Browsing by Subject "GTPase-Activating Proteins"

Now showing 1 - 8 of 8
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    ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry.
    (PLoS pathogens, 2015-09-15) Shu, Qian; Lennemann, Nicholas J; Sarkar, Saumendra N; Sadovsky, Yoel; Coyne, Carolyn B
    Interferon stimulated genes (ISGs) target viruses at various stages of their infectious life cycles, including at the earliest stage of viral entry. Here we identify ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2) as a gene upregulated by type I IFN treatment in a STAT1-dependent manner. ADAP2 functions as a GTPase-activating protein (GAP) for Arf6 and binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and PI(3,4)P2. We show that overexpression of ADAP2 suppresses dengue virus (DENV) and vesicular stomatitis virus (VSV) infection in an Arf6 GAP activity-dependent manner, while exerting no effect on coxsackievirus B (CVB) or Sendai virus (SeV) replication. We further show that ADAP2 expression induces macropinocytosis and that ADAP2 strongly associates with actin-enriched membrane ruffles and with Rab8a- and LAMP1-, but not EEA1- or Rab7-, positive vesicles. Utilizing two techniques--light-sensitive neutral red (NR)-containing DENV and fluorescence assays for virus internalization--we show that ADAP2 primarily restricts DENV infection at the stage of virion entry and/or intracellular trafficking and that incoming DENV and VSV particles associate with ADAP2 during their entry. Taken together, this study identifies ADAP2 as an ISG that exerts antiviral effects against RNA viruses by altering Arf6-mediated trafficking to disrupt viral entry.
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    Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.
    (J Cell Biol, 1996-11) Kuehn, MJ; Schekman, R; Ljungdahl, PO
    In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.
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    ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.
    (J Cell Biol, 2005-01-17) Lee, SY; Yang, JS; Hong, W; Premont, RT; Hsu, VW
    Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.
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    ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.
    (J Cell Biol, 2002-10-14) Yang, JS; Lee, SY; Gao, M; Bourgoin, S; Randazzo, PA; Premont, RT; Hsu, VW
    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 GTPgammaS 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.
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    beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.
    (Proc Natl Acad Sci U S A, 1998-11-24) Premont, RT; Claing, A; Vitale, N; Freeman, JL; Pitcher, JA; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ
    G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.
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    Consensus nomenclature for the human ArfGAP domain-containing proteins.
    (J Cell Biol, 2008-09-22) Kahn, Richard A; Bruford, Elspeth; Inoue, Hiroki; Logsdon, John M; Nie, Zhongzhen; Premont, Richard T; Randazzo, Paul A; Satake, Masanobu; Theibert, Anne B; Zapp, Maria L; Cassel, Dan
    At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.
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    Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.
    (J Cell Sci, 2010-07-01) Jones, Whitney M; Chao, Anna T; Zavortink, Michael; Saint, Robert; Bejsovec, Amy
    Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.
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    Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.
    (Proc Natl Acad Sci U S A, 2000-02-01) Claing, A; Perry, SJ; Achiriloaie, M; Walker, JK; Albanesi, JP; Lefkowitz, RJ; Premont, RT
    Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.