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.

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.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

1997 ◽  
Vol 273 (1) ◽  
pp. F129-F135 ◽  
Author(s):  
J. M. Arthur ◽  
G. P. Collinsworth ◽  
T. W. Gettys ◽  
L. D. Quarles ◽  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Activation ◽  
Protein Activation ◽  
A Cell ◽  
Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

G-protein Activation Decreases Isoflurane Inhibition of N-type Ba2+Currents

2003 ◽  
Vol 99 (2) ◽  
pp. 392-399 ◽  
Author(s):  
Igor M. Nikonorov ◽  
Thomas J. J. Blanck ◽  
Esperanza Recio-Pinto
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Volatile Anesthetic ◽  
Cell Voltage ◽  
Dependent Manner ◽  
Protein Activation ◽  
G Protein Activation ◽  
Voltage Dependent

Background G-protein activation mediates inhibition of N-type Ca2+ currents. Volatile anesthetics affect G-protein pathways at various levels, and activation of G-proteins has been shown to increase the volatile anesthetic potency for inhibiting the electrical-induced contraction in ileum. The authors investigated whether isoflurane inhibition of N-type Ba2+ currents was mediated by G-protein activation. Methods N-type Ba2+ currents were measured in the human neuronal SH-SY5Y cell line by using the whole cell voltage-clamp method. Results Isoflurane was found to have two effects on N-type Ba2+ currents. First, isoflurane reduced the magnitude of N-type Ba2+ currents to a similar extent (IC50 approximately 0.28 mm) in the absence and presence of GDPbetaS (a nonhydrolyzable GDP analog). Interestingly, GTPgammaS (a nonhydrolyzable GTP analog and G-protein activator) in a dose-dependent manner reduced the isoflurane block; 120 microm GTPgammaS completely eliminated the block of 0.3 mm isoflurane and reduced the apparent isoflurane potency by approximately 2.4 times (IC50 approximately 0.68 mm). Pretreatment with pertussis toxin or cholera toxin did not eliminate the GTPgammaS-induced protection against the isoflurane block. Furthermore, isoflurane reduced the magnitude of voltage-dependent G-protein-mediated inhibition of N-type Ba2+ currents, and this effect was eliminated by pretreatment with pertussis toxin or cholera toxin. Conclusions It was found that activation of G-proteins in a neuronal environment dramatically reduced the isoflurane potency for inhibiting N-type Ba2+ currents and, in turn, isoflurane affected the G-protein regulation of N-type Ba2+ currents.

Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae

1996 ◽  
Vol 320 (2) ◽  
pp. 459-466 ◽  
Author(s):  
GRANT Karen R. ◽  
Margaret M. HARNETT ◽  
Graeme MILLIGAN ◽  
William HARNETT
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Eukaryotic Cells ◽  
Heterotrimeric G Proteins ◽  
Protein Subunits ◽  
Acanthocheilonema Viteae ◽  
Affinity Labelling ◽  
Α Subunits

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [α-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the β1-and/or β2-like subunits and α-subunits of the Gs, Gi, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

scholarly journals Pertussis Toxin-insensitive Activation of the Heterotrimeric G-proteins Gi/Goby the NG108-15 G-protein Activator

2002 ◽  
Vol 277 (52) ◽  
pp. 50223-50225 ◽  
Author(s):  
Catalina Ribas ◽  
Aya Takesono ◽  
Motohiko Sato ◽  
John D. Hildebrandt ◽  
Stephen M. Lanier
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Heterotrimeric G Proteins ◽  
Protein Activator

scholarly journals Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins

1997 ◽  
Vol 321 (3) ◽  
pp. 561-571 ◽  
Author(s):  
Timothy A. FIELDS ◽  
Patrick J. CASEY
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Biochemical Properties ◽  
Signalling Pathways ◽  
Heterotrimeric G Proteins ◽  
Kinase C ◽  
Regulate Cell Growth ◽  
Dependent Processes ◽  
Α Subunits

Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein α subunits of the Gi family, and this modification prevents the occurrence of the receptorŐG-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as ‘PTX-resistant’ due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivohave not been clearly established. Since two of these proteins, G12α and Gzα, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC.

