scholarly journals Active Arf6 Recruits ARNO/Cytohesin GEFs to the PM by Binding Their PH Domains

2007 ◽  
Vol 18 (6) ◽  
pp. 2244-2253 ◽  
Author(s):  
Lee Ann Cohen ◽  
Akira Honda ◽  
Peter Varnai ◽  
Fraser D. Brown ◽  
Tamas Balla ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Family Member ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Biochemical Assays ◽  
Inositol Phospholipids ◽  
Sec7 Domain

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions

2002 ◽  
Vol 115 (14) ◽  
pp. 2867-2879 ◽  
Author(s):  
Valérie Derrien ◽  
Carole Couillault ◽  
Michel Franco ◽  
Stéphanie Martineau ◽  
Philippe Montcourrier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Coiled Coil ◽  
Terminal Region ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Sec7 Domain

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

scholarly journals Promiscuity of the catalytic Sec7 domain within the guanine nucleotide exchange factor GBF1 in ARF activation, Golgi homeostasis, and effector recruitment

2019 ◽  
Vol 30 (12) ◽  
pp. 1523-1535 ◽  
Author(s):  
Jay M. Bhatt ◽  
William Hancock ◽  
Justyna M. Meissner ◽  
Aneta Kaczmarczyk ◽  
Eunjoo Lee ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Downstream Effector ◽  
Nucleotide Exchange Factor ◽  
Sec7 Domain ◽  
Golgi Network ◽  
The Impact

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.

scholarly journals The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane

2005 ◽  
Vol 281 (5) ◽  
pp. 2506-2514 ◽  
Author(s):  
Yu Li ◽  
Sirisha Asuri ◽  
John F. Rebhun ◽  
Ariel F. Castro ◽  
Nivanka C. Paranavitana ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

scholarly journals Structure of the C-terminal guanine nucleotide exchange factor module of Trio in an autoinhibited conformation reveals its oncogenic potential

2019 ◽  
Vol 12 (569) ◽  
pp. eaav2449 ◽  
Author(s):  
Sumit J. Bandekar ◽  
Nadia Arang ◽  
Ena S. Tully ◽  
Brittany A. Tang ◽  
Brenna L. Barton ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Independent Manner ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Factor Module

The C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC) transfers signals from the Gαq/11subfamily of heterotrimeric G proteins to the small guanosine triphosphatase (GTPase) RhoA, enabling Gαq/11-coupled G protein–coupled receptors (GPCRs) to control downstream events, such as cell motility and gene transcription. This conserved signal transduction axis is crucial for tumor growth in uveal melanoma. Previous studies indicate that the GEF activity of the TrioC module is autoinhibited, with release of autoinhibition upon Gαq/11binding. Here, we determined the crystal structure of TrioC in its basal state and found that the pleckstrin homology (PH) domain interacts with the Dbl homology (DH) domain in a manner that occludes the Rho GTPase binding site, thereby suggesting the molecular basis of TrioC autoinhibition. Biochemical and biophysical assays revealed that disruption of the autoinhibited conformation destabilized and activated the TrioC module in vitro. Last, mutations in the DH-PH interface found in patients with cancer activated TrioC and, in the context of full-length Trio, led to increased abundance of guanosine triphosphate–bound RhoA (RhoA·GTP) in human cells. These mutations increase mitogenic signaling through the RhoA axis and, therefore, may represent cancer drivers operating in a Gαq/11-independent manner.

scholarly journals Identification of a Plasma Membrane-associated Guanine Nucleotide Exchange Factor for ARF6 in Chromaffin Cells

2000 ◽  
Vol 275 (21) ◽  
pp. 15637-15644 ◽  
Author(s):  
Anne-Sophie Caumont ◽  
Nicolas Vitale ◽  
Marc Gensse ◽  
Marie-Christine Galas ◽  
James E. Casanova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

N-terminal targeting of guanine nucleotide exchange factors (GEF) for ADP ribosylation factors (ARF) to the Golgi

2000 ◽  
Vol 113 (11) ◽  
pp. 1883-1889 ◽  
Author(s):  
S.Y. Lee ◽  
B. Pohajdak
Keyword(s):  
Coiled Coil ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Adp Ribosylation ◽  
Guanine Nucleotide Exchange ◽  
Coiled Coil Domain ◽  
Nucleotide Exchange Factor ◽  
N Terminus ◽  
Point Mutagenesis

B2-1 (cytohesin-1) is a member of a group of proteins (including ARNO and ARNO3) that are all of similar size and domain composition. The three proteins contain an N-terminal coiled-coil domain, followed by a Sec7 and a pleckstrin homology (PH) domain. While it is well established that the Sec7 domain functions as a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factors (ARFs) and the PH domain anchors the proteins to membrane phosphoinositols, the function of the N-terminal domain is unknown. Here we show that the N terminus of B2-1 (residues 1–54) is necessary and sufficient to target the protein to the Golgi. The Sec7+PH domains of B2-1 (residues 55–398) are not sufficient for Golgi localization. Further deletion analysis and point mutagenesis indicate that the coiled-coil domain within the N terminus is responsible for Golgi targeting. Furthermore, ARNO and ARNO3 N termini also have the same capability of targeting to the Golgi. We conclude that the N-terminal, (α)-helical, coiled-coil domain is used to target this family of proteins to the Golgi complex.

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