Arf6 plays an early role in platelet activation by collagen and convulxin

Abstract Small GTPases play critical roles in hemostasis, though the roster of such molecules in platelets is not complete. In this study, we report the presence of Ras-related GTPases of the ADP-ribosylation factor (Arf) family. Platelets contain Arf1 or 3 and Arf6, with the latter being predominantly membrane associated. Using effector domain pull-down assays, we show, counter to other GTPases, that Arf6-GTP is present in resting platelets and decreases rapidly upon activation with collagen or convulxin. This decrease does not completely rely on secondary agonists (ADP and thromboxane A2) or require integrin signaling. The decrease in free Arf6-GTP temporally precedes activation of Rho family GTPases (RhoA, Cdc42, and Rac1). Using a membrane-permeant, myristoylated peptide, which mimics the N-terminus of Arf6, we show that the Arf6-GTP decrease is essential for collagen- and convulxin-induced aggregation, platelet adherence, and spreading on collagen-coated glass. Treatment with this peptide also affects the activation of Rho family GTPases, but has little effect on RalA and Rap1 or on agonist-induced calcium mobilization. These data show that Arf6 is a key element in activation through GPVI, and is required for activation of the Rho family GTPases and the subsequent cytoskeletal rearrangements needed for full platelet function.

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Rho Family of Ras-Like GTPases in Early-Branching Animals

Cells ◽

10.3390/cells9102279 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2279

Author(s):

Silvestar Beljan ◽

Maja Herak Bosnar ◽

Helena Ćetković

Keyword(s):

Current Knowledge ◽

Molecular Switches ◽

Cellular Responses ◽

Small Gtpases ◽

Rho Family Gtpases ◽

History Of ◽

Rho Family ◽

Major Branch ◽

External Signals ◽

Insight Into

Non-bilaterian animals consist of four phyla; Porifera, Cnidaria, Ctenophora, and Placozoa. These early-diverging animals are crucial for understanding the evolution of the entire animal lineage. The Rho family of proteins make up a major branch of the Ras superfamily of small GTPases, which function as key molecular switches that play important roles in converting and amplifying external signals into cellular responses. This review represents a compilation of the current knowledge on Rho-family GTPases in non-bilaterian animals, the available experimental data about their biochemical characteristics and functions, as well as original bioinformatics analysis, in order to gain a general insight into the evolutionary history of Rho-family GTPases in simple animals.

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EXC-4/CLIC, Gα, and Rho/Rac signaling regulate tubulogenesis in C. elegans

10.1101/2021.06.21.449267 ◽

2021 ◽

Author(s):

Anthony F Arena ◽

Daniel D Shaye

Keyword(s):

G Protein ◽

Small Gtpases ◽

Loss Of Function ◽

C Elegans ◽

C Terminus ◽

Evolutionarily Conserved ◽

Rho Family Gtpases ◽

Rho Family ◽

Vessel Development ◽

G Protein Coupled

The Rho-family of small GTPases, which play crucial roles in development and disease, are regulated by many signal-transduction cascades, including G-protein-coupled receptor (GPCR)-heterotrimeric G-protein (Gα/β/γ) pathways. Using genetic approaches in C. elegans we identified a new role for Gα and Rho/Rac signaling in cell outgrowth during tubulogenesis and show that the Chloride Intracellular Channel (CLIC) protein EXC-4 is an evolutionarily-conserved player in this pathway. The gene exc-4 was identified by its role in tubulogenesis of the excretory canal (ExCa) cell: a unicellular tube required for osmoregulation and fluid clearance. We identified a new exc-4 loss-of-function allele that affects an evolutionarily conserved residue in the C-terminus. Using this mutant we identified genetic interactions between exc-4, Gα's and Rho-family GTPases, defining novel roles for Gα-encoding genes (gpa-12/Gα12/13, gpa-7/Gαi, egl-30/Gαq, and gsa-1/Gαs) and the Rho-family members ced-10/Rac and mig-2/RhoG in ExCa outgrowth. EXC-4 and human CLICs have conserved functions in tubulogenesis, and CLICs and Gα-Rho/Rac signaling regulate tubulogenesis during blood vessel development. Therefore, our work defines a primordial role for EXC-4/CLICs in Gα-Rho/Rac-signaling during tubulogenesis.

