GTP-binding protein associated with amino acid binding proteins from olfactory epithelium of skate,Dasyatis pastinaca

FEBS Letters ◽  
1988 ◽  
Vol 234 (2) ◽  
pp. 471-474 ◽  
Author(s):  
V.I. Novoselov ◽  
L.D. Krapivinskaya ◽  
G.B. Krapivinsky ◽  
E.E. Fesenko
Keyword(s):  
Amino Acid ◽  
Olfactory Epithelium ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Amino Acid 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.

scholarly journals The Growth-Promoting and Stress Response Activities of the Bacillus subtilis GTP Binding Protein Obg Are Separable by Mutation

2008 ◽  
Vol 190 (20) ◽  
pp. 6625-6635 ◽  
Author(s):  
Shrin Kuo ◽  
Borries Demeler ◽  
W. G. Haldenwang
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Binding Protein ◽  
Normal Growth ◽  
Gtp Binding Protein ◽  
Missense Change ◽  
Gtp Binding ◽  
Growth Promoting ◽  
Carboxy Terminal

ABSTRACT Bacillus subtilis Obg is a ribosome-associating GTP binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (GSR). It is unclear whether the roles of Obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. The present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for Obg in each process. The first allele [obg(G92D)] encodes a missense change in the protein's highly conserved “obg fold” region. This mutation impairs cell growth and the ability of Obg to associate with ribosomes but fails to block sporulation or the induction of the GSR. The second obg mutation [obg(Δ22)] replaces the 22-amino-acid carboxy-terminal sequence of Obg with an alternative 26-amino-acid sequence. This Obg variant cofractionates with ribosomes and allows normal growth but blocks sporulation and impairs the induction of the GSR. Additional experiments revealed that the block on sporulation occurs early, preventing the activation of the essential sporulation transcription factor Spo0A, while inhibition of the GSR appears to involve a failure of the protein cascade that normally activates the GSR to effectively catalyze the reactions needed to activate the GSR transcription factor (σB).

scholarly journals Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 486-492 ◽  
Author(s):  
BA Miller ◽  
K Foster ◽  
JD Robishaw ◽  
CF Whitfield ◽  
L Bell ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Binding Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanosine Triphosphate ◽  
Gtp Binding Protein ◽  
Gtp Binding

Abstract Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat- inactivated PT. These observations provided strong evidence that a PT- sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein

1990 ◽  
Vol 10 (8) ◽  
pp. 4116-4122
Author(s):  
Y Matsui ◽  
A Kikuchi ◽  
S Araki ◽  
Y Hata ◽  
J Kondo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Reaction ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Bovine Brain ◽  
Subunit Structure ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Ras P21

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

A pertussis toxin-sensitive GTP-binding protein couples endothelin to phospholipase C in rat mesangial cells

1991 ◽  
Vol 260 (3) ◽  
pp. F347-F352
Author(s):  
C. P. Thomas ◽  
M. Kester ◽  
M. J. Dunn
Keyword(s):  
Phospholipase C ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Mesangial Cells ◽  
Binding Protein ◽  
Glomerular Mesangial Cells ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Gtp Binding ◽  
Stimulation Of

The mechanisms of stimulation of phospholipase C (PLC) by endothelin, specifically the role of guanine nucleotide-binding proteins (GTP-binding proteins) in coupling the endothelin receptor to PLC, were investigated in rat mesangial cells. Endothelin-1 (ET) synergistically released inositol polyphosphates in the presence of the stimulatory GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in permeabilized cells. In addition, in intact cells, pertussis toxin partially inhibited the stimulation of total inositol phosphates (IPn) by ET. Pertussis toxin also reduced the peak ET-stimulated intracellular free calcium level ([Ca2+]i) in these cells, both in the presence and absence of extracellular calcium. Pertussis toxin induced ADP ribosylation of a 41- to 43-kDa protein in mesangial cell membranes, and this effect was inhibited by prior exposure to ET and augmented by the inhibitory GDP analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). Thus a pertussis toxin-sensitive GTP-binding protein is involved in the activation of PLC by ET in glomerular mesangial cells.

