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.

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 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 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.

scholarly journals Molecular Determinants of the Interaction between Coxsackievirus Protein 3A and Guanine Nucleotide Exchange Factor GBF1

2007 ◽  
Vol 81 (10) ◽  
pp. 5238-5245 ◽  
Author(s):  
Els Wessels ◽  
Daniël Duijsings ◽  
Kjerstin H. W. Lanke ◽  
Willem J. G. Melchers ◽  
Catherine L. Jackson ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Binding Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Molecular Determinants ◽  
N Terminus ◽  
Insight Into

ABSTRACT The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding domain, and the homology upstream of Sec7 domain are required for the interaction with 3A. In contrast to that of full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.

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