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

2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês

10.1074/jbc.r300026200

ISSN

1083-351X

Autores

Julie G. Donaldson,

Tópico(s)

Ion channel regulation and function

Resumo

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. Biol. 2001; 11: 1969-1974Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), wound healing (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar), and Fc-mediated phagocytosis (29Zhang Q. Cox D. Tseng C.C. Donaldson J.G. Greenberg S. J. Biol. Chem. 1998; 273: 19977-19981Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 30Niedergang F. Colucci-Guyon E. Dubois T. Raposo G. Chavrier P. J. Cell Biol. 2003; 161: 1143-1150Crossref PubMed Scopus (150) Google Scholar). The requirement was demonstrated by inhibition of these activities upon expression of the GTP-binding defective, dominant negative mutant of Arf6, T27N, and in some cases by the stimulation of these activities by overexpression of an Arf6 GEF (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar, 31Franco M. Peters P.J. Boretto J. van Donselaar E. Neri A. D'Souza-Schorey C. Chavrier P. EMBO J. 1999; 18: 1480-1491Crossref PubMed Scopus (236) Google Scholar, 32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar). It is important to note, however, that expression of Arf6Q67L, the constitutively active mutant, generally blocks all of the above actin activities (29Zhang Q. Cox D. Tseng C.C. Donaldson J.G. Greenberg S. J. Biol. Chem. 1998; 273: 19977-19981Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar), consistent with the requirement that Arf6 cycles between active and inactive forms to function properly. In many cases it appears that Arf6 changes the actin structure at the PM through activation of Rac, the Rho protein implicated in membrane ruffling. Arf6 is required for and enhances the ability of Rac to form PM ruffles (24Radhakrishna H. Al-Awar O. Khachikian Z. Donaldson J.G. J. Cell Sci. 1999; 112: 855-866Crossref PubMed Google Scholar). Furthermore, expression of an Arf6-specific GEF, EFA6, in cells generates protrusions and ruffles through activation of Arf6 (31Franco M. Peters P.J. Boretto J. van Donselaar E. Neri A. D'Souza-Schorey C. Chavrier P. EMBO J. 1999; 18: 1480-1491Crossref PubMed Scopus (236) Google Scholar, 32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar) and this activity appears to require Rac activation downstream of Arf6 activation (31Franco M. Peters P.J. Boretto J. van Donselaar E. Neri A. D'Souza-Schorey C. Chavrier P. EMBO J. 1999; 18: 1480-1491Crossref PubMed Scopus (236) Google Scholar). Indeed, direct evidence that Arf6-GTP leads to activation of Rac has been obtained (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar), although the mechanism has not been identified. Interestingly, both Arf6 and Rac bind to a common effector protein Arfaptin 2/Partner of Rac (33D'Souza-Schorey C. Boshans R.L. McDonough M. Stahl P.D. Van Aelst L. EMBO J. 1997; 16: 5445-5454Crossref PubMed Scopus (205) Google Scholar). Unfortunately, the function of Arfaptin 2 is not known, and it may show more specificity for Arf1 (34Shin O.H. Couvillon A.D. Exton J.H. Biochemistry. 2001; 40: 10846-10852Crossref PubMed Scopus (48) Google Scholar). Another identified partner of Arf6, Arfophilin, appears to be more selective for Arf6 than for Arf1 (34Shin O.H. Couvillon A.D. Exton J.H. Biochemistry. 2001; 40: 10846-10852Crossref PubMed Scopus (48) Google Scholar). This protein binds to Switch I and II regions of Arf6 but recognizes the N-terminal portion of Arf5 (34Shin O.H. Couvillon A.D. Exton J.H. Biochemistry. 2001; 40: 10846-10852Crossref PubMed Scopus (48) Google Scholar). Intriguingly, Arfophilin is identical to a Rab11 effector protein, FIP3, suggesting common effectors that might bridge Arf6 and Rab11 functions (35Hickson G.R. Matheson J. Riggs B. Maier V.H. Fielding A.B. Prekeris R. Sullivan W. Barr F.A. Gould G.W. Mol. Biol. Cell. 2003; 14: 2908-2920Crossref PubMed Scopus (115) Google Scholar). The effects of Arf6 on membrane lipid composition can also lead to changes in cortical actin cytoskeleton, perhaps working synergistically with activation of Rac. Phosphoinositides, and in particular, PIP2, can recruit and influence the activity of a number of actin-binding proteins that lead to changes in the cortical actin network (see Ref. 16Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2003; 65: 761-789Crossref PubMed Scopus (567) Google Scholar for review). PIP2, PIP5-kinase, and Arf6 are present together on the PM and endosomal membranes. Acute stimulation of Arf6 activation leads to protrusions of PIP2-rich PM driven by actin polymerization (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, 32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar) and stimulated membrane internalization and rapid recycling of the membrane back to the PM (32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar) (Fig. 1B). At low expression levels, PIP5-kinase and Arf6 act synergistically to form protrusions (32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar), and in CHO cells expression of either Arf6Q67L or PIP5-kinase generates actin comet-propelled endosomal membranes (36Schafer D.A. D'Souza-Schorey C. Cooper J.A. Traffic. 