scholarly journals A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes

1994 ◽  
Vol 299 (2) ◽  
pp. 399-407 ◽  
Author(s):  
L A Berven ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Single Cells ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Inositol Polyphosphates ◽  
Gtp Binding ◽  
Inositol 1,4,5 Trisphosphate ◽  
Epidermal Growth

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or guanosine 5′-[beta gamma-imido]triphosphate to the cells, but not adenosine 5′-[gamma-thio]triphosphate (ATP[S]) or guanosine 5′-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.

Regulation of Na+-K+-ATPase activity in kidney proximal tubules: involvement of GTP binding proteins

1989 ◽  
Vol 256 (1) ◽  
pp. F57-F62 ◽  
Author(s):  
A. Bertorello ◽  
A. Aperia
Keyword(s):  
Atpase Activity ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
Proximal Tubules ◽  
Regulatory Proteins ◽  
Free Access ◽  
Gtp Binding

This study evaluates the involvement of GTP-dependent regulatory proteins (G-proteins) in the regulation of Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments. Single PCT segments were dissected from rat kidney and permeabilized to allow nucleotides and medium free access to the interior of the cell. A GDP analogue that blocks GTP-dependent activation of the G-protein, GDP beta S (400 microM) significantly inhibited PCT Na+-K+-ATPase activity when Na in the medium (Nam) was greater than or equal to 70 mM. The inhibition was attenuated when Nam was 55 and 35 mM and was no longer significant when Nam was 25 mM. GDP beta S had no inhibitory effect on the activity of purified Na+-K+-ATPase. A nonhydrolyzable GTP analogue, GppNHp (50 microM) significantly increased Na+-K+-ATPase activity when Nam was 25 and 35 mM, but not when Nam was 55-140 mM. Dopamine (DA) and DA1 plus DA2 agonists significantly inhibit Na+-K+-ATPase activity. DA inhibition was competitively abolished by GppNHp. In PCT segments from rats pretreated with pertussis toxin, DA and DA1 plus DA2 agonist inhibition of Na+-K+-ATPase activity was abolished. In PCT segments from rats pretreated with cholera toxin, basal Na+-K+-ATPase activity was increased, but DA significantly inhibited Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments is regulated via a G-protein that stimulates Na+-K+-ATPase activity and a DA-activated pertussis toxin-sensitive G-protein that inhibits Na+-K+-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein

1991 ◽  
Vol 280 (3) ◽  
pp. 753-760 ◽  
Author(s):  
T F J Martin ◽  
J E Lewis ◽  
J A Kowalchyk
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Gh3 Cells ◽  
Phospholipid Vesicles ◽  
Protein Tyrosine Phosphorylation ◽  
Gtp Binding ◽  
Gamma Subunits ◽  
Gh3 Cell

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.

scholarly journals Erratum

1991 ◽  
Vol 11 (4) ◽  
pp. 706-706
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Equilibrium State ◽  
Rat Brain ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Adp Ribosylation ◽  
Gtp Binding ◽  
Reconstituted System ◽  
Α Subunits

Ischemia of Rat Brain Decreases Pertussis Toxin-Catalyzed [32P] ADP Ribosylation of GTP-Binding Proteins (Gi1 and G0) in Membranes Katsunobu Takenaka, Yasunori Kanaho, Koh-ichi Nagata, Noboru Sakai, Hiromu Yamada, Yoshinori Nozawa [ Originally published in Journal of Cerebral Blood Flow and Metabolism 1991;11:155–160] On page 158 of the above, arrows were erroneously deleted from the equation in the following passage: Heterotrimers of G proteins that bind GDP to α subunits seem to be the preferred substrates for PTcatalyzed ADP ribosylation since guanine nucleotides (GDP and GTP) and 13'Y subunits stimulate ADP ribosylation in the reconstituted system and in membranes (Tsai et aI., 1984). These results indicate that the G proteins may exist at the equilibrium state as shown below: This omission was the result of a typesetting error, which the publisher regrets.

