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.

scholarly journals Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1α when constrained within an α2A-adrenoceptor-Gi1α fusion protein

1997 ◽  
Vol 325 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Alan WISE ◽  
I. Craig CARR ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Pertussis Toxin ◽  
Gtpase Activity ◽  
Α Subunit ◽  
Fusion Construct ◽  
High Affinity ◽  
C Terminus ◽  
Adrenoceptor Agonist ◽  
Stimulation Of

A fusion protein was generated between the porcine α2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351 → Gly) variant of the α subunit of Gi1α by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the α2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min-1 for Gi1α when the α2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37±0.07 μM. Co-expression of excess β1γ2 along with the α2A-adrenoceptor-Gi1α fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1±0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with βγ complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.

scholarly journals Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and α2C10 adrenoceptor-transfected Rat 1 fibroblasts

1994 ◽  
Vol 298 (2) ◽  
pp. 493-497 ◽  
Author(s):  
C Carr ◽  
M Grassie ◽  
G Milligan
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Lysophosphatidic Acid ◽  
Gtpase Activity ◽  
Response Curves ◽  
Protein Activation ◽  
Adp Ribosylation ◽  
High Affinity ◽  
Stimulation Of

Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 microM LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a Gi-like G-protein. LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor. The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells. LPA was also able to promote cholera toxin-catalysed [32P]ADP-ribosylation of Gi. This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin. LPA-stimulated cholera toxin-catalysed [32P]ADP-ribosylation of Gi in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304. Dose-response curves for LPA in the two assays of G-protein activation were coincident. The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of Gi.

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.

Phospholipase D is activated by G protein and not by calcium ions in vascular smooth muscle

1996 ◽  
Vol 270 (3) ◽  
pp. H1031-H1037
Author(s):  
E. F. LaBelle ◽  
R. M. Fulbright ◽  
R. J. Barsotti ◽  
H. Gu ◽  
E. Polyak
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
G Protein ◽  
Phospholipase D ◽  
Pertussis Toxin ◽  
Alpha Toxin ◽  
Tail Artery ◽  
Agonist Stimulation ◽  
Rat Tail ◽  
Stimulation Of

We assessed the sensitivity of phospholipase D (PLD) activity in vascular smooth muscle to cytosolic Ca2+ by increasing cytosolic Ca2+ levels independently of agonist stimulation. When rat tail artery was preloaded with the Ca2+ indicator fluo 3 pentaacetoxymethyl ester, the addition of high extracellular K+, caffeine, or norepinephrine rapidly enhanced cytosolic Ca2+ levels. Neither increased extracellular K+ nor caffeine addition increased phosphatidylethanol production, indicating that cytosolic Ca2+ elevation alone did not stimulate PLD. In contrast, norepinephrine stimulated phosphatidylethanol production in this tissue. In strips of tail artery permeabilized with alpha-toxin and incubated in solutions containing free Ca2+ concentrations observed during physiological stimulation (pCa 6.4), PLD was not stimulated, whereas incubation with guanosine 5'-O-(3-thiotriphosphate) at pCa 7.0 activated this enzyme. Aluminum fluoride (AlF4-) stimulated PLD, and this activity was insensitive to pertussis toxin after stimulation by either norepinephrine or AlF4-. These results indicate that PLD in vascular smooth muscle is activated by norepinephrine via stimulation of a pertussis toxin-insensitive G protein and not via an increase in intracellular Ca2+ levels.

scholarly journals Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins

1988 ◽  
Vol 251 (1) ◽  
pp. 201-205 ◽  
Author(s):  
S J Butler ◽  
E C H Kelly ◽  
F R McKenzie ◽  
S B Guild ◽  
M J O Wakelam ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Hybrid Cell ◽  
Calf Serum ◽  
Opioid Peptide ◽  
Individual Species ◽  
Foetal Calf Serum ◽  
Maximal Velocity ◽  
Gtpase Activity ◽  
High Affinity

