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

Abstract

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 I