2000; 1: 892-903Crossref PubMed Scopus (7) Google Scholar). Recent reports have implicated Arf6 in determining polarized structures in neuronal cells and in yeast and in the disassembly of polarized epithelium. Arf6 can regulate dendritic branching in hippocampal neurons (37Hernandez-Deviez D.J. Casanova J.E. Wilson J.M. Nat. Neurosci. 2002; 5: 623-624Crossref PubMed Scopus (95) Google Scholar) and neurite outgrowth in PC12 cells (38Albertinazzi C. Za L. Paris S. De Curtis I. Mol. Biol. Cell. 2003; 14: 1295-1307Crossref PubMed Scopus (90) Google Scholar). In yeast, the Arf6 homologue, Arf3, localizes to the growing bud and is important for polarized growth and bud site selection (39Huang C.F. Liu Y.W. Tung L. Lin C.H. Lee F.J. Mol. Biol. Cell. 2003; (DOI 10.1091/mbc.03-01-0013)Google Scholar). The stimulation of migration of MDCK cells in response to growth factor (26Palacios F. Price L. Schweitzer J. Collard J.G. D'Souza-Schorey C. EMBO J. 2001; 20: 4973-4986Crossref PubMed Scopus (250) Google Scholar, 40Palacios F. D'Souza-Schorey C. J. Biol. Chem. 2003; 278: 17395-17400Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) or during wound healing (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar) involves the transition from an epithelium with cells held together by adhesive interactions to more motile cells freed of these adhesions (Fig. 1C). The transformation and conversion to a motile cell required the activation of Arf6, as these processes were inhibited by expression of Arf6T27N (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). In response to hepatocyte growth factor (HGF), Arf6 is continuously activated whereas Rac is initially inactivated, leading to the internalization of adherens junction proteins (40Palacios F. D'Souza-Schorey C. J. Biol. Chem. 2003; 278: 17395-17400Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Active Arf6 binds to Nm23-H1, a nucleoside diphosphate kinase that has been implicated in both the inhibition of Rac activation and dynamin-dependent endocytosis, and could account for the observations at early times of HGF treatment (41Palacios F. Schweitzer J.K. Boshans R.L. D'Souza-Schorey C. Nat. Cell Biol. 2002; 4: 929-936Crossref PubMed Scopus (268) Google Scholar). At later times, levels of Rac-GTP increase and the cells begin to scatter (40Palacios F. D'Souza-Schorey C. J. Biol. Chem. 2003; 278: 17395-17400Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Another study demonstrated that the activation of Arf6 induced by expression of ARNO, an Arf6 GEF, could initiate cell migration in the absence of HGF stimulation and enhance wound healing in MDCK cells (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar). The activation of Arf6 led to two downstream effects, one involving Rac activation and one involving PLD (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar). Although PLD activity was not required for the activation of Rac, both the Rac and PLD pathway were required for the stimulation of cell migration (27Santy L.C. Casanova J.E. J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar). In addition to roles in stimulating migration of epithelial cells, Arf6 is also required in leukocytes for chemokine-stimulated migration across endothelial cells (28Weber K.S. Weber C. Ostermann G. Dierks H. Nagel W. Kolanus W. Curr. Biol. 2001; 11: 1969-1974Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). It is difficult to describe Arf6 actions on cell morphology, polarity, and the actin cytoskeleton without including its effects on membrane traffic. In addition to its PM localization, Arf6 is associated with endosomal membranes in many cells (42Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). In kidney proximal tubules, Arf6 localizes to specialized apical endosomes (43Maranda B. Brown D. Bourgoin S. Casanova J.E. Vinay P. Ausiello D.A. Marshansky V. J. Biol. Chem. 2001; 276: 18540-18550Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and in CHO cells there