Multiple Roles for Arf6: Sorting, Structuring, and Signaling at the Plasma Membrane

The ADP-ribosylation factors (Arfs) 1The abbreviations used are: Arf, ADP-ribosylation factor; PM, plasma membrane; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; PIP5-kinase, phosphatidylinositol 4-phosphate 5-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; CHO, Chinese hamster ovary; MDCK, Madin-Darby canine kidney; PLD, phospholipase D; HIV, human immunodeficiency virus; PA, phosphatidic acid; HGF, hepatocyte growth factor; CPE, carboxypeptidase E; MHCI, major histocompatibility complex class I. are a family of Ras-related, low molecular mass (∼20 kDa), GTP-binding proteins that are expressed in all eukaryotes. There are six mammalian Arfs and many more Arf-like proteins. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound, active states. In the active state, Arfs interact with proteins and other effector molecules to carry out their functions. Although Arf1 and its activities at the Golgi complex have been extensively studied, Arf6 has been the subject of increased attention over the past 5 years. Arf6 influences membrane trafficking and the actin cytoskeleton at the plasma membrane (PM). The goal of this review is to summarize these recent findings and provide a cellular context for understanding Arf6 function. There are homologues of mammalian Arf6 in almost all eukaryotes including Xenopus laevis (97% amino acid sequence identity), Drosophila melanogaster (97%), Caenorhabditis elegans (88%), Schizosaccharomyces pombe (75%), and Saccharomyces cerevisiae (60%). All Arfs are N-terminally myristoylated, and all Arf6 homologues are basic proteins with predicted pIs in the range of 8.5–9.5. It is this characteristic and a signature dipeptide sequence (Gln-Ser) (1Al-Awar O. Radhakrishna H. Powell N.N. Donaldson J.G. Mol. Cell. Biol. 2000; 20: 5998-6007Crossref PubMed Scopus (70) Google Scholar) adjacent to the effector domain interaction site, Switch I, that allow homologues of Arf6 to be identified. By contrast, other Arf isoforms have predicted pIs in the range of 6.0–7.0. It is likely that its positive surface charge and N-terminal myristoylation target Arf6 to the plasma membrane. This may explain why, during the GTPase cycle, Arf6-GDP, unlike Arf1-GDP, is retained on membranes to a large extent (2Cavenagh M.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 3Song J. Khachikian Z. Radhakrishna H. Donaldson J.G. J. Cell Sci. 1998; 111: 2257-2267Crossref PubMed Google Scholar) although release of Arf6-GDP to the cytosol cannot be ruled out (4Gaschet J. Hsu V.W. J. Biol. Chem. 1999; 274: 20040-20045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The absence of an Arf6 homologue in Arabadopsis or other plant species suggests that Arf6 is not present in plants. Arf6 activation and inactivation are catalyzed by guanine nucleotide exchange factors (GEFs) that facilitate GTP binding and GTPase-activating proteins (GAPs) that catalyze GTP hydrolysis. In general, Arf6 GEFs are not inhibited by the fungal metabolite brefeldin A, in contrast with other Arf GEFs (5Jackson C.L. Casanova J.E. Trends Cell Biol. 2000; 10: 60-67Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). The ARNO/cytohesin and EFA6 families of Arf6 GEFs contain a catalytic Sec7 homology domain and a pleckstrin homology domain thought to be involved in membrane targeting (5Jackson C.L. Casanova J.E. Trends Cell Biol. 2000; 10: 60-67Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Arf-GEP100, another Arf6-specific GEF, also contains an IQ motif and localizes to endosomal membranes (6Someya A. Sata M. Takeda K. Pacheco-Rodriguez G. Ferrans V.J. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2413-2418Crossref PubMed Scopus (93) Google Scholar). Candidate Arf6 GAPs are even more plentiful. These multidomain proteins can contain, in addition to the Arf GAP domain, pleckstrin homology, Src homology 2 and 3, and proline-rich domains capable of interacting with a multitude of signaling molecules that impact the actin cytoskeleton. As these regulators will not be further discussed, the reader is referred to two reviews in this area (5Jackson C.L. Casanova J.E. Trends Cell Biol. 2000; 10: 60-67Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 7Jackson T.R. Kearns B.G. Theibert A.B. Trends Biochem. Sci. 2000; 25: 489-495Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The molecular structures of both GDP- and GTP-bound Arf6 have been published, and the differences and similarities with the Arf1 structures provide some insight into mechanisms of activation and interaction with effector proteins (8Pasqualato S. Menetrey J. Franco M. Cherfils J. EMBO Rep. 2001; 2: 234-238Crossref PubMed Scopus (96) Google Scholar). The effector domain regions, Switch I and Switch II, are mostly identical in Arf6 and Arf1 and hence