scholarly journals Involvement of GTP-binding proteins in actin polymerization in human neutrophils.

1990 ◽  
Vol 87 (8) ◽  
pp. 2921-2925 ◽  
Author(s):  
T. Bengtsson ◽  
E. Sarndahl ◽  
O. Stendahl ◽  
T. Andersson
Keyword(s):  
Actin Polymerization ◽  
Binding Proteins ◽  
Human Neutrophils ◽  
Gtp Binding Proteins ◽  
Gtp Binding

scholarly journals Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton

2000 ◽  
Vol 347 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Juan A. ROSADO ◽  
Stewart O. SAGE
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Cytochalasin D ◽  
Dependent Manner ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Prenylated Proteins

We have investigated the mechanism of Ca2+ entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca2+ concentration ([Ca2+]i) in the presence, but not in the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca2+ entry evoked by the depletion of intracellular Ca2+ stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca2+ entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca2+ entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca2+ entry, indicating a cytoskeleton-independent component in the regulation of Ca2+ entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca2+ entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.

scholarly journals Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

1994 ◽  
Vol 126 (4) ◽  
pp. 1005-1015 ◽  
Author(s):  
J C Norman ◽  
L S Price ◽  
A J Ridley ◽  
A Hall ◽  
A Koffer
Keyword(s):  
Mast Cells ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Small Gtpases ◽  
Secretory Cells ◽  
Cortical Region ◽  
Permeabilized Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

scholarly journals Rac GTPase interacts specifically with phosphatidylinositol 3-kinase

1996 ◽  
Vol 315 (3) ◽  
pp. 775-779 ◽  
Author(s):  
Gary M. BOKOCH ◽  
Chris J. VLAHOS ◽  
Yan WANG ◽  
Ulla G. KNAUS ◽  
Alexis E. TRAYNOR-KAPLAN
Keyword(s):  
Growth Factor ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Specific Interaction ◽  
Bound State ◽  
Rac Gtpase ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Rho Family

The Rac GTP-binding proteins are members of the Rho family and regulate growth factor-stimulated actin assembly in a variety of cells. The formation of phosphorylated inositol lipids has been implicated in control of the processes initiating and regulating such actin polymerization. Associations of Rho family GTP-binding proteins with enzymes involved in lipid metabolism have been described. Here we demonstrate a direct and specific interaction of Rac proteins with phosphatidylinositol (PI) 3-kinase. This interaction is dependent upon Rac being in a GTP-bound state and requires an intact Rac effector domain. In contrast, direct binding of RhoA to PI 3-kinase could not be detected. Rac–GTP also bound to PI 3-kinase in Swiss 3T3 fibroblast and human neutrophil lysates, and increased PI 3-kinase activity became associated with Rac–GTP in platelet-derived growth factor-stimulated cells. Interaction of Rac–GTP with PI 3-kinase in vitro stimulated the activity of the enzyme by 2–9-fold. A specific interaction of active Rac with PI 3-kinase might be important in regulation of the actin cytoskeleton.

Subcellular distribution and characterization of GTP-binding proteins in human neutrophils

1990 ◽  
Vol 1054 (2) ◽  
pp. 237-245 ◽  
Author(s):  
Leili Khachatrian ◽  
Jeffrey B. Rubins ◽  
Eric C. Manning ◽  
David Dexter ◽  
Alfred I. Tauber ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Binding Proteins ◽  
Human Neutrophils ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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