 normally 106. BRAG2 has been linked to alterations in synaptic content during long term depression (LTD). Signalling through AMPA-type glutamate receptors facilitate LTD, and donwregulation of activated AMPA receptors is normally regulated by AMPA receptor-mediated recruitment of BRAG2, which in turn activates Arf6 and endocytosis. 107. Thus, BRAG GEFs and Arf6 are vital for neuronal development and learning.
Cytohesin/Arno GEFs may affect signalling through epidermal growth factor (EGF)/ErbB receptor tyrosine kinase receptors independently of their GEF activity. EGFRs undergo ligand-induced dimerization and subsequent trans-phosphorylation, mediated by conformational changes in their cytoplasmic portion. Cytohesins bind directly to these cytoplasmic domains and promote conformational changes that increase phosphorylation96. Furthermore, treatment of an EGF receptor-dependent lung cancer cell line with the Cytohesin/Arno inhibitor SecinH3 reduced proliferation96. Surprisingly, this function of the Cytohesins does not require their GEF activity. Similarly, in C. elegans, the GEF EFA-6 regulates microtubule dynamics at the cell cortex independently of its substrate Arf6 108. Furthermore, essential functions of GBF1 in poliovirus replication are independent of Arf1 activation 109. The extent to which other Arf GEFs may have broader roles beyond Arf activation warrants further investigation. There are also suggestions that some multi-domain Arf GAP proteins have functions that are independent of their GAP activity.
All Arf GAPs contain the conserved Zinc finger Arf GAP catalytic domain in addition to other domains responsible for membrane recruitment, regulation of GAP activity and other scaffolding functions (Table 2). ArfGAP1, the first Arf GAP cloned 110, is Golgi-localized, and together with ArfGAP2 and 3, these GAPs mediate most Arf-bound GTP hydrolysis at the Golgi. The complex, multi-domain structure of the other Arf GAP families has stimulated much research. Here we highlight a few examples of how these multi-domain Arf GAPs, by recognizing the GTP-bound form of their substrate Arf, act as downstream effectors in addition to signal terminators. Information about other Arf GAPs can be found in an excellent review article 111.
The ASAP proteins are the prototypical multi-domain GAPs that interact with many signalling molecules including Src and focal adhesion kinase (Table 2) 111. ASAP1 resides in focal adhesions but, in response to Src activation, it facilitates formation of podosomes 112, discrete actin-based structures formed at the cell substratum to degrade matrix. The crystal structure of Arf6 in complex with the catalytic domain of ASAP3 revealed that a catalytic arginine finger of ASAP3 is responsible for GTP hydrolysis 113, consistent with an earlier structure of the GAP domain of ASAP2 114 and similarly to many other GAPs. There is also some evidence that calcium might bind to the complex and regulate GAP activity 113, although this needs to be confirmed with full-length ASAP3 and Arf6 and in cells. The ASAPs all have amino terminal BAR domains that can induce membrane curvature and tubule formation in transfected cells and in cell-free systems. The BAR domain in ASAP1 negatively regulates its GAP activity towards Arf1 115, and binding of the Rab11 effector FIP3 to the BAR domain of ASAP1 stimulates its GAP activity 116. As mentioned earlier, ASAP1 also promotes ciliary targeting together with Arf4 and FIP3 20 (Fig. 2b). ASAP1 (also called AMAP1) is up-regulated in breast, pancreatic and colorectal cancer 58. CIN85, a Cbl-interacting protein, binds to ASAP1, recruiting the E3 ubiquitin ligase Cbl, to trigger the monoubiquitylation of ASAP1; this modification is important for invasion of breast cancer cells 117 but the role for ubiquitinylation of ASAP in cell invasion is not known. One caveat to observations made when ASAP is expressed in cells is that a study designed to systematically look at Arf GAP function and Arf specificity failed to detect an effect of ASAP1 expression on either Arf1- or Arf6-GTP levels in cells 118. This raises the possibility that the GAP activity of ASAP1 might not always be critical for some of ASAP1's specific functions.
