== Structure of ARNO constructs used in this study. regulate ARF dependent processes by biasing ARF signaling toward particular outputs. == INTRODUCTION == ARFs (ADP ribosylation factors) are members of the Ras superfamily of small GTPases. The six mammalian ARFs are divided into three classes Verbascoside based upon sequence similarity. ARFs 13 make up class I, ARFs 4 and 5 comprise class II, and ARF6 is the sole class III member. The class I and II ARFs recruit vesicle coats and promote vesicle budding in the secretory system. ARF6 is located predominantly in the cell periphery. ARF6 regulates trafficking between the plasma membrane and endosomal systems (Donaldson and Honda, 2005). In addition to regulating the endocytosis and recycling of plasma membrane proteins ARF6 also regulates the cortical actin cytoskeleton (Radhakrishnaet al., 1996;Franket al., 1998a,b;Songet al., 1998;Radhakrishnaet al., 1999;Boshanset al., 2000;Santy, 2002). ARF6-dependent actin rearrangements are critical during cell spreading, migration, and phagocytosis (Songet al., 1998;Zhanget al., 1998;Palacioset al., 2001;Santy and Casanova, 2001;Beemilleret al., 2006). Previous work has demonstrated cross-talk between ARF6 and the Rho-family GTPase Rac (Radhakrishnaet al., 1999;Zhanget al., 1999;Santy and Casanova, Verbascoside 2001;Palacios and D’Souza-Schorey, 2003;Santyet al., 2005;Kooet al., 2007). These two small GTPases coordinate to regulate the cortical actin cytoskeleton and to alter cell shape. Like all GTPases, ARF6 cycles between an inactive GDP-bound state and an active GTP-bound state. Interconversion between these two states requires the actions of accessory proteins. Verbascoside Guanine nucleotide exchange factors (GEFs) promote the binding of GTP and the activation of the GTPase. GTPase-activating proteins (GAPs), on the other hand, induce the hydrolysis of the bound GTP, thereby inactivating the GTPase. Although there are only six ARFs, the human genome encodes 15 ARF GEFs and 20 ARF GAPs. This discrepancy suggests that ARFs are regulated by different GEFs and GAPs at particular subcellular locations or during particular processes (Donaldson and Honda, 2005;Casanova, 2007). There are five families of ARF-GEFs: the GBF/BIG family, cytohesins, EFA6s, BRAGs, and Fbox8. TNFRSF8 Three of these families, the cytohesins, EFA6s, and BRAGs, have been reported to act in the cell periphery and to regulate endocytosis, recycling, and cell shape (Casanova, 2007). The cytohesins in particular have been implicated in the regulation of the actin cytoskeleton and cell shape. Cytohesins are recruited to the plasma membrane in response to growth factor signaling and induce rearrangements in the cortical actin cytoskeleton (Klarlundet al., 1997,1998;Franket al., 1998a,b;Venkateswarluet al., 1998a,b). We have previously shown that overexpression of ARNO/cytohesin 2 in Madin-Darby canine kidney (MDCK) cells promotes migration of these cells (Santy and Casanova, 2001). ARNO expression produces a scattering phenotype that resembles the action of the motility-promoting growth factor HGF. Enhanced migration in response to ARNO expression requires ARF activation and the subsequent downstream activation of phospholipase D (PLD) and Rac (Santy and Casanova, 2001). ARNO-induced Rac activation can be blocked by coexpression of dominant negative mutants of Dock180 and Elmo (Santyet al., 2005). Dock180 and Elmo act together as a bipartite Rac-GEF that has been extensively implicated as an activator of Rac during migration and phagocytosis (Hasegawaet al., 1996;Ericksonet al., 1997;Nolanet al., 1998;Wu and Horvitz, 1998;Reddien and Horvitz, 2000;Gumiennyet al., 2001;Brugneraet al., 2002). How ARNO and Dock180/Elmo coordinate to promote ARF-to-Rac cross-talk remains unclear. Recent work on kinase signaling cascades has demonstrated that the output downstream of a particular kinase can be biased by proteinprotein interactions. Scaffold proteins assemble the components of a particular signaling pathway into a complex and thereby promote signaling through that pathway (Morrison and Davis, 2003;Kolch, 2005;Dard and Peter, 2006;Pullikuth and Catling, 2007). We wondered whether similar processes bias ARF signaling downstream of ARNO toward Rac activation and motility. We tested the hypothesis that proteinprotein interactions direct ARF-dependent signaling downstream of ARNO toward Rac activation and motility. ARNO, like all the cytohesins, is made up of four distinct domains (seeFigure 1). The sec7 domain is the catalytic ARF-GEF domain. The pleckstrin homology (PH) and polybasic domains mediate binding to membrane surfaces. The polybasic domain also functions as an intramolecular inhibitory domain (DiNittoet al., 2007), and the PH domain also interacts with other proteins. The coiled-coil domain promotes dimerization and interacts with a number of other proteins (Casanova, 2007). The coiled-coil domain of ARNO has previously been shown to interact with several small scaffold proteins that contain multiple proteinprotein interacting domains. These include GRASP/Tamalin (Nevrivyet al.,.