the two Arfs may share many interacting proteins. However, the glutamine and serine residues unique to Arf6 have been shown to confer distinct guanine nucleotide binding properties on Arf6 (9Menetrey J. Macia E. Pasqualato S. Franco M. Cherfils J. Nat. Struct. Biol. 2000; 7: 466-469Crossref PubMed Scopus (73) Google Scholar) and to be required for the actin rearrangement activities of Arf6 observed in cells (1Al-Awar O. Radhakrishna H. Powell N.N. Donaldson J.G. Mol. Cell. Biol. 2000; 20: 5998-6007Crossref PubMed Scopus (70) Google Scholar). Mutations of these two residues and others in the effector domain of Arf6 will be useful for sorting out Arf6-specific functions. For example, expression of Arf6 (T175A), a rapidly cycling Arf6 mutant (based on a similar mutation in Ras), caused increased PM ruffling and cell migration (10Santy L.C. J. Biol. Chem. 2002; 277: 40185-40188Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Additionally, Arf6 mutants defective in GTP binding (T27N) and GTP hydrolysis (Q67L) have been used to identify locations where active Arf6 is needed and to define the consequences of constitutively active Arf6, respectively. However, observations obtained with these inactive and active mutants should be interpreted with caution as Arf6 function normally depends on its GTPase cycle, and expression of any mutant that blocks the cycle may block Arf6 function (see below). By contrast, exogenous expression of wild type Arf6 has no discernible effect on cells in most cases (3Song J. Khachikian Z. Radhakrishna H. Donaldson J.G. J. Cell Sci. 1998; 111: 2257-2267Crossref PubMed Google Scholar). The many cellular functions ascribed to Arf6 indicate that the activities of Arf6 at the PM are complex. It is likely that Arf6 gets activated and inactivated at many locations along the PM where it can influence the sorting of membrane proteins, endocytic pathways, and the structure of the plasma membrane (Fig. 1). This is reminiscent of Arf1 function at the Golgi complex where multiple sites of action of Arf1 influence many membrane trafficking steps into and out of the Golgi and the structure of the Golgi complex. Arfs are thought to act through 1) the recruitment of cytosolic coat proteins onto membranes to facilitate sorting and vesicle formation, 2) the activation of lipid-modifying enzymes, and 3) the modulation of actin structures. The ability of active Arf1 to recruit a variety of cytosolic coat proteins onto Golgi membranes is well documented in vitro and in cells (11Donaldson J.G. Jackson C.L. Curr. Opin. Cell Biol. 2000; 12: 475-482Crossref PubMed Scopus (319) Google Scholar). By contrast, there are as yet no identified coat proteins that are recruited to membranes by active Arf6, although the binding of Arf6-GTP to adaptor protein 1 and other cytosolic coat proteins has been demonstrated in vitro (12Austin C. Boehm M. Tooze S.A. Biochemistry. 2002; 41: 4669-4677Crossref PubMed Scopus (45) Google Scholar, 13Takatsu H. Yoshino K. Toda K. Nakayama K. Biochem. J. 2002; 365: 369-378Crossref PubMed Scopus (95) Google Scholar). Rather, Arf6 is more closely associated with membrane lipid modifications and modulation of the actin cytoskeleton (Fig. 2). Although all Arfs activate phosphatidylinositol 4-phosphate 5-kinase (PIP5-kinase) in vitro, in cells it is Arf6 that localizes with, and activates, PIP5-kinase (14Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). PIP5-kinase is responsible for generating phosphatidylinositol 4,5-bisphosphate (PIP2), a major PM phosphoinositide involved in membrane traffic and actin rearrangements (15Czech M.P. Annu. Rev. Physiol. 2003; 65: 791-815Crossref PubMed Scopus (136) Google Scholar, 16Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2003; 65: 761-789Crossref PubMed Scopus (567) Google Scholar). Therefore, this has provided a key to understanding cellular activities of Arf6. Furthermore, a biophysical study demonstrating that Arf6 binding to PIP2 vesicles alters bilayer structure (17Ge M. Cohen J.S. Brown H.A. Freed J.H. Biophys. J. 2001; 81: 994-1005Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) suggests another way that Arf6 might affect membrane structure. Arfs also activate phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA), and in cells, PLD1 is activated by many agonists. Although the intracellular mediators in the pathway are not clear, accumulating evidence implicates Arf6, as it can directly bind to and activate PLD (18Melendez A.J. Harnett M.M. Allen J.M. Curr. Biol. 2001; 11: 869-874Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19Powner D.J. Hodgkin M.N. Wakelam M.J. Mol. Biol. Cell. 2002; 13: 1252-1262Crossref PubMed Scopus (66) Google Scholar) leading to regulated secretion (20Caumont A.S. Galas M.C. Vitale N. Aunis D. Bader M.F. J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), stimulated membrane