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

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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Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae

Biochemical Journal ◽

10.1042/bj3200459 ◽

1996 ◽

Vol 320 (2) ◽

pp. 459-466 ◽

Cited By ~ 2

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.

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

Biochemical Journal ◽

10.1042/bj3210561 ◽

1997 ◽

Vol 321 (3) ◽

pp. 561-571 ◽

Cited By ~ 190

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00677.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. H940-H945 ◽

Cited By ~ 45

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.

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Regulation of renal cortical Na-HCO3 cotransporter. II. Role of G proteins

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.3.f461 ◽

1995 ◽

Vol 268 (3) ◽

pp. F461-F467 ◽

Cited By ~ 1

Author(s):

O. S. Ruiz ◽

Y. Y. Qiu ◽

L. J. Wang ◽

J. A. Arruda

Keyword(s):

Cholera Toxin ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Inhibitory Effect ◽

Adp Ribosylation ◽

Protein Activator ◽

The Difference ◽

22Na Uptake ◽

Gamma S

We examined the regulation of the renal cortical basolateral Na-HCO3 cotransporter by G proteins. Na-HCO3 cotransporter activity was measured in highly purified rabbit renal cortical basolateral membranes (BLMV) as the difference in 22Na uptake in presence of HCO3- and gluconate. HCO(3-)-dependent 22Na uptake was significantly inhibited by 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a G protein activator. In contrast, addition of 50 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), an inhibitor of G protein, prevented the inhibition of the Na-HCO3 cotransporter activity by GTP gamma S. AlF4-, another G protein activator, also inhibited the activity of the Na-HCO3 cotransporter. This inhibitory effect of G protein on the Na-HCO3 cotransporter activity was not prevented by dideoxyadenosine, an adenylate cyclase inhibitor, or by the protein kinase A inhibitor, suggesting a direct effect of G protein on the cotransporter. To identify the G proteins that mediate the regulation of the Na-HCO3 cotransporter, purified BLMV were ADP ribosylated in presence of cholera toxin or pertussis toxin. Autoradiograms of BLMV incubated with [32P]NAD showed that cholera and pertussis toxins caused ADP ribosylation of 42- and 41-kDa G proteins, respectively. To determine whether the ADP ribosylation by cholera or pertussis toxin was associated with alterations of the Na-HCO3 cotransporter activity, we measured HCO(3-)-dependent 22Na uptake in BLMV treated with 20 micrograms/ml cholera toxin or with 100 ng/ml pertussis toxin. Na-HCO3 cotransporter activity was significantly decreased by both cholera and pertussis toxins.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Membranes ◽

10.3390/membranes11030222 ◽

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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Signaling from G Protein-coupled Receptors to ERK5/Big MAPK 1 Involves Gαqand Gα12/13Families of Heterotrimeric G Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m002410200 ◽

2000 ◽

Vol 275 (28) ◽

pp. 21730-21736 ◽

Cited By ~ 67

Author(s):

Shigetomo Fukuhara ◽

Maria Julia Marinissen ◽

Mario Chiariello ◽

J. Silvio Gutkind

Keyword(s):

G Protein ◽

G Proteins ◽

G Protein Coupled Receptors ◽

Heterotrimeric G Proteins ◽

G Protein Coupled

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

Journal of Endocrinology ◽

10.1677/joe.0.1720095 ◽

2002 ◽

Vol 172 (1) ◽

pp. 95-104 ◽

Cited By ~ 24

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.

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Physiological Regulation of G Protein-Linked Signaling

Physiological Reviews ◽

10.1152/physrev.1999.79.4.1373 ◽

1999 ◽

Vol 79 (4) ◽

pp. 1373-1430 ◽

Cited By ~ 307

Author(s):

Andrew J. Morris ◽

Craig C. Malbon

Keyword(s):

G Protein ◽

G Proteins ◽

Chemokine Receptors ◽

Transcriptional Control ◽

Heterotrimeric G Proteins ◽

Protein Prenylation ◽

Physiological Control ◽

Physiological Regulation ◽

Surface Receptors ◽

Genes Encoding

Heterotrimeric G proteins in vertebrates constitute a family molecular switches that transduce the activation of a populous group of cell-surface receptors to a group of diverse effector units. The receptors include the photopigments such as rhodopsin and prominent families such as the adrenergic, muscarinic acetylcholine, and chemokine receptors involved in regulating a broad spectrum of responses in humans. Signals from receptors are sensed by heterotrimeric G proteins and transduced to effectors such as adenylyl cyclases, phospholipases, and various ion channels. Physiological regulation of G protein-linked receptors allows for integration of signals that directly or indirectly effect the signaling from receptor→G protein→effector(s). Steroid hormones can regulate signaling via transcriptional control of the activities of the genes encoding members of G protein-linked pathways. Posttranscriptional mechanisms are under physiological control, altering the stability of preexisting mRNA and affording an additional level for regulation. Protein phosphorylation, protein prenylation, and proteolysis constitute major posttranslational mechanisms employed in the physiological regulation of G protein-linked signaling. Drawing upon mechanisms at all three levels, physiological regulation permits integration of demands placed on G protein-linked signaling.

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Heterotrimeric G-Proteins Are Indispensable for FLT3-ITD autophosphorylation and Oncogenic Function

Blood ◽

10.1182/blood.v128.22.2712.2712 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2712-2712

Author(s):

Maike Rehage ◽

Susanne Wingert ◽

Nadine Haetscher ◽

Sabrina Bothur ◽

Hubert Serve ◽

...

Keyword(s):

Tyrosine Kinase ◽

G Protein ◽

G Proteins ◽

B Cell Lymphoma ◽

Physical Interaction ◽

Heterotrimeric G Proteins ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Oncogenic Function

Abstract Heterotrimeric G-proteins transmit signals of G-protein coupled receptors and regulate many basic cellular functions. However, their role in normal and malignant hematopoietic stem cells remains obscure. Activating mutations in the heterotrimeric G-protein Gaq were found in various cancers and its expression is enhanced in diffuse large B-cell lymphoma and T-ALL. Our previous data suggested the involvement of heterotrimeric G-proteins in Flt3-ITD-mediated leukemic transformation. FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is a frequent oncoprotein in acute myeloid leukemia causing constitutive active STAT5 signaling. Here, we investigated a novel role of Gaq in Flt3-ITD-induced leukemic transformation. We could show that Gaq is indispensable for aberrant FLT3-ITD activation and oncogenic function as Gaq activity is necessary to maintain the autophosphorylation of FLT3-ITD. Gaq abrogation resulted in diminished cell proliferation and colony formation as well as delayed leukemogenesis in vivo of Flt3-ITD leukemic cells. Importantly, the growth inhibition could be rescued by addition of IL3 and did not occur in the presence of FLT3 ligand-activated FLT3 wildtype receptor, demonstrating the specificity of Gaq requirement for FLT3-ITD oncogenic signaling. Interestingly, co-immunoprecipitations revealed a direct physical interaction between FLT3-ITD and Gaq which did not require phosphorylation of the receptor tyrosine kinase. Hence, FLT3-ITD hyperphosphorylation seems to be rather a consequence of the interaction than a prerequisite. Flt3-ITD-induced transformation of murine hematopoietic stem/progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 double knock-out mice delayed leukemic burden. These findings of an unexpected, yet critical, role of Gaq place the molecule as an important target for antileukemic strategies. Disclosures No relevant conflicts of interest to declare.

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