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 Oocyte Maturation in Starfish

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 476
Author(s):  
Kazuyoshi Chiba
Keyword(s):  
Plasma Membrane ◽  
Oocyte Maturation ◽  
Binding Protein ◽  
Germinal Vesicle ◽  
Phosphatidylinositol 3 Kinase ◽  
Gtp Binding Protein ◽  
Germinal Vesicle Breakdown ◽  
Α Subunit ◽  
Gtp Binding ◽  
Dependent Protein

Oocyte maturation is a process that occurs in the ovaries, where an immature oocyte resumes meiosis to attain competence for normal fertilization after ovulation/spawning. In starfish, the hormone 1-methyladenine binds to an unidentified receptor on the plasma membrane of oocytes, inducing a conformational change in the heterotrimeric GTP-binding protein α-subunit (Gα), so that the α-subunit binds GTP in exchange of GDP on the plasma membrane. The GTP-binding protein βγ-subunit (Gβγ) is released from Gα, and the released Gβγ activates phosphatidylinositol-3 kinase (PI3K), followed by the target of rapamycin kinase complex2 (TORC2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-dependent phosphorylation of serum- and glucocorticoid-regulated kinase (SGK) of ovarian oocytes. Thereafter, SGK activates Na+/H+ exchanger (NHE) to increase the intracellular pH (pHi) from ~6.7 to ~6.9. Moreover, SGK phosphorylates Cdc25 and Myt1, thereby inducing the de-phosphorylation and activation of cyclin B–Cdk1, causing germinal vesicle breakdown (GVBD). Both pHi increase and GVBD are required for spindle assembly at metaphase I, followed by MI arrest at pHi 6.9 until spawning. Due to MI arrest or SGK-dependent pHi control, spawned oocytes can be fertilized normally

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 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.

134 Analysis of the role of small GTP binding protein Rab in intracellular melanosome transport

1996 ◽  
Vol 12 (2) ◽  
pp. 204
Author(s):  
K. Araki ◽  
T. Horikawa ◽  
K. Nakagawa ◽  
Y. Funasaka ◽  
M. Ichihashi
Keyword(s):  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding

Photoreceptor rod outer segment 48-kDa protein has ATPase activity

1990 ◽  
Vol 5 (6) ◽  
pp. 585-589 ◽  
Author(s):  
Ari Sitaramayya ◽  
Shereen Hakki
Keyword(s):  
Atpase Activity ◽  
Cyclic Gmp ◽  
Slow Rate ◽  
Binding Protein ◽  
Rod Outer Segments ◽  
Light Activation ◽  
Visual Transduction ◽  
Gtp Binding Protein ◽  
Gtp Binding

AbstractThe role of 48-kDa protein in Visual transduction remains unresolved. Two hypotheses for its role in quenching the light activation of cyclic GMP cascade suggest that the protein binds to either phosphodiesterase or phosphorylated rhodopsin. Since the protein is also reported to bind ATP, we anticipated that the protein may have ATP hydrolyzing activity, and in analogy with the GTP-binding protein of the rod outer segments, such activity may be greatly enhanced by the elements of transduction cyclic GMP cascade, permitting the protein to function cyclically as GTP-binding protein does. We found that purified 48-kDa protein hydrolyzes ATP but at a slow rate of 0.04–0.05 per min. The Km for ATP is about 45–65 μM. The activity is inhibited noncompetitively by ADP with a Ki of about 50 μM. The ATPase activity of 48-kDa protein is not affected by rhodopsin, bleached rhodopsin, phosphorylated rhodopsin, unactivated cyclic GMP phosphodiesterase, or phosphodiesterase (PDE) activated by GMP PNP-bound G-protein. These data show that although 48-kDa protein has ATPase activity, lack of regulation of this activity by the elements of visual transduction makes it unlikely for this activity to have a role in quenching the light activation of cyclic GMP cascade.

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 Evidence for regulation of human platelet adenylate cyclase by phosphorylation. Inhibition by ATP and guanosine 5′-[β-thio]diphosphate occur by distinct mechanisms

1991 ◽  
Vol 276 (3) ◽  
pp. 621-630 ◽  
Author(s):  
I A Wadman ◽  
R W Farndale ◽  
B R Martin
Keyword(s):  
Creatine Kinase ◽  
Adenylate Cyclase ◽  
Human Platelet ◽  
Binding Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Phosphoryl Transfer ◽  
Activation Process ◽  
Gtp Binding Protein ◽  
Gtp Binding

1. Incubation of human platelet membranes with guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5′-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5′-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.

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