is partial overlap between Arf6-associated endosomes and endosomes containing the transferrin receptor (44D'Souza-Schorey C. van Donselaar E. Hsu V.W. Yang C. Stahl P.D. Peters P.J. J. Cell Biol. 1998; 140: 603-616Crossref PubMed Scopus (196) Google Scholar). In HeLa and other types of epithelial cells, Arf6 is associated with a distinct endosomal compartment that contains integral membrane proteins that are endocytosed into cells independently of adaptor protein 2 and clathrin (45Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (417) Google Scholar, 46Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell. 2003; 14: 417-431Crossref PubMed Scopus (219) Google Scholar). This separate endosomal system that exists in parallel with the classical, transferrin-containing endosomal system is depicted in Fig. 1D and is the trafficking route followed by a number of PM proteins including the major histocompatibility complex class I proteins (MHCI) (45Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (417) Google Scholar, 46Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell. 2003; 14: 417-431Crossref PubMed Scopus (219) Google Scholar), integrins (32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar), and E-cadherin (47Paterson A.D. Parton R.G. Ferguson C. Stow J.L. Yap A.S. J. Biol. Chem. 2003; 278: 21050-21057Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). After internalization, these proteins can either recycle back to the PM (45Radhakrishna H. Donaldson J.G. J. Cell Biol. 1997; 139: 49-61Crossref PubMed Scopus (417) Google Scholar, 48Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. EMBO J. 2002; 21: 2557-2567Crossref PubMed Scopus (244) Google Scholar) or fuse with the Rab5-associated endosomal system (46Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell. 2003; 14: 417-431Crossref PubMed Scopus (219) Google Scholar). The crossover from the Arf6 endosome to the Rab5 endosome is apparently a trafficking route followed by the M2-muscarinic acetylcholine receptor (49Delaney K.A. Murph M.M. Brown L.M. Radhakrishna H. J. Biol. Chem. 2002; 277: 33439-33446Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and may be part of the mechanism for the down-regulation of surface MHCI induced by Nef, an HIV-encoded protein (50Blagoveshchenskaya A.D. Thomas L. Feliciangeli S.F. Hung C.H. Thomas G. Cell. 2002; 111: 853-866Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Nef stimulates the activation of Arf6, enhances endocytosis of MHCI, inhibits MHCI recycling, and causes MHCI to traffic to the Golgi complex, presumably from the Rab5 early endosome (50Blagoveshchenskaya A.D. Thomas L. Feliciangeli S.F. Hung C.H. Thomas G. Cell. 2002; 111: 853-866Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Although Arf6 is associated with these endosomal membranes in HeLa cells, Arf6 activation is not required for internalization and trafficking through this pathway. Inactivation of Arf6, however, is a requirement. When Arf6 activation is stimulated and protrusions form, increased membrane is taken up by macropinocytosis and then recycled back to the PM, provided that Arf6 can be inactivated by GAP (32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar). If, however, Arf6Q67L is overexpressed, membrane is internalized but not recycled, and cells accumulate PIP2 and F-actin-coated endosomal structures (Fig. 1D) that contain only clathrin-independent cargo proteins such as MHCI (32Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (361) Google Scholar, 46Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell. 2003; 14: 417-431Crossref PubMed Scopus (219) Google Scholar). Expression of the peripheral myelin protein, Pmp22, in HeLa cells appears to activate Arf6 and induces the formation of similar endocytic structures (51Chies R. Nobbio L. Edomi P. Schenone A. Schneider C. Brancolini C. J. Cell Sci. 2003; 116: 987-999Crossref PubMed Scopus (32) Google Scholar), suggesting a role for Arf6 in the formation of the myelin sheath, a process that is poorly understood. Another membrane trafficking function attributed to Arf6 is its role in certain regulated secretory events (Fig. 1E). In PC12 cells, Arf6 is associated with the dense core granules that are docked at the PM (52Galas M.C. Helms J.B. Vitale N. Thierse D. Aunis D. Bader M.F. J. Biol. Chem. 1997; 272: 2788-2793Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and upon calcium stimulation, Arf6 is activated and increases PLD activity resulting in membrane fusion (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, 53Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (102) Google Scholar). Expression