The Arf GAP Git1, originally identified as a G protein-coupled receptor (GPCR) kinase interacting protein, can coordinate signalling by acting as a scaffold. Git1 and its substrate Arf6 affect ligand-stimulated endocytosis of several GPCRs through either clathrin-dependent or clathrin-independent endocytic pathways 119. Among the proteins interacting with Git1 are the Cdc42 and Rac GEF Pix, focal adhesion kinase and also paxillin. Git1, similarly to ASAP1, is sometimes observed in focal adhesions and its influence on activation of Cdc42 and Rac suggests that Arf inactivation and Rac activation are coordinated (Fig. 3). Drosophila Git is required for muscle morphogenesis 120 and the Git1 knockout mouse is defective in fear learning 121 and dendritic spine formation 122. Rac3 interacts with Git1, disrupting Git1 binding to paxillin; this in turn stimulates Git1 GAP activity, presumably towards Arf6 123, and inhibits cell spreading and neuritogenesis. In endothelial cells, Robo4 interacts with paxillin, which recruits Git1 to inactivate Arf6 and this leads to vascular stability, blocking cellular protrusions and neovascular leak 55. Thus, these examples provide insight into how modular Arf GAPs promote spatially and temporally restricted assembly of signalling complexes, and allow a precise physiological output in response to a signal.
Intracellular pathogens can use a fascinating GAP-dependent mechanism to rewire the host cell's signalling network for their own purposes. Enterohaemorrhagic E. coli produce the EspG protein, which binds to GTP-bound Arf1 and Arf6, blocking their GAP activity, and disrupting the function of both early Golgi and recycling endosomes124. Moreover, EspG simultaneously binds to p21-activated kinase (PAK), an effector of a distinct G protein family member, Cdc42, and promotes PAK localization at Golgi membranes rather than at the plasma membrane . This raises the possibility that EspG assembles its own signalling complex on intracellular membranes to subvert membrane trafficking and polarity processes in host cells.
Arf activity is regulated in a spatiotemporal manner by the GEFs and GAPs, underlining the importance of precise localization of these regulators. In the case of cytohesins, such specificity can be achieved through a coincidence detection mechanism, requiring both an activating Arf or Arl protein and a specific lipid composition. This example also reveals the existence of Arf family activation cascades and how relief of autoinhibition can be coupled to precise spatial cues. It will be interesting to see how widespread these mechanisms are among Arf family members. Arf cascades, similarly to those demonstrated for Rab G proteins, could transform one membrane domain into another during highly dynamic membrane trafficking. These transformations involve coordinate changes in both the lipid and protein composition of each membrane domain, a specialty of many Arf family members, which recruit both lipid-modifying enzymes and protein effectors such as coats and tethers. The signature feature of Arf family proteins, their N-terminal membrane-binding amphipathic helix, ensures that they are closely associated with the lipid bilayer in their GTP-bound form. Future studies on how Arf family proteins function will therefore require in vitro reconstitution on model membranes. There appears to be a particularly important link between Arf1 function and PI4P, a lipid that has a central role in the function of the Golgi, which parallels the coordination of membrane trafficking and PI(4,5)P2 signaling by Arf6 at the plasma membrane.
The GAPs and GEFs for the Arf family proteins are multidomain proteins that can assemble signalling complexes and so place the Arfs and Arls into larger networks. These networks include cytoskeleton regulators, and it appears that some Arl proteins (Arl2, for example) have evolved exclusively to regulate the cytoskeleton. The role of Arf6 in networks linking membrane trafficking to the actin cytoskeleton also involves interaction of Arf6 with GEFs and GAPs of the Rac and Rho small G proteins, actin cytoskeleton regulators. Another emerging concept is that some Arf family members remain membrane-bound in their GDP-bound form, where they can interact with signalling complexes and promote alternative signalling pathways. Ultimately, these Arf family signalling networks will need to be studied through systems level analysis.