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.

scholarly journals Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates

1988 ◽  
Vol 249 (3) ◽  
pp. 653-659 ◽  
Author(s):  
F R McKenzie ◽  
E C H Kelly ◽  
C G Unson ◽  
A M Spiegel ◽  
G Milligan
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
High Affinity ◽  
C Terminus ◽  
Inhibitory G Protein ◽  
Guanine Nucleotide Binding

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.

scholarly journals δ-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2

1990 ◽  
Vol 267 (2) ◽  
pp. 391-398 ◽  
Author(s):  
F R McKenzie ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Opioid Receptor ◽  
Protein S ◽  
Guanine Nucleotide ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
High Affinity ◽  
Guanine Nucleotide Binding

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.

scholarly journals Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins

1999 ◽  
Vol 342 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Chee Wai FONG ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
G Proteins ◽  
Fusion Proteins ◽  
Hek293 Cells ◽  
Gtpase Activity ◽  
Prostanoid Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Stimulation Of

Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with Gsα. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the α subunits of its cognate G-protein Gs, Gi1, a G-protein which it fails to activate in co-expression studies, and a chimaeric Gi1-Gs6 (a form of Gi1 in which the C-terminal six amino acids were replaced with the equivalent sequence of Gs) were stably expressed in HEK293 cells. These were detected by [3H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-Gsα fusion protein, and both constructs were shown to interact with and activate endogenously expressed Gsα. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-Gi1α fusion. However, the fusion proteins containing either Gsα or Gi1-Gs6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-Gi1-Gs6α fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the Gsα-containing fusion protein and a 9-fold improvement when using the fusion protein containing Gi1-Gs6α to detect G-protein activation compared with expression of the isolated receptor.

scholarly journals Gi3 does not contribute to the inhibition of adenylate cyclase when stimulation of an α2-adrenergic receptor causes activation of both Gi2 and Gi3

1992 ◽  
Vol 284 (2) ◽  
pp. 565-568 ◽  
Author(s):  
S J McClue ◽  
E Selzer ◽  
M Freissmuth ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
G Proteins ◽  
Adrenergic Receptor ◽  
Genomic Dna ◽  
Gtpase Activity ◽  
Adrenergic Agonists ◽  
Dna Encoding ◽  
Receptor Stimulation ◽  
High Affinity ◽  
Stimulation Of

Agonist occupancy of the alpha 2-C10 adrenergic receptor in a stable clone (1C) of Rat 1 fibroblasts produced by transfection of cells with genomic DNA encoding this receptor causes the activation of both of the pertussis-toxin-sensitive G-proteins Gi2 and Gi3 [Milligan, Carr, Gould, Mullaney & Lavan (1991) J. Biol. Chem. 266, 6447-6455]. An IgG fraction from an antiserum (I3B) which identifies the C-terminal decapeptide of Gi3 alpha only was able to inhibit partially receptor stimulation of high-affinity GTPase activity. An equivalent fraction from an antiserum (AS7) able to identify the C-terminal decapeptide of Gi1 alpha + Gi2 alpha, but not Gi3 alpha, was also able to inhibit partially receptor stimulation of GTPase activity, and the effects of the two antisera were additive. By contrast, agonist-mediated inhibition of forskolin-amplified adenylate cyclase activity was abolished completely by the IgG fraction of antiserum AS7, but was not decreased by treatment with antiserum 13B. Based on the proportion of agonist-stimulated high-affinity GTPase which was prevented by each antiserum and on the measured membrane levels of Gi2 and Gi3, calculations indicated that essentially all of the cellular Gi3, but only 15% of the available Gi2, can be activated by the alpha 2-C10 adrenergic receptor in these cells. These results demonstrate that, although Gi3 is activated by alpha 2-adrenergic agonists in membranes of clone 1C cells, it does not contribute to the transduction of receptor-mediated inhibition of adenylate cyclase.

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