scholarly journals Identification and characterization of a novel Rho-specific guanine nucleotide exchange factor

2000 ◽  
Vol 352 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Andrea BLOMQUIST ◽  
Guntram SCHWÖRER ◽  
Helge SCHABLOWSKI ◽  
Amalia PSOMA ◽  
Michaela LEHNEN ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Transcriptional Activation ◽  
Bound States ◽  
Membrane Receptors ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Rho Gef

Rho GTPases are implicated in a multitude of cellular processes regulated by membrane receptors, such as cytoskeletal rearrangements, gene transcription and cell growth and motility. Activation of these GTPases is under the direct control of guanine nucleotide exchange factors (GEFs), the Dbl family proteins. By searching protein databases we have identified a novel Rho-GEF, termed p114-Rho-GEF, which similarly to other Rho-GEFs contains a Dbl homology domain followed by a pleckstrin homology domain. p114-Rho-GEF interacted specifically with RhoA, in its nucleotide-free and guanosine 5′-[γ-thio]triphosphate-bound states, but not with Rac1 and Cdc42, and efficiently catalysed guanine nucleotide exchange of RhoA. Consistent with these results in vitro was our finding that the overexpression of p114-Rho-GEF in J82 and HEK-293 cells induced the formation of actin stress fibres and stimulated serum-response-factor-mediated gene transcription in a Rho-dependent manner. Rho-mediated transcriptional activation induced by M3 muscarinic acetylcholine and lysophosphatidic acid receptors was enhanced by p114-Rho-GEF, suggesting that the activity of this novel Rho-GEF, which is widely expressed in human tissues, can be controlled by G-protein-coupled receptors.

scholarly journals A mammalian Rho-specific guanine-nucleotide exchange factor (p164-RhoGEF) without a pleckstrin homology domain

2002 ◽  
Vol 366 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Ulrich RÜMENAPP ◽  
Andrea FREICHEL-BLOMQUIST ◽  
Burkhard WITTINGHOFER ◽  
Karl H. JAKOBS ◽  
Thomas WIELAND
Keyword(s):  
Gene Transcription ◽  
Rho Gtpases ◽  
Serum Response Factor ◽  
Full Length ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Pleckstrin Homology ◽  
Guanine Nucleotide Exchange ◽  
Dh Domain

Rho GTPases, which are activated by specific guanine-nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a recently cloned human cDNA, namely KIAA0337, encoding a protein containing 1510 amino acids (p164). It contains a RhoGEF-specific Dbl homology (DH) domain but lacks their typical pleckstrin homology domain. The expression of the mRNA encoding p164 was found to be at least 4-fold higher in the heart than in other tissues. Recombinant p164 interacted with and induced GDP/GTP exchange at RhoA but not at Rac1 or Cdc42. p164-ΔC and p164-ΔN are p164 mutants that are truncated at the C- and N-termini respectively but contain the DH domain. In contrast with the full-length p164, expression of p164-ΔC and p164-ΔN strongly induced actin stress fibre formation and activated serum response factor-mediated and Rho-dependent gene transcription. Interestingly, p164-ΔN2, a mutant containing the C-terminus but having a defective DH domain, bound to p164-ΔC and suppressed the p164-ΔC-induced gene transcription. Overexpression of the full-length p164 inhibited M3 muscarinic receptor-induced gene transcription, whereas co-expression with Gβ1γ2 dimers induced transcriptional activity. It is concluded that p164-RhoGEF is a Rho-specific GEF with novel structural and regulatory properties and predominant expression in the heart. Apparently, its N- and C-termini interact with each other, thereby inhibiting its GEF activity.

scholarly journals Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

2019 ◽  
Author(s):  
Nathalie R. Reinhard ◽  
Sanne van der Niet ◽  
Anna Chertkova ◽  
Marten Postma ◽  
Peter L. Hordijk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Actin Dynamics ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere

2002 ◽  
Vol 115 (3) ◽  
pp. 629-640 ◽  
Author(s):  
Michel Souchet ◽  
Elodie Portales-Casamar ◽  
David Mazurais ◽  
Susanne Schmidt ◽  
Isabelle Léger ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.

scholarly journals MicroRNA Regulation of the Small Rho GTPase Regulators—Complexities and Opportunities in Targeting Cancer Metastasis