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Abl Tyrosine Kinase Promotes Dendrogenesis by Inducing Actin Cytoskeletal Rearrangements in Cooperation with Rho Family Small GTPases in Hippocampal Neurons

Journal of Neuroscience ◽

10.1523/jneurosci.1264-04.2004 ◽

2004 ◽

Vol 24 (39) ◽

pp. 8510-8521 ◽

Cited By ~ 34

Author(s):

S. B. Jones

Keyword(s):

Tyrosine Kinase ◽

Hippocampal Neurons ◽

Small Gtpases ◽

Abl Tyrosine Kinase ◽

Rho Family ◽

Cytoskeletal Rearrangements

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p120 catenin affects cell motility via modulation of activity of Rho-family GTPases: a link between cell-cell contact formation and regulation of cell locomotion

Journal of Cell Science ◽

10.1242/jcs.114.4.695 ◽

2001 ◽

Vol 114 (4) ◽

pp. 695-707 ◽

Cited By ~ 10

Author(s):

I. Grosheva ◽

M. Shtutman ◽

M. Elbaum ◽

A.D. Bershadsky

Keyword(s):

Cell Motility ◽

Small Gtpases ◽

Cell Junctions ◽

P120 Catenin ◽

Contact Formation ◽

Cell Locomotion ◽

Rho Family Gtpases ◽

Rho Family ◽

E Cadherin ◽

Cell Cell

The molecular basis for contact inhibition of cell locomotion is still largely unknown. Cadherins, the major receptors mediating cell-cell adhesion, associate in the cytoplasm with armadillo family proteins, including beta- and gamma-catenin and p120 catenin (p120ctn). E-cadherin-mediated contact formation was shown to inhibit cellular motility. We examine whether p120ctn may have a role in this regulation. We show here that overexpression of p120ctn in fibroblasts and epithelial cells induces pronounced changes in cell shape, motility and adhesion to the extracellular matrix. p120ctn-transfected cells display increased filopodial/lamellipodial activity, decreased contractility and focal adhesion formation, and augmented migratory ability. These effects of p120ctn are mediated by small GTPases of the Rho family. Direct assessment of the activity of these GTPases in cells expressing a 5-fold higher level of p120ctn as compared to non-transfected control cells revealed significant augmentation of Cdc42 and Rac activity. Moreover, co-transfection of p120ctn with dominant-negative Cdc42 and Rac, or constitutively active Rho suppressed morphological effects of p120ctn. Confocal immunofluorescence visualization of the distribution of endogenous p120ctn in dense cultures showed that formation of cadherin-mediated cell-cell contacts is accompanied by sequestering of p120ctn to the junction regions. In sparse cultures p120ctn is distributed over the cytoplasm. Co-transfection with an excess of E-cadherin leads to sequestration of exogenous p120ctn to cell-cell junctions or to small cadherin-containing vesicles, and abolishes p120ctn effects on cell morphology. Thus, p120ctn may couple the formation and disruption of cadherin-mediated contacts with regulation of cell motility by triggering pathway(s) affecting Rho family GTPases.

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Rho-Family Small GTPases: From Highly Polarized Sensory Neurons to Cancer Cells

Cells ◽

10.3390/cells8020092 ◽

2019 ◽

Vol 8 (2) ◽

pp. 92 ◽

Cited By ~ 8

Author(s):