ruffling (21O'Luanaigh N. Pardo R. Fensome A. Allen-Baume V. Jones D. Holt M.R. Cockcroft S. Mol. Biol. Cell. 2002; 13: 3730-3746Crossref PubMed Scopus (85) Google Scholar), and other consequences associated with PLD activity (22Dana R.R. Eigsti C. Holmes K.L. Leto T.L. J. Biol. Chem. 2000; 275: 32566-32571Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Because PA can also activate PIP5-kinase, regulation of both PLD and PIP5-kinase by Arf6 can greatly amplify a PIP2-mediated signal (Fig. 2). Thus, changes in membrane lipid composition and structure may mediate Arf6 alterations of the cortical actin cytoskeleton and regulation of membrane traffic and signal transduction. The ability of Arf6 to affect the cortical actin cytoskeleton, cell shape, and cell migration is now well recognized (Fig. 1A). In 1996, however, it was unexpected to find that an Arf protein could, upon activation, generate protrusive structures (23Radhakrishna H. Klausner R.D. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar). These observations were later extended to include a requirement for Arf6 activity for cell spreading (3Song J. Khachikian Z. Radhakrishna H. Donaldson J.G. J. Cell Sci. 1998; 111: 2257-2267Crossref PubMed Google Scholar), Rac-induced ruffling (24Radhakrishna H. Al-Awar O. Khachikian Z. Donaldson J.G. J. Cell Sci. 1999; 112: 855-866Crossref PubMed Google Scholar, 25Boshans R.L. Szanto S. van Aelst L. D'Souza-Schorey C. Mol. Cell. Biol. 2000; 20: 3685-3694Crossref PubMed Scopus (152) Google Scholar), cell migration (26Palacios F. Price L. Schweitzer J. Collard J.G. D'Souza-Schorey C. EMBO J. 2001; 20: 4973-4986Crossref PubMed Scopus (250) Google Scholar, 27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar, 28Weber K.S. Weber C. Ostermann G. Dierks H. Nagel W. Kolanus W. Curr. 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Physiol. 2003; 65: 761-789Crossref PubMed Scopus (567) Google for PIP2, PIP5-kinase, and Arf6 are present on the PM and endosomal of Arf6 activation to of PM by actin (14Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar, A.L. H.L. T. Donaldson J.G. J. Cell Biol. 2001; 154: PubMed Scopus Google Scholar) and stimulated membrane and of the membrane to the PM A.L. H.L. T. Donaldson J.G. J. Cell Biol. 2001; 154: PubMed Scopus Google Scholar) (Fig. low expression PIP5-kinase and Arf6 act to A.L. H.L. T. Donaldson J.G. J. Cell Biol. 2001; 154: PubMed Scopus Google Scholar), and in cells expression of or PIP5-kinase actin endosomal membranes D'Souza-Schorey C. J.A. 2000; PubMed Scopus Google Scholar). have Arf6 in structures in cells and in and in the of Arf6 can in D.J. Casanova J.E. J.M. 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Frohman M.A. Bader M.F. J. Cell Biol. 2002; PubMed Scopus Google Scholar). In another example, of with to release a of expression of blocks this release A. J.M. J.G. A. M.P. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, M.J. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). recent study the of Arf6 in Fc-mediated that vesicles in cells that activation of Arf6 to the of vesicles required to F. E. T. G. J. Cell Biol. 2003; PubMed Scopus Google Scholar). In addition to its on cortical actin and Arf6 regulation of PIP2 can influence other PM activities (Fig. PIP2 is required for the recruitment of onto is rapidly upon vesicle recent study demonstrated that to PIP5-kinase the and to binding to a vesicle membrane M. M. M.R. K. V. J. Cell Biol. 2003; PubMed Scopus Google Scholar). an study an effect of expression of Arf6 mutants on the of at the surface of cells Y. S. L. K. S. F. G. K. J. Cell Biol. 1999; PubMed Scopus Google Scholar). of have for Arf6 in sorting of PM proteins to facilitate their The of and Arf6 In both of the to activation of Arf6 S. Kahn R.A. Bader M.F. H. M.M. Casanova J.E. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. W. Vitale N. Moss J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) the release of to allow S. A. Bader M.F. L. M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (54) Google Scholar). another for Arf6 the of carboxypeptidase a sorting for regulated is to the Golgi complex. This of amino acid residues into the and with Arf6-GTP I. Jackson C.L. Al-Awar O. Donaldson J.G. Mol. Biol. Cell. 2003; Google Scholar). expression of blocks the of the PM to the and mutation of the six residues in for Arf6 binding also I. Jackson C.L. Al-Awar O. Donaldson J.G. Mol. Biol. Cell. 2003; Google Scholar). Although are to the of Arf6 the of more proteins that interact with Arf6 is needed to a molecular for understanding Arf6 function. The multiple sites of action of Arf6 at the PM the of and regulation of Arf6 by to and Although Arf6 may act in a in cells, it is subject to through signal on Arf6 function will on the complex between signal membrane and the cytoskeleton. I and of for on the