Heterotrimeric G protein Giis involved in a signal transduction pathway for ATP release from erythrocytes

2004 ◽  
Vol 286 (3) ◽  
pp. H940-H945 ◽  
Author(s):  
Jeffrey J. Olearczyk ◽  
Alan H. Stephenson ◽  
Andrew J. Lonigro ◽  
Randy S. Sprague
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Atp Release ◽  
Signal Transduction Pathway ◽  
Protein G ◽  
Transduction Pathway ◽  
Heterotrimeric G Proteins ◽  
Heterotrimeric G Protein

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein Gsresulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the Gisubtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins Gαi1, Gαi2, and Gαi3but not Gαowere identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples Gi/ofrom their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 μM) or Mas-7 (1 μM), which are compounds that directly activate Giproteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 μM), which is an inactive Mas analog. In separate experiments, Mas (10 μM) but not Mas-17 (10 μM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein Giis a component of a signal transduction pathway for ATP release from erythrocytes.

The Ca2+-sensing receptor couples to Gα12/13 to activate phospholipase D in Madin-Darby canine kidney cells

2004 ◽  
Vol 286 (1) ◽  
pp. C22-C30 ◽  
Author(s):  
Chunfa Huang ◽  
Kristine M. Hujer ◽  
Zhenzhen Wu ◽  
R. Tyler Miller
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Phospholipase D ◽  
Pertussis Toxin ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gαi to inhibit the activity of adenylyl cyclase and activate ERK, Gαq to stimulate phospholipase C and phospholipase A2, and Gβγ to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Gα12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Gα12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796W as a negative control, prelabeled these cells with [3H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [3H]phosphatidylethanol (PEt). The formation of [3H]PEt increased in a time-dependent manner in the cells that overexpress the CaRWT but not the CaRR796W. Treatment of the cells with C3 exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Gα12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate Gαi or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gαi and Gαq, and a p115RhoGEF construct containing the RGS domain for Gα12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT inhibited extracellular Ca2+-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to Gα12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of Gαi and Gαq. This suggests that the CaR may regulate cytoskeleton via Gα12/13, Rho, and PLD.

scholarly journals Regulators of G protein Signaling (RGS) proteins (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Mohammed Alqinyah ◽  
Christopher Bodle ◽  
Josephine Bou Dagher ◽  
Bandana Chakravarti ◽  
Shreoshi P. Choudhuri ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
G Protein ◽  
G Proteins ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
G Protein Signalling ◽  
Rgs Domain

Regulators of G protein signalling (RGS) proteins display a common RGS domain that interacts with the GTP-bound Gα subunits of heterotrimeric G proteins, enhancing GTP hydrolysis by stabilising the transition state [29, 419, 418], leading to a termination of GPCR signalling. Interactions through protein:protein interactions of many RGS proteins have been identified for targets other than heteromeric G proteins. Sequence analysis of the 20 RGS proteins suggests four families of RGS: RZ, R4, R7 and R12 families. Many of these proteins have been identified to have effects other than through targetting G proteins. Included here is RGS4 for which a number of pharmacological inhibitors have been described.

scholarly journals Interactions of the α2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants

1997 ◽  
Vol 321 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Alan WISE ◽  
Marie-Ange WATSON-KOKEN ◽  
Stephen REES ◽  
Melanie LEE ◽  
Graeme MILLIGAN
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Resistant Mutant ◽  
Cysteine Residue ◽  
Gtpase Activity ◽  
Wild Type ◽  
High Affinity ◽  
Agonist Stimulation ◽  
Stimulation Of

The α2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of ‘Gi-like’ pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine α2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1α, Gi2α and Gi3α or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The α2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5ƀ-[γ-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein α-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1α, Gi2α and Gi3α resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys → Gly mutations of Gi1α, Gi2α and Gi3α were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1a was correlated highly both with the level of expression of this G-protein and with the level of expression of the α2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys → Gly mutants of Gi1α, Gi2α and Gi3α required 10Ő15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the α2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

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