of a mutant of Arf6 (N48I) that cannot activate PLD inhibits secretion, demonstrating that activation of PLD is a major downstream effect of Arf6 that leads to secretion (53Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (102) Google Scholar). In another example, stimulation of adipocytes with endothelin acts via Gαq to release a pool of Glut4-containing vesicles, but expression of Arf6T27N blocks this release (54Bose A. Cherniack A.D. Langille S.E. Nicoloro S.M. Buxton J.M. Park J.G. Chawla A. Czech M.P. Mol. Cell. Biol. 2001; 21: 5262-5275Crossref PubMed Scopus (57) Google Scholar, 55Lawrence J.T. Birnbaum M.J. Mol. Cell. Biol. 2001; 21: 5276-5285Crossref PubMed Scopus (39) Google Scholar). A recent study examining the role of Arf6 in Fc-mediated phagocytosis found that vesicles accumulated in cells expressing Arf6T27N, suggesting that activation of Arf6 leads to the exocytosis of vesicles required to complete phagocytosis (30Niedergang F. Colucci-Guyon E. Dubois T. Raposo G. Chavrier P. J. Cell Biol. 2003; 161: 1143-1150Crossref PubMed Scopus (150) Google Scholar). In addition to its effects on cortical actin and clathrin-independent endocytosis, Arf6 regulation of PIP2 synthesis can influence other PM activities (Fig. 1F). PIP2 is required for the recruitment of AP2/clathrin onto forming clathrin-coated pits but is rapidly lost upon vesicle fission. A recent study demonstrated that Arf6Q67L binds to PIP5-kinase I-γ, activates the kinase, and leads to enhanced AP2/clathrin binding to a synaptic vesicle membrane preparation (56Krauss M. Kinuta M. Wenk M.R. De Camilli P. Takei K. Haucke V. J. Cell Biol. 2003; 162: 113-124Crossref PubMed Scopus (235) Google Scholar). Interestingly, an earlier study reported an effect of expression of Arf6 mutants on the morphology of clathrin-coated pits at the apical surface of MDCK cells (57Altschuler Y. Liu S. Katz L. Tang K. Hardy S. Brodsky F. Apodaca G. Mostov K. J. Cell Biol. 1999; 147: 7-12Crossref PubMed Scopus (119) Google Scholar). Two areas of investigation have uncovered roles for Arf6 in sorting of PM proteins to facilitate their internalization. The agonist-induced down-regulation of β2-adrenergic and luteinizing hormone receptors requires Arf6 activity. In both cases, engagement of the receptor leads to activation of Arf6 (58Salvador L.M. Mukherjee S. Kahn R.A. Lamm M.L. Fazleabas A.T. Maizels E.T. Bader M.F. Hamm H. Rasenick M.M. Casanova J.E. Hunzicker-Dunn M. J. Biol. Chem. 2001; 276: 33773-33781Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 59Claing A. Chen W. Miller W.E. Vitale N. Moss J. Premont R.T. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 42509-42513Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) facilitating the release of sequestered arrestin to allow internalization via clathrin-mediated endocytosis (60Mukherjee S. Gurevich V.V. Preninger A. Hamm H.E. Bader M.F. Fazleabas A.T. Birnbaumer L. Hunzicker-Dunn M. J. Biol. Chem. 2002; 277: 17916-17927Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Yet another role for Arf6 involves the transmembrane form of carboxypeptidase E (CPE), a sorting receptor for regulated secretory granules that, after secretion, is recycled back to the Golgi complex. This form of CPE extends about 6 amino acid residues (SETLNF) into the cytoplasm and interacts specifically with Arf6-GTP (61Arnaoutova I. Jackson C.L. Al-Awar O. Donaldson J.G. Loh Y.P. Mol. Biol. Cell. 2003; (DOI 10.1091/mbc.02-11-0758)PubMed Google Scholar). Remarkably, expression of Arf6-T27N blocks the recycling of CPE from the PM back to the Golgi, and mutation of the six residues in CPE important for Arf6 binding also inhibits recycling (61Arnaoutova I. Jackson C.L. Al-Awar O. Donaldson J.G. Loh Y.P. Mol. Biol. Cell. 2003; (DOI 10.1091/mbc.02-11-0758)PubMed Google Scholar). Although we are beginning to understand the breadth of Arf6 activities, the identification of more proteins that interact with Arf6 is needed to develop a molecular framework for understanding Arf6 function. The multiple sites of action of Arf6 at the PM highlight the importance of spatial and temporal regulation of Arf6 by connecting to specific GEFs, GAPs, and effectors. Although Arf6 may act in a constitutive capacity in resting cells, it is subject to acute stimulation through signal transduction pathways. Further studies on Arf6 function will shed light on the complex interplay between signal transduction, membrane traffic, and the cytoskeleton. I thank Rob Donaldson, Cathy Jackson, Ed Korn, and members of my laboratory for comments on the manuscript.

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