So far, no GEFs and only two GAPs specific for an Arl have been identified. Several Arl proteins affect ciliogenesis and, in some cases, ciliopathies; other Arls function in neurons and have been associated with neurodegenerative disorders. Hence, increased understanding of Arls and their regulators should inform both fundamental questions in cell biology and disease mechanisms.
Finally, the use of model organisms to complement studies in mammalian cells has already provided valuable insights into the physiological roles of Arf family proteins. This approach holds great promise for uncovering the unknown functions of most Arls, as well as defining the full spectrum of activities of all Arf and Arl proteins.
Plants have numerous Arfs that are homologous to human Arf1 125 and were originally thought to lack Class III Arf6-like proteins. However, in Arabidopsis thaliana, ARFB (also called ARFB1a) localizes to the plasma membrane and lacks the Golgi targeting motif (MxxE) found in other ARF1 homologues in plants 126 and in mammals 17. Nevertheless, there appear to be only GBF and BIG subfamilies of Arf GEFs in plants, which function in both endocytic and Golgi trafficking pathways 127. Arabidopsis GNOM is a homologue of mammalian GBF1 but acts at endosomes and the plasma membrane during the polar transport of the plant hormone auxin during development127, 128. Another GBF-like protein in Arabidopsis, GNL1, functions at the Golgi similarly to mammalian GBF1, but is also involved in endosomal trafficking129. BIG5/BEN1/AtMIN7 was identified in a screen for Arabidopsis mutants defective for internalization of the PIN auxin transporter from the plasma membrane. This Arf GEF is most closely related to BIG1 and BIG2 in mammalian cells, localizes predominantly to the TGN and early endosomes, and is involved in early endosomal trafficking 130. Interestingly, BIG5 is targeted for degradation by a plant bacterial pathogen, Pseudomonas syringae, to protect the latter from host defense systems at the cell wall131.
Arabidopsis Arf GAPs include four members of a family of mammalian ACAP homologues, known as Van3-like for the first member characterized 125. VAN3, also known as Scarface, regulates formation of plant vascular networks 132, 133. In addition to their roles on endosomes, VAN3 cooperates with GNOM during clathrin-mediated endocytosis of the PIN auxin transporter128. Another Arf GAP in Arabidopsis, AGD5, is a homologue of yeast Age2p, and localizes to the TGN134. The NEVERSHED Arf GAP in Arabidopsis (an orthologue of yeast Age1, with a paralogue in Arabidopsis (AGD15), and homologues in C. elegans and mammals (SMAP family GAPs) is required for floral organ cell separation125 and regulates membrane trafficking though TGN-early endosomal compartments to trigger organ abscission 135.
Interestingly, the protozoan parasite Trypanosoma bruccei expresses a single Arf protein that has characteristics of both Arf1 and Arf6. TbArf1 is a basic protein with a calculated pI value of 9.1 similar to that of human Arf6, but TbArf1 contains the Golgi-targeting motif MxxE136 found in human Arf1 and 3 17. Depletion of TbArf1 by small-interferring RNA causes a major decrease in endocytosis and the formation of intracellular flagella, but the Golgi remains intact 136. Trypanosomes also express an Arl2 homologue, involved in microtubule biogenesis and cytokinesis 137, and an Arl1 homologue, which is important for Golgi structure and exocytosis of GPI-anchored proteins 138. Arf and Arl proteins in trypanosomes are myristoylated, a modification required for their activity. Trypanosomes cause African Sleeping Sickness, a disease with no successful therapy. A selective inhibitor of trypanosomal N-myristoyl transferase has been shown to be effective in blocking trypanosome viability in a mouse model of this disease 139.
We apologize to authors whose work we could not cite due to space limitations. We thank Craig Eyster, Lymarie Maldonado-Baez, Julie Ménétrey and Christophe Le Clainche for critical reading of the manuscript. Work in our laboratories is supported by the Division of Intramural Research in the National Heart, Lung, and Blood Institute, NIH (JGD) and grants from the Agence Nationale de la Recherche and the CNRS, France (CLJ).