G-proteins in mammalian gametes: an immunocytochemical study

1988 ◽  
Vol 91 (1) ◽  
pp. 21-31
Author(s):  
N.B. Garty ◽  
D. Galiani ◽  
A. Aharonheim ◽  
Y.K. Ho ◽  
D.M. Phillips ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Cumulus Cells ◽  
Bovine Brain ◽  
Regulatory Proteins ◽  
Immunocytochemical Study ◽  
Cell Cytoplasm ◽  
Sperm Tail ◽  
Immunoreactive Material

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

scholarly journals Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production

2002 ◽  
Vol 172 (1) ◽  
pp. 95-104 ◽  
Author(s):  
AM Ronco ◽  
PF Moraga ◽  
MN Llanos
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Leydig Cells ◽  
Inhibitory Effect ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Camp Production

We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of PLA(2) and G proteins in the release of AA from hCG-stimulated Leydig cells, and under particular conditions, regulation of cAMP production by this fatty acid in these cells.

Immunoprecipitation of a pertussis toxin substrate of the Go family from rat islets of Langerhans

1992 ◽  
Vol 12 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Nicholas S. Berrow ◽  
Roger D. Hurst ◽  
Susan L. F. Chan ◽  
Noel G. Morgan
Keyword(s):  
Molecular Weight ◽  
Insulin Secretion ◽  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Islets Of Langerhans ◽  
Pertussis Toxin ◽  
Inhibitory Receptors ◽  
Rat Islets ◽  
Rat Islets Of Langerhans

Rat islets express a pertussis toxin sensitive G-protein involved in receptor-mediated inhibition of insulin secretion. This has been assumed previously to represent “Gi” which couples inhibitory receptors to adenylate cyclase. Incubation of islet G-proteins with32P-NAD and pertussis toxin resulted in the labelling of a band of molecular weight 40,000. This band was very broad and did not allow resolution of individual components. Incubation of the radiolabelled proteins with an anti-Go antiserum resulted in specific immunoprecipitation of a32P-labelled band. These results demonstrate that the complement of pertussis toxin sensitive G-proteins in rat islets includes Go.

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling

Development ◽  
2001 ◽  
Vol 128 (5) ◽  
pp. 645-654
Author(s):  
X. Shi ◽  
S. Amindari ◽  
K. Paruchuru ◽  
D. Skalla ◽  
H. Burkin ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Cytoplasmic Domain ◽  
Protein Activation ◽  
Acrosomal Exocytosis ◽  
G Protein Activation ◽  
Galactosyltransferase I

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.

Abscisic acid induction of GTP hydrolysis in maize coleoptile plasma membranes

1998 ◽  
Vol 25 (5) ◽  
pp. 539 ◽  
Author(s):  
Helen R. Irving
Keyword(s):  
Abscisic Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Gtpase Activity ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
Maize Coleoptile

Since receptor-coupled G proteins increase GTP hydrolysis (GTPase) activity upon ligands binding to the receptor, a study was undertaken to determine if abscisic acid (ABA) induced such an effect. Plasma membranes isolated from etiolated maize (Zea mays L.) coleoptiles were enriched in GTPase activity relative to microsomal fractions. Vanadate was included in the assay to inhibit the high levels of vanadate sensitive low affinity GTPases present. Under these conditions, GTPase activity was enhanced by Mg2+, stimulated by mastoparan, and inhibited by GTPγS indicating the presence of either monomeric or heterotrimeric G proteins. The combination of NaF and AlCl3 is expected to inhibit heterotrimeric G protein activity but had little effect on GTPase activity in maize coleoptile membranes. Cholera toxin enhanced basal GTPase activity, confirming the presence of heterotrimeric G proteins in maize plasma membranes. Pertussis toxin also slightly enhanced basal GTPase activity in maize membranes. Abscisic acid enhanced GTPase activity optimally at 5 mmol/L Mg2+ in a concentration dependent manner by 1.5-fold at 10 µmol/L and up to three-fold at 100 µmol/L ABA. Abscisic acid induced GTPase activity was inhibited by GTPγS, the combination of NaF and AlCl3, and pertussis toxin. Overall, these results are typical of a receptor-coupled G protein responding to its ligand.

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