Takehiko Ueyama

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Small Gtpases ◽

In Vivo Studies ◽

Signal Pathways ◽

Ros Production ◽

Oxygen Species ◽

Rho Family Gtpases ◽

Rho Family

The small GTPases of the Rho-family (Rho-family GTPases) have various physiological functions, including cytoskeletal regulation, cell polarity establishment, cell proliferation and motility, transcription, reactive oxygen species (ROS) production, and tumorigenesis. A relatively large number of downstream targets of Rho-family GTPases have been reported for in vitro studies. However, only a small number of signal pathways have been established at the in vivo level. Cumulative evidence for the functions of Rho-family GTPases has been reported for in vivo studies using genetically engineered mouse models. It was based on different cell- and tissue-specific conditional genes targeting mice. In this review, we introduce recent advances in in vivo studies, including human patient trials on Rho-family GTPases, focusing on highly polarized sensory organs, such as the cochlea, which is the primary hearing organ, host defenses involving reactive oxygen species (ROS) production, and tumorigenesis (especially associated with RAC, novel RAC1-GSPT1 signaling, RHOA, and RHOBTB2).

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Regulators and Effectors of Small GTPases: Rho Family

10.1016/s0076-6879(06)x1068-6 ◽

2006 ◽

Keyword(s):

Small Gtpases ◽

Rho Family

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Properties of Washed Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649230 ◽

1977 ◽

Vol 37 (02) ◽

pp. 291-308 ◽

Cited By ~ 88

Author(s):

Raelene L Kinlough-Rathbone ◽

J Fraser Mustard ◽

Marian A Packham ◽

Dennis W Perry ◽

Hans-Joachim Reimers ◽

...

Keyword(s):

Major Effect ◽

Human Platelets ◽

Immune Serum ◽

Immune Serum Globulin ◽

Serum Globulin ◽

Platelet Adherence ◽

Low Concentrations ◽

Induced Aggregation ◽

Extensive Release ◽

Protein Free Medium

SummaryWe have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentrations of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A 23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.

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Deficiency of (33P)2MeS-ADP Binding Sites on Platelets with Secretion Defect, Normal Granule Stores and Normal Thromboxane A2 Production

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656090 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0986-0990 ◽

Cited By ~ 66

Author(s):

Marco Cattaneo ◽

Rossana Lombardi ◽

Maddalena L Zighetti ◽

Christian Gachet ◽

Philippe Ohlmann ◽

...

Keyword(s):

Binding Sites ◽

Specific Binding ◽

Thromboxane A2 ◽

Normal Subjects ◽

Congenital Defects ◽

Normal Production ◽

Adp Receptor ◽

Partial Deficiency ◽

Induced Aggregation ◽

Platelet Secretion

SummaryBy the term “Primary Secretion Defect” (PSD), we mean a common heterogeneous group of congenital defects of platelet secretion, characterized by a normal primary wave of platelet aggregation induced by ADP and other agonists, a normal concentration of platelet granule contents, and normal production of thromboxane A2. The biochemical abnormalities responsible for PSD are not well known. Since a secretion defect similar to PSD is found in platelets that are severely deficient of binding sites for the ADP analogue 2MeS-ADP and do not aggregate in response to ADP, we tested the hypothesis that PSD platelets have moderately decreased 2MeS-ADP binding sites, which may be sufficient for normal ADP-induced aggregation but not for potentiating platelet secretion. The specific binding of [33P]2MeS-ADP to platelets from 3 PSD patients (347,443 and 490 sites/platelet; KD 2.8-3.9 nM) was lower than to platelets from 24 normal subjects (647 [530-1102]; KD = 3.8 [2.3-7.3]) (median [range]). Normal values were found in a fourth PSD patient (710; KD 3.7). The degree of inhibition of PGE1- induced cAMP increase by 0.1 |μM ADP was lower in patients than in controls. The secretion induced by the endoperoxide analogue U46619 from normal, acetylsalicylic acid-treated platelets under conditions that prevented the formation of large aggregates was potentiated by 1 fimol/1 ADP and inhibited by apyrase. These findings indicate that a partial deficiency of the platelet ADP receptor(s) might be responsible for the defect of platelet secretion in some PSD patients and that ADP potentiates platelet secretion independently of the formation of large aggregates and thromboxane A2 production.

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