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1092 ◽  
Author(s):  
Brock A. Humphries ◽  
Zhishan Wang ◽  
Chengfeng Yang
Keyword(s):  
Transcriptional Activation ◽  
Rho Gtpases ◽  
Cancer Metastasis ◽  
Rho Gtpase ◽  
Critical Role ◽  
Large Family ◽  
Actin Dynamics ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange

The small Rho GTPases regulate important cellular processes that affect cancer metastasis, such as cell survival and proliferation, actin dynamics, adhesion, migration, invasion and transcriptional activation. The Rho GTPases function as molecular switches cycling between an active GTP-bound and inactive guanosine diphosphate (GDP)-bound conformation. It is known that Rho GTPase activities are mainly regulated by guanine nucleotide exchange factors (RhoGEFs), GTPase-activating proteins (RhoGAPs), GDP dissociation inhibitors (RhoGDIs) and guanine nucleotide exchange modifiers (GEMs). These Rho GTPase regulators are often dysregulated in cancer; however, the underlying mechanisms are not well understood. MicroRNAs (miRNAs), a large family of small non-coding RNAs that negatively regulate protein-coding gene expression, have been shown to play important roles in cancer metastasis. Recent studies showed that miRNAs are capable of directly targeting RhoGAPs, RhoGEFs, and RhoGDIs, and regulate the activities of Rho GTPases. This not only provides new evidence for the critical role of miRNA dysregulation in cancer metastasis, it also reveals novel mechanisms for Rho GTPase regulation. This review summarizes recent exciting findings showing that miRNAs play important roles in regulating Rho GTPase regulators (RhoGEFs, RhoGAPs, RhoGDIs), thus affecting Rho GTPase activities and cancer metastasis. The potential opportunities and challenges for targeting miRNAs and Rho GTPase regulators in treating cancer metastasis are also discussed. A comprehensive list of the currently validated miRNA-targeting of small Rho GTPase regulators is presented as a reference resource.

scholarly journals The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Regulates Assembly of Titin at the Z-Disk through Interactions with Ran Binding Protein 9

2008 ◽  
Vol 19 (9) ◽  
pp. 3782-3792 ◽  
Author(s):  
Amber L. Bowman ◽  
Dawn H. Catino ◽  
John C. Strong ◽  
William R. Randall ◽  
Aikaterini Kontrogianni-Konstantopoulos ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Striated Muscle ◽  
Binding Protein ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Binding Region ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rho Gef ◽  
Signaling Domains

Obscurin is an ∼800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including α-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 μM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.

scholarly journals The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

2015 ◽  
Vol 112 (5) ◽  
pp. 1404-1409 ◽  
Author(s):  
Ned Van Eps ◽  
Celestine J. Thomas ◽  
Wayne L. Hubbell ◽  
Stephen R. Sprang
Keyword(s):  
G Protein ◽  
Bound States ◽  
Guanine Nucleotide Exchange Factor ◽  
Nucleotide Binding ◽  
Conformational Ensemble ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.

scholarly journals Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32

2021 ◽  
Author(s):  
Chenchen Mi ◽  
Li Zhang ◽  
Shan Sun ◽  
Guoqiang Huang ◽  
Guangcan Shao ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Bound States ◽  
Structural Basis ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Nucleotide Binding Domain ◽  
Closed Conformation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Protein Particle

Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and have three forms- TRAPPI, TRAPPII and TRAPPIII, which share a core of six TRAPPI proteins. TRAPPII facilitates intra-Golgi and endosome-to-Golgi transports by activating GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor (GEF) in yeast. Here we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle shaped monomers, while the monomer in the apo structure exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation. Located in the interior of the monomer, Ypt32 binds with both TRAPPI and Trs120 via its nucleotide binding domain and binds with Trs31 of TRAPPI via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.

scholarly journals G1/S Cyclin-dependent Kinase Regulates Small GTPase Rho1p through Phosphorylation of RhoGEF Tus1p inSaccharomyces cerevisiae

2008 ◽  
Vol 19 (4) ◽  
pp. 1763-1771 ◽  
Author(s):  
Keiko Kono ◽  
Satoru Nogami ◽  
Mitsuhiro Abe ◽  
Masafumi Nishizawa ◽  
Shinichi Morishita ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Cyclin Dependent Kinase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Temporal And Spatial ◽  
Cdk Phosphorylation

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.

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