A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1.
ABSTRACT: ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.
Project description:ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1-6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy.
Project description:Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport. More recently, the same protein has been reported to be a regulatory factor for the alphaLbeta2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function. ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP). Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP. Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated. Cytohesin-1 enhanced binding of 35S-labeled guanosine 5'-[gamma-thio]triphosphate [35S]GTP[gammaS] or [3H]GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with [35S]GTP[gammaS] bound, accelerated [35S]GTP[gammaS] release to a similar degree in the presence of unlabeled GDP or GTP[gammaS] and to a lesser degree with GDP[betaS]; release was negligible without added nucleotide. Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function. In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.
Project description:The integrity of the Golgi and trans-Golgi network (TGN) is disrupted by brefeldin A (BFA), which inhibits the Golgi-localized BFA-sensitive factor (GBF1) and brefeldin A-inhibited guanine nucleotide-exchange factors (BIG1 and BIG2). Using a cellular replacement assay to assess GBF1 functionality without interference from the BIGs, we show that GBF1 alone maintains Golgi architecture; facilitates secretion; activates ADP-ribosylation factor (ARF)1, 3, 4, and 5; and recruits ARF effectors to Golgi membranes. Unexpectedly, GBF1 also supports TGN integrity and recruits numerous TGN-localized ARF effectors. The impact of the catalytic Sec7 domain (Sec7d) on GBF1 functionality was assessed by swapping it with the Sec7d from ARF nucleotide-binding site opener (ARNO)/cytohesin-2, a plasma membrane GEF reported to activate all ARFs. The resulting chimera (GBF1-ARNO-GBF1 [GARG]) targets like GBF1, supports Golgi/TGN architecture, and facilitates secretion. However, unlike GBF1, GARG activates all ARFs (including ARF6) at the Golgi/TGN and recruits additional ARF effectors to the Golgi/TGN. Our results have general implications: 1) GEF's targeting is independent of Sec7d, but Sec7d influence the GEF substrate specificity and downstream effector events; 2) all ARFs have access to all membranes, but are restricted in their distribution by the localization of their activating GEFs; and 3) effector association with membranes requires the coincidental presence of activated ARFs and specific membrane identifiers.
Project description:ADP-ribosylation factor (ARF) 6 regulates plasma membrane trafficking and cortical actin formation by cycling between inactive GDP and active GTP-bound conformations. Here we show that agonist stimulation of phosphatidylinositide 3-kinase (PI 3-kinase) activates a pathway that leads to ARF6 activation. We also describe experiments that propose a central role in this pathway for the PI 3-kinase-dependent plasma membrane recruitment of the cytohesin-1 family of PtdIns(3,4,5)P(3)-binding ARF-exchange factors.
Project description:ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.
Project description:The small GTP-binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP-ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full-length, non-myristoylated human Arf6 bound to GTPgammaS. Unlike their GDP-bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP-bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.
Project description:The maturation of glutamatergic synapses in the CNS is regulated by NMDA receptors (NMDARs) that gradually change from a GluN2B- to a GluN2A-dominated subunit composition during postnatal development. Here we show that NMDARs control the activity of the small GTPase ADP-ribosylation factor 6 (Arf6) by consecutively recruiting two related brefeldin A-resistant Arf guanine nucleotide exchange factors, BRAG1 and BRAG2, in a GluN2 subunit-dependent manner. In young cortical cultures, GluN2B and BRAG1 tonically activated Arf6. In mature cultures, Arf6 was activated through GluN2A and BRAG2 upon NMDA treatment, whereas the tonic Arf6 activation was not detectable any longer. This shift in Arf6 regulation and the associated drop in Arf6 activity were reversed by a knockdown of BRAG2. Given their sequential recruitment during development, we examined whether BRAG1 and BRAG2 influence synaptic currents in hippocampal CA1 pyramidal neurons using patch clamp recordings in acute slices from mice at different ages. The number of AMPA receptor (AMPAR) miniature events was reduced by depletion of BRAG1 but not by depletion of BRAG2 during the first 2 weeks after birth. In contrast, depletion of BRAG2 during postnatal weeks 4 and 5 reduced the number of AMPAR miniature events and compromised the quantal sizes of both AMPAR and NMDAR currents evoked at Schaffer collateral synapses. We conclude that both Arf6 activation through GluN2B-BRAG1 during early development and the transition from BRAG1- to BRAG2-dependent Arf6 signaling induced by the GluN2 subunit switch are critical for the development of mature glutamatergic synapses.
Project description:<h4>Unlabelled</h4>To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections.<h4>Importance</h4>To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP family in Salmonella invasion. Surprisingly, we found that Arf1 and Arf6 GAPs cooperate with their Arf GEF counterparts to facilitate cycles of Arf GTPase activation and inactivation, which direct pathogen invasion. This report illustrates that GAP proteins promote actin-dependent processes and are not necessarily restricted to negatively regulating cellular signaling. It uncovers a remarkable interplay between Arf GEFs and GAPs that is exploited by Salmonella to establish infection and expands our understanding of Arf GTPase-regulated cytoskeleton remodeling.
Project description:Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H(+)-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1-17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1-17) and its amino acids Phe(5), Met(10), and Gln(14) involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1-17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1-a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.
Project description:Proteins of the cytohesin/Arno/Grp1 family of Arf activators are positive regulators of the insulin-signaling pathway and control various remodeling events at the plasma membrane. Arno has a catalytic Sec7 domain, which promotes GDP to GTP exchange on Arf, followed by a pleckstrin homology (PH) domain. Previous studies have revealed two functions of the PH domain: inhibition of the Sec7 domain and membrane targeting. Interestingly, the Arno PH domain interacts not only with a phosphoinositide (phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate) but also with an activating Arf family member, such as Arf6 or Arl4. Using the full-length membrane-bound forms of Arf1 and Arf6 instead of soluble forms, we show here that the membrane environment dramatically affects the mechanism of Arno activation. First, Arf6-GTP stimulates Arno at nanomolar concentrations on liposomes compared with micromolar concentrations in solution. Second, mutations in the PH domain that abolish interaction with Arf6-GTP render Arno completely inactive when exchange reactions are reconstituted on liposomes but have no effect on Arno activity in solution. Third, Arno is activated by its own product Arf1-GTP in addition to a distinct activating Arf isoform. Consequently, Arno activity is strongly modulated by competition with Arf effectors. These results show that Arno behaves as a bistable switch, having an absolute requirement for activation by an Arf protein but, once triggered, becoming highly active through the positive feedback effect of Arf1-GTP. This property of Arno might provide an explanation for its function in signaling pathways that, once triggered, must move forward decisively.