THE ROLE OF INOSITIDES, PHOSPHOLIPASE C AND G-PROTEINS IN RECEPTOR TRANSDUCTION

1987 ◽  
Author(s):  
Eduardo G Lapetina
Keyword(s):  
Phospholipase C ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Cytosolic Phospholipase ◽  
Gtp Binding ◽  
Membrane Bound ◽  
Stimulation Of

It is now widely recognized that the activation of phospholipase C by specific agonists leads to the formation of two second messengers: (1) inositol trisphosphate, which releases Ca2+ from the endoplasmic reticulum to the cytosol and (2) 1,2- diacylglycerol, which stimulates protein kinase C. In the past few years, GTP-binding proteins have been associated with the regulation of phospholipase C. However, the identity of the GTP-binding protein involved and the type of association with phospholipase C is not yet known. It is now recognized that there are two types of phospholipase C enzymes: (a) a soluble enzyme that has been characterized in several tissues and does not preferentially hydrolyze polyphospholinositides and (b) membrane-bound enzymes that are coupled to the receptors, specifically hydrolyzing polyphosphoinositides and activated by membrane guanine nucleotide-binding proteins. Recent reports have tried to assess the involvement of GTP-binding proteins in the agonist-induced stimulation of phospholipase C, and various related aspects have been reported. These are concerned with: (a) detection of various GTP-binding proteins in platelets, (b) the effects of known inhibitors of GTP-binding proteins such as GDPgS or pertussis toxin on the agonist-induced stimulation of phospholipase C, (c) the direct effects of stimulators of GTP-binding proteins such as GTP, GTP-analogs and fluoride on phospholipase C activity, (d) the possible association of GTP-binding proteins to cytosolic phospholipase C that would then lead to degradation of the membrane-bound inositides and (e) cytosolic phospholipase C response to the activation of cell surface receptors. The emerging information has had contradictory conclusions. (1) Pretreatment of saponin-permeabilized platelets with pertussis toxin has been shown to enhance and to inhibit the thrombin-induced activation of phospholipase C. Therefore, it is not clear if a G protein that is affected by pertussis toxin in a manner similar to Gi or Go plays a central role in activation of phospholipase C. (2) Studies on the effect of GDPβ;S are also conflicting indicating that there may be GTP-independent and/or -dependent pathways for the activation of phosphoinositide hydrolysis. (3) A cytosolic phospholipase C is activated by GTP, and it has been advanced that this activity might trigger the hydrolysis of membrane-bound inositides. A cytosolic GTP-binding protein might be involved in this action, and it is speculated that an α-subunit might be released to the cytoplasm by a receptor-coupled mechanism to activate phospholipase C. However, no direct evidence exists to support this conclusion. Moreover, the exact contribution of phospholipase C from the membranes or the cytosol to inositide hydrolysis in response to cellular agonists and the relationship of those activites to membrane-bound or soluble GTP-binding proteins are unknown. Our results indicate that the stimulation of phospholipase C in platelets by GDPβS and thrombin are affected differently by GDPβS. GDPgSinhibits the formation of inositol phosphates produced by GTPγS but not that induced by thrombin. Thrombin, therefore, can directly stimulate phospholipase C without the involvement of a “stimulatory” GTP-binding protein, such as Gs, for the agonist stimulation of adenylate cyclase. However, an “inhibitory” GTP-binding protein might have some influence on thrombin-stimulated phospholipase C, since in the presence of GDPγS thrombin produces a more profound stimulation of phospholipase C.This “inhibitory” GTP-binding protein might be ADP-ribosylated by pertussis toxin because pertussis toxin can also enhance thrombin action on phospholipase C activity. Therefore, phospholipase C that responds to thrombin could be different from the one that responds to GTPγS. Cytosolic phospholipase C can be activated by GTP or GTP analogs, and the one that responds to thrombin should be coupled to the receptors present in the plasma membrane. The initial action of thrombin is to directly activate the plasma membrane-bound phospholipase C and the mechanism of this activation is probably related to the proteolytic action of thrombin or the activation of platelet proteases by thrombin. In agreement with this, trypsin can also directly activate platelet phospholipase C and, subsequently, GTPyS produces further activation of phospholipase C. If these two mechanisms are operative in platelets, the inhibition of cytosolic phospholipase C by GDPβS would allow a larger fraction of inositides for degradation of the thrombin-stimulated phospholipase C, as our results show.

scholarly journals 45-KDa GTP-binding Protein from Rat Olfactory Epithelium: Purification, Characterization and Localization

1996 ◽  
Vol 21 (2) ◽  
pp. 181-188 ◽  
Author(s):  
V.I. Novoselov ◽  
I.V. Peshenko ◽  
V.A. Evdokimov ◽  
J.V. Nikolaev ◽  
E.A. Matveeva ◽  
...  
Keyword(s):  
Olfactory Epithelium ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding

scholarly journals Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

1990 ◽  
Vol 10 (8) ◽  
pp. 4116-4122 ◽  
Author(s):  
Y Matsui ◽  
A Kikuchi ◽  
S Araki ◽  
Y Hata ◽  
J Kondo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Reaction ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Bovine Brain ◽  
Subunit Structure ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Ras P21

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

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