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.

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.

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 Plant hormone perception and action: a role for G–protein signal transduction?

1998 ◽  
Vol 353 (1374) ◽  
pp. 1425-1430 ◽  
Author(s):  
Richard Hooley
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Plant Hormone ◽  
Higher Plants ◽  
Signalling Pathways ◽  
Heterotrimeric G Proteins ◽  
Extracellular Signals ◽  
G Protein Signalling ◽  
Auxin Signalling

Plants perceive and respond to a profusion of environmental and endogenous signals that influence their growth and development. The G–protein signalling pathway is a mechanism for transducing extracellular signals that is highly conserved in a range of eukaryotes and prokaryotes. Evidence for the existence of G–protein signalling pathways in higher plants is reviewed, and their potential involvement in plant hormone signal transduction evaluated. A range of biochemical and molecular studies have identified potential components of G–protein signalling in plants, most notably a homologue of the G–protein coupled receptor superfamily ( GCR1 ) and the G α and G β subunits of heterotrimeric G–proteins. G–protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate heterotrimeric G–proteins in gibberellin and possibly auxin signalling. Antisense suppression of GCR1 in Arabidopsis leads to a phenotype which supports a role for this receptor in cytokinin signalling. These observations suggest that higher plants have at least some of the components of G–protein signalling pathways and that these might be involved in the action of certain plant hormones.

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

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.

scholarly journals Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins

1996 ◽  
Vol 320 (1) ◽  
pp. 87-91 ◽  
Author(s):  
Jin-Lin WANG ◽  
Somasundaram KALYANARAMAN ◽  
Michael DE VIVO ◽  
Narasimhan GAUTAM
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Signalling Pathways ◽  
3T3 Cells ◽  
Atpase Inhibitor ◽  
Protein Subunit ◽  
Stable Transfectants ◽  
Bombesin Receptor ◽  
Nih 3T3

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca2+-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca2+-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type αq showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.

scholarly journals Biochemical characterization of RGS14: RGS14 activity towards G-protein α subunits is independent of its binding to Rap2A

2006 ◽  
Vol 394 (1) ◽  
pp. 309-315 ◽  
Author(s):  
Vivek Mittal ◽  
Maurine E. Linder
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Biochemical Characterization ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Subunit Dissociation ◽  
Α Subunits

RGS (regulators of G-protein signalling) modulate signalling by acting as GAPs (GTPase-activating proteins) for α subunits of heterotrimeric G-proteins. RGS14 accelerates GTP hydrolysis by Giα family members through its RGS domain and suppresses guanine nucleotide dissociation from Giα1 and Giα3 subunits through its C-terminal GoLoco domain. Additionally, RGS14 binds the activated forms of the small GTPases Rap1 and Rap2 by virtue of tandem RBDs (Raf-like Ras/Rap binding domains). RGS14 was identified in a screen for Rap2 effectors [Traver, Splingard, Gaudriault and De Gunzburg (2004) Biochem. J. 379, 627–632]. In the present study, we tested whether Rap binding regulates RGS14's biochemical activities. We found that RGS14 activity towards heterotrimeric G-proteins, as either a GAP or a GDI (guanine nucleotide dissociation inhibitor), was unaffected by Rap binding. Extending our biochemical characterization of RGS14, we also examined whether RGS14 can suppress guanine nucleotide exchange on Giα1 in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated Giα1 and prenylated Gβγ is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free Gα and suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of Gβγ-mediated signalling pathways.

Guanine-nucleotide binding regulatory proteins as targets for novel drugs

1992 ◽  
Vol 99 (1-2) ◽  
pp. 27-36 ◽  
Author(s):  
N. J. Pyne
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Guanine Nucleotide ◽  
Kinase C ◽  
Pde Inhibitors ◽  
Guanine Nucleotide Binding

Summary:Guanine-nucleotide regulatory binding proteins (G-proteins) serve to transduce information from agonist-bound receptor complexes to either effector enzymes or ion-channels. Drugs that perturb the function of G-proteins may do so by one of four mechanisms, (i) They may exert negative intrinsic activity toward the G-protein. For instance, we have shown that incubation of isolated plasma-membranes with the β-adrenoceptor blocking drug sotalol blocked both GTP-stimulated and isoprenaline-stimulated adenylyl cyclase. This suggests that the empty β-adrenoceptor is capable of tonically stimulating Gsα, and therefore adenylyl cyclase; that is, empty β-adrenoceptors promote GDP-GTP exchange, (ii) They may perturb the GDP-GTP exchange reaction. For instance, certain PDE inhibitors, including SKF 94836 and rolipram, stimulate a marked increase in the pertussis toxin-catalysed NAD+-dependent ADP-ribosylation of Giα. This effect is similar to that of GDP, which promotes stabilisation of the αβγ holomer of Gi. The effect of these PDE inhibitors is completely reversed by GppNHp, which triggers afly dissociation by binding to the guanine-nucleotide binding domain of the G-protein. PDE inhibitors may serve as a class of drugs which perturb GDP-GTP exchange, (iii) They may trigger uncoupling of receptor-G-protein complexes. For instance, the polycationic drug mastoparan binds to the C-terminal end of the G-protein and mimics the effect of receptor activation by promoting GTP-γ-S binding, a reduction in pertussis toxin-catalysed ADP-ribosylation, and inhibition of adenylyl cyclase activity. Other agents, such as polyanionic drugs, bind to the receptor to promote uncoupling of receptor-mediated activation of certain G-proteins. (iv) They may alter the cross-talk mechanisms that operate between different receptor signalling systems. For instance, protein kinase C promotes the phosphorylation and inactivation of Gi. This leads to an unopposed stimulation of adenylyl cyclase via Gs and, therefore, enhanced sensitivity to agents such as glucagon. Protein kinase C inhibitors may be usefully exploited to modulate these processes which appear to be abberant in certain disease states.

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

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