scholarly journals ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment

2015 ◽  
Vol 26 (23) ◽  
pp. 4265-4279 ◽  
Author(s):  
Joseph C. Salem ◽  
Marta M. Reviriego-Mendoza ◽  
Lorraine C. Santy
Keyword(s):  
Adhesion Formation ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Recycling Endosomes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Migration ◽  
Regulated Trafficking ◽  
Migration Response

When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras–dependent effects on spreading and adhesion formation.

scholarly journals The DHR1 Domain of DOCK180 Binds to SNX5 and Regulates Cation-independent Mannose 6-phosphate Receptor Transport

2008 ◽  
Vol 19 (9) ◽  
pp. 3823-3835 ◽  
Author(s):  
Shigeo Hara ◽  
Etsuko Kiyokawa ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
Thomas Wassmer ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Retrograde Transport ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Sorting Nexins ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mannose 6 Phosphate

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

scholarly journals The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

1997 ◽  
Vol 139 (3) ◽  
pp. 797-807 ◽  
Author(s):  
Frank N. van Leeuwen ◽  
Hendrie E.T. Kain ◽  
Rob A. van der Kammen ◽  
Frits Michiels ◽  
Onno W. Kranenburg ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Neuronal Morphology ◽  
Laminin Receptor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Neurite Formation

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.

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 ARHGEF7 (β-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function

2020 ◽  
Vol 31 (5) ◽  
pp. 996-1008 ◽  
Author(s):  
Jun Matsuda ◽  
Mirela Maier ◽  
Lamine Aoudjit ◽  
Cindy Baldwin ◽  
Tomoko Takano
Keyword(s):  
Knockout Mice ◽  
Critical Role ◽  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Glomerular Function ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

BackgroundPrevious studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown.MethodsWe used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange factors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specific ARHGEF7 (commonly known as β-PIX) knockout mice by crossing β-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with β-PIX knockdown and their controls.ResultsWe identified β-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific β-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific β-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific β-PIX knockout mice and cultured mouse podocytes with β-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of β-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated protein.ConclusionsThese findings indicate that β-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho–guanine nucleotide exchange factor plays a critical role in podocytes.

scholarly journals Calcium-RasGRP2-Rap1 signaling mediates CD38-induced migration of chronic lymphocytic leukemia cells

2018 ◽  
Vol 2 (13) ◽  
pp. 1551-1561 ◽  
Author(s):  
Silvia Mele ◽  
Stephen Devereux ◽  
Andrea G. Pepper ◽  
Elvira Infante ◽  
Anne J. Ridley
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Cd38 Expression ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Key Points ◽  
And Migration

Key Points Basal intracellular Ca2+ levels and migration increase with higher CD38 expression in CLL cells. Rap1 and the Rap1 guanine-nucleotide exchange factor RasGRP2 are required for CLL migration and regulated by CD38 levels.

scholarly journals Plekhg4 Is a Novel Dbl Family Guanine Nucleotide Exchange Factor Protein for Rho Family GTPases

2013 ◽  
Vol 288 (20) ◽  
pp. 14522-14530 ◽  
Author(s):  
Meghana Gupta ◽  
Elena Kamynina ◽  
Samantha Morley ◽  
Stacey Chung ◽  
Nora Muakkassa ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Developmentally Regulated ◽  
Nih3t3 Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Bona Fide

Mutations in the PLEKHG4 (puratrophin-1) gene are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. However, the biochemical functions of this gene product have not been described. We report here that expression of Plekhg4 in the murine brain is developmentally regulated, with pronounced expression in the newborn midbrain and brainstem that wanes with age and maximal expression in the cerebellar Purkinje neurons in adulthood. We show that Plekhg4 is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones Hsc70 and Hsp90 and by the ubiquitin ligase CHIP. On the functional level, we demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor (GEF) that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. Overexpression of Plekhg4 in NIH3T3 cells induces rearrangements of the actin cytoskeleton, specifically enhanced formation of lamellopodia and fillopodia. These findings indicate that Plekhg4 is an aggregation-prone member of the Dbl family GEFs and that regulation of GTPase signaling is critical for proper cerebellar function.

scholarly journals PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

2008 ◽  
Vol 180 (1) ◽  
pp. 187-203 ◽  
Author(s):  
Yangmi Lim ◽  
Ssang-Taek Lim ◽  
Alok Tomar ◽  
Margaret Gardel ◽  
Joie A. Bernard-Trifilo ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cellular Contact

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

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