scholarly journals Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane

1994 ◽  
Vol 298 (3) ◽  
pp. 585-591 ◽  
Author(s):  
A Abo ◽  
M R Webb ◽  
A Grogan ◽  
A W Segal
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Molecular Mass ◽  
Binding Protein ◽  
Superoxide Production ◽  
Superoxide Generation ◽  
Gtp Binding Protein ◽  
Whole Cells ◽  
Gtp Binding ◽  
Gdp Dissociation Inhibitor

Activation of the NADPH oxidase of phagocytes involves the small GTP-binding protein p21rac. In this paper we report that neutrophil cytosol contains predominantly p21rac2 rather than p21rac1, and that the P21rac2 is almost entirely complexed with rhoGDI (GDP dissociation inhibitor) to form a heterodimer with a molecular mass of 45-50 kDa. Activation of superoxide production by phorbol 12-myristate 13-acetate or formylmethionyl-leucyl-phenylalanine in whole cells, and by SDS in the cell-free assay, led to the dissociation of some of the p21rac2 from rhoGDI and its movement to the plasma membrane together with p47phox and p67phox. The appearance of these proteins at the plasma membrane was related to the dose of the agonist and to the rate of superoxide generation. The nucleotide bound to p21rac2 in this complex following isolation was almost exclusively GDP, with less than 2% GTP, and the complex was active in the cell-free assay. Although the rac/GDI complex could activate the NADPH oxidase in the absence of exogenous GTP, the rate of superoxide production was increased 3-fold by the addition of GTP and was almost completely inhibited by GDP. Our findings confirm that rhoGDI serves as GDP dissociation inhibitor and that the release of p21rac2 from this inhibitor is an important step in activation of the NADPH oxidase.

scholarly journals Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

1992 ◽  
Vol 281 (2) ◽  
pp. 325-331 ◽  
Author(s):  
E Corvazier ◽  
J Enouf ◽  
B Papp ◽  
J de Gunzburg ◽  
A Tavitian ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Intracellular Membranes ◽  
Gtp Binding ◽  
Dependent Protein

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

scholarly journals Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

1994 ◽  
Vol 124 (1) ◽  
pp. 43-53 ◽  
Author(s):  
BP Jena ◽  
FD Gumkowski ◽  
EM Konieczko ◽  
GF von Mollard ◽  
R Jahn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Binding Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Apical Plasma Membrane ◽  
Gtp Binding Protein ◽  
Regulated Exocytosis ◽  
Gtp Binding

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

GTP analogues cause release of the alpha subunit of the GTP binding protein, GO, from the plasma membrane of NG108-15 cells

1988 ◽  
Vol 152 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Hayley McArdle ◽  
Ian Mullaney ◽  
Anthony Magee ◽  
Cecilia Unson ◽  
Graeme Milligan
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Alpha Subunit ◽  
Gtp Binding Protein ◽  
Gtp Binding

scholarly journals A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.

1991 ◽  
Vol 11 (5) ◽  
pp. 2909-2912 ◽  
Author(s):  
T Sasaki ◽  
K Kaibuchi ◽  
A K Kabcenell ◽  
P J Novick ◽  
Y Takai
Keyword(s):  
Exchange Reaction ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Constitutive Secretion ◽  
Gdp Dissociation Inhibitor ◽  
Exchange Protein

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.

A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast

Cell ◽  
1988 ◽  
Vol 53 (5) ◽  
pp. 753-768 ◽  
Author(s):  
Bruno Goud ◽  
Antti Salminen ◽  
Nancy C. Walworth ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Secretory Vesicles ◽  
Gtp Binding Protein ◽  
Gtp Binding

scholarly journals The small GTP-binding protein rab6 functions in intra-Golgi transport.

1994 ◽  
Vol 127 (6) ◽  
pp. 1575-1588 ◽  
Author(s):  
O Martinez ◽  
A Schmidt ◽  
J Salaméro ◽  
B Hoflack ◽  
M Roa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Wild Type ◽  
Gtp Binding Protein ◽  
Anterograde Transport ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Hemagglutinin Protein

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Géraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.

scholarly journals The inhibitory GTP-binding protein (Gi) occurs in rat Leydig cells and is differentially modified by lutropin and 12-O-tetradecanoylphorbol 13-acetate

1988 ◽  
Vol 253 (3) ◽  
pp. 895-899 ◽  
Author(s):  
E A Platts ◽  
D Schulster ◽  
B A Cooke
Keyword(s):  
Plasma Membrane ◽  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
Pertussis Toxin ◽  
Leydig Cells ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Subsequent Response

Luteinizing-hormone (LH)-stimulated cyclic AMP production in rat testis Leydig cells was desensitized by both LH and 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA, but not LH, enhanced the subsequent response to cholera toxin. Treatment of the cells with pertussis toxin potentiated cyclic AMP production in both control and LH-desensitized cells, but did not potentiate further the responses obtained by TPA pretreatment. The results implicate the presence of an inhibitory GTP-binding protein (Gi), which may be inhibited by TPA. The presence of a Gi-like protein within the plasma membrane of Leydig cells was demonstrated by pertussis-toxin-catalysed [32P]ADP-ribosylation of a Mr-40000-41000 protein.

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