NOVEL GTP-BINDING PROTEINS OF CYTOSOLIC AND MEMBRANE FRACTIONS OF HUMAN PLATELETS

1987 ◽  
Author(s):  
Eduardo G Lapetina ◽  
Bryan R Reep ◽  
Luis Molina Y Vedia
Keyword(s):  
Phospholipase C ◽  
Binding Proteins ◽  
Binding Protein ◽  
Human Platelets ◽  
Trypsin Treatment ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Sds Page ◽  
Membrane Bound

We have assessed the binding of (α-32P)GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with (α-32p)GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP or GTPyS and by 100 nM GDP; binding was unaffected by 1 nM-1 μM ATP. One main GTP-binding protein (29.5 KDa) was detected in the membrane fraction while three others (29, 27, and 21 KDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 KDa) were degraded by trypsin; another cytosolic protein (21 KDa) and the membrane-bound protein (29.5 KDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of (α-32P)GTP to the membrane-bound protein. GTPyS still stimulates phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTPyS. We have started to purify the membrane-bound, trypsin-resistant, GTP-binding protein. Purification includes 1 M NaCl extraction and the use of an FPLC system with successive phenyl superose and superose 12 columns.

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.

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.

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.

scholarly journals Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-β and GTP-binding protein Gq

1993 ◽  
Vol 291 (1) ◽  
pp. 235-240 ◽  
Author(s):  
J J Baldassare ◽  
A P Tarver ◽  
P A Henderson ◽  
W M Mackin ◽  
B Sahagan ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Binding Proteins ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Phosphoinositide Hydrolysis ◽  
Arachidonic Acid Metabolite ◽  
Thromboxane A2 Receptor ◽  
Gtp Binding Proteins ◽  
Phosphoinositide Breakdown ◽  
Gtp Binding

Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through phosphoinositide-specific phospholipase C-catalysed hydrolysis of phosphoinositides. We have established conditions to reconstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated phosphoinositide hydrolysis was observed in the presence of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced phosphoinositide hydrolysis in human platelets.

scholarly journals Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+

1994 ◽  
Vol 301 (1) ◽  
pp. 41-47 ◽  
Author(s):  
M M L Davidson ◽  
R J Haslam
Keyword(s):  
Binding Protein ◽  
Human Platelets ◽  
Dense Granule ◽  
Actin Binding ◽  
Light Chains ◽  
Ionophore A23187 ◽  
Peptide Fragments ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Sds Page

In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.

scholarly journals Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and βγ-subunits of GTP-binding proteins

1996 ◽  
Vol 317 (2) ◽  
pp. 475-480 ◽  
Author(s):  
Taro OKADA ◽  
Osamu HAZEKI ◽  
Michio UI ◽  
Toshiaki KATADA
Keyword(s):  
Binding Proteins ◽  
Kinase Activity ◽  
Binding Protein ◽  
Combined Treatment ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Phosphorylated Peptide ◽  
Stimulation Of

Stimulation of differentiated THP-1 cells by insulin led to rapid accumulation of PtdIns(3,4,5)P3, a product of PtdIns 3-kinase. Stimulation of the GTP-binding-protein-linked receptor by N-formylmethionyl-leucyl-phenylalanine (fMLP) also induced the accumulation of PtdIns(3,4,5)P3 in the cells. The effect of insulin was, while that of fMLP was not, accompanied by increased PtdIns 3-kinase activity in the anti-phosphotyrosine immunoprecipitate. The combination of insulin and fMLP induced more PtdIns(3,4,5)P3 production than the sum of the individual effects. The insulin-induced recruitment of PtdIns 3-kinase activity in the anti-phosphotyrosine immunoprecipitate was unaffected by the combined treatment with fMLP. To investigate the mechanism underlying the synergistic accumulation of PtdIns(3,4,5)P3, we separated the cytosolic proteins of THP-1 cells on a Mono Q column. PtdIns 3-kinase activities were eluted in two peaks, and one of the peaks markedly increased on the addition of βγ-subunits of GTP-binding proteins (Gβγ). The other peak was affected only slightly by Gβγ, but was synergistically increased by Gβγ and a tyrosine-phosphorylated peptide which was synthesized according to the amino acid sequence of insulin receptor substrate-1. The activity in the latter fraction was completely immunoprecipitated by an antibody against the regulatory subunit of PtdIns 3-kinase (p85). These results suggest that the conventional PtdIns 3-kinase (p85/p110), which has been implicated in insulin-induced cellular events, or a closely related isoenzyme is controlled by a combination of a tyrosine-phosphorylated protein and a GTP-binding protein in intact cells.

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