Paul A. Randazzo

The GGAs Promote ARF-Dependent Recruitment of Clathrin to the TGN

The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.

Arf and its many interactors

Arf GTP-binding proteins regulate membrane traffic and actin remodeling. Similar to other GTP-binding proteins, a complex of Arf-GTP with an effector protein mediates Arf function. Arf interacts with at least three qualitatively different types of effectors. First, it interacts with structural proteins, the vesicle coat proteins. The second type of effector is lipid-metabolizing enzymes, and the third comprises those proteins that bind to Arf-GTP but whose biochemical or biological functions are not yet clearly defined. Arf interacts with two other families of proteins, the exchange factors and the GTPase-activating proteins. Recent work examining the functional relationships among the diverse Arf interactors has led to reconsideration of the prevailing paradigms for Arf action.

ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

ABSTRACT Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPγS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

ARAP1 : a point of convergence for Arf and Rho signaling

We have identified ARAP1 and ARAP2 and examined ARAP1 as a possible link between phosphoinositide-, Arf-, and Rho-mediated cell signaling. ARAP1 contains Arf GAP, Rho GAP, Ankyrin repeat, Ras-associating, and five PH domains. In vitro, ARAP1 had Rho GAP and phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Arf GAP activity. ARAP1 associated with the Golgi. The Rho GAP activity mediated cell rounding and loss of stress fibers when ARAP1 was overexpressed. The Arf GAP activity mediated changes in the Golgi apparatus and the formation of filopodia, the latter a consequence of increased cellular activity of Cdc42. The Arf GAP and Rho GAP activities both contributed to inhibiting cell spreading. Thus, ARAP1 is a PIP3-dependent Arf GAP that regulates Arf-, Rho-, and Cdc42-dependent cell activities.

Arf GAPs and their interacting proteins.

Membrane trafficking and remodeling of the actin cytoskeleton are critical activities contributing to cellular events that include cell growth, migration and tumor invasion. ADP‐ribosylation factor (Arf)‐directed GTPase activating proteins (GAPs) have crucial roles in these processes. The Arf GAPs function in part by regulating hydrolysis of GTP bound to Arf proteins. The Arf GAPs, which have multiple functional domains, also affect the actin cytoskeleton and membranes by specific interactions with lipids and proteins. A description of these interactions provides insights into the molecular mechanisms by which Arf GAPs regulate physiological and pathological cellular events. Here we describe the Arf GAP family and summarize the currently identified protein interactors in the context of known Arf GAP functions.

Arf GAPs and membrane traffic

The selective transfer of material between membrane-delimited organelles is mediated by protein-coated vesicles. In many instances, formation of membrane trafficking intermediates is regulated by the GTP-binding protein Arf. Binding and hydrolysis of GTP by Arf was originally linked to the assembly and disassembly of vesicle coats. Arf GTPase-activating proteins (GAPs), a family of proteins that induce hydrolysis of GTP bound to Arf, were therefore proposed to regulate the disassembly and dissociation of vesicle coats. Following the molecular identification of Arf GAPs, the roles for GAPs and GTP hydrolysis have been directly examined. GAPs have been found to bind cargo and known coat proteins as well as directly contribute to vesicle formation, which is consistent with the idea that GAPs function as subunits of coat proteins rather than simply Arf inactivators. In addition, GTP hydrolysis induced by GAPs occurs largely before vesicle formation and is required for sorting. These results are the primary basis for modifications to the classical model for the function of Arf in transport vesicle formation, including a recent proposal that Arf has a proofreading, rather than a structural, role.

Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras.

ABSTRACT Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.

Molecular Aspects of the Cellular Activities of ADP-Ribosylation Factors

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arfs effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arfs role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events. The adenosine diphosphate-ribosylation factor (Arf) proteins, first identified as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of Gs, are a family of guanosine triphosphate-binding proteins that regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. Arf activity is in part mediated by binding and recruitment of vesicle coat proteins. Arf proteins also bind to and activate phospholipase D and phosphatidylinositol 4-phosphate 5-kinase, producing phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, respectively. These two lipids contribute to the effects of Arf on membrane traffic and the actin cytoskeleton. In addition, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate are involved in the regulation of Arf, interacting with both guanine nucleotide exchange factors and guanosine triphosphatase-activating proteins. Additional Arf-binding proteins have been identified through two-hybrid screens. The molecular mechanisms by which Arf-binding proteins and phospholipids contribute to Arfs physiologic functions are being discovered. Abbreviations: Arf, ADP-ribosylation factor; BFA, brefeldin A; COP, coat protein complex; FA, focal adhesion; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate.