scholarly journals Concurrent down-regulation of IP prostanoid receptors and the α-subunit of the stimulatory guanine-nucleotide-binding protein (Gs) during prolonged exposure of neuroblastoma × glioma cells to prostanoid agonists. Quantification and functional implications

1992 ◽  
Vol 285 (2) ◽  
pp. 529-536 ◽  
Author(s):  
E J Adie ◽  
I Mullaney ◽  
F R McKenzie ◽  
G Milligan
Keyword(s):  
Membrane Protein ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Prolonged Exposure ◽  
Specific Binding ◽  
Down Regulation ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Prostanoid Receptor ◽  
Gs Alpha

Neuroblastoma x glioma hybrid NG108-15 cells express a high-affinity IP prostanoid receptor. Saturation binding analysis of this receptor, using [3H]prostaglandin E1 ([3H]PGE1) as ligand, indicated that it was present at some 1.5 pmol/mg of membrane protein and displayed a dissociation constant for this ligand of 30-40 nM. Prolonged exposure of these cells either to PGE1 or to iloprost, which is a stable analogue of prostacyclin, caused a 40-70% decrease in levels of the receptor. The remaining receptors were capable of interacting with the stimulatory G-protein (Gs) of the adenylate cyclase cascade, as saturation analysis of the binding of [3H]PGE1 indicated that they had a similar affinity for the 3H-labelled ligand, and because the specific binding of [3H]PGE1 to these receptors was still sensitive to the presence of poorly hydrolysed analogues of GTP. We have recently demonstrated that prolonged exposure of NG108-15 ells to PGE1 causes a cyclic AMP-independent loss of Gs alpha-subunit (Gs alpha) from these cells [McKenzie & Milligan (1990) J. Biol. Chem. 265, 17084-17093]. Steady-state concentration of the larger 45 kDa form of Gs alpha (which is the predominant form expressed in these cells) was assessed to be 9.6 pmol/mg of membrane protein, and treatment with iloprost decreased levels of this polypeptide to some 3.0 pmol/mg of protein. Time courses of iloprost-mediated down-regulation of the IP prostanoid receptor, loss of Gs alpha protein as assessed by immunoblotting and loss of Gs alpha activity as assessed by the reconstitution of NaF stimulation of adenylate cyclase activity to membranes of S49 cyc- cells by sodium cholate extracts of NG108-15 cells were identical, suggesting that the loss of the IP prostanoid receptor and G-protein occurred in parallel. Each of these effects was half-maximal between 2 and 3 h of exposure to the agonist. Stoichiometry of loss of Gs alpha and IP prostanoid receptor was unchanged by the percentage receptor occupancy, and quantification indicated the loss of some 7-10 mol of Gs alpha/mol of receptor. This is the first report to demonstrate the temporal concurrence of loss of Gs alpha and of a receptor which interacts with this G-protein. Chronic activation of the IP prostanoid receptor on these cells results in the development of a heterologous form of desensitization to agents which function to activate adenylate cyclase [Kelly, Keen, Nobbs & MacDermot (1990) Br. J. Pharmacol. 99, 306-316]. Agonist regulation of Gs alpha levels in these cells may contribute to this process.

scholarly journals Agonist regulation of cellular Gs α-subunit levels in neuroblastoma × glioma hybrid NG108-15 cells transfected to express different levels of the human β 2 adrenoceptor

1994 ◽  
Vol 300 (3) ◽  
pp. 709-715 ◽  
Author(s):  
E J Adie ◽  
G Milligan
Keyword(s):  
Membrane Protein ◽  
G Protein ◽  
Response Curves ◽  
Down Regulation ◽  
Α Subunit ◽  
Prostanoid Receptor ◽  
Secretin Receptor ◽  
Stimulatory G Protein ◽  
The Individual ◽  
Gs Alpha

Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.

scholarly journals Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma × glioma hybrid cells is due to a toxin-induced decrease in opioid receptor levels

1991 ◽  
Vol 275 (1) ◽  
pp. 175-181 ◽  
Author(s):  
F R McKenzie ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
G Protein ◽  
Opioid Receptor ◽  
Alpha Subunit ◽  
Intrinsic Activity ◽  
Delta Opioid Receptor ◽  
Down Regulation ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding

Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5′-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and ‘Gi’. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.

Nitric oxide donor SIN-1 mediated down-regulation of the G-protein α-subunit in C6 glioma cells

Life Sciences ◽  
1997 ◽  
Vol 60 (15) ◽  
pp. 1279-1285 ◽  
Author(s):  
L.Trevor Young ◽  
Caroline M. Woods ◽  
Jun-Feng Wang ◽  
Vida Asghari
Keyword(s):  
Nitric Oxide ◽  
G Protein ◽  
Glioma Cells ◽  
C6 Glioma ◽  
Nitric Oxide Donor ◽  
C6 Glioma Cells ◽  
Down Regulation ◽  
Α Subunit ◽  
G Protein Α Subunit

G PROTEIN REGULATORS OF PHOSPHOLIPASE C AND ADENYLATE CYCLASE IN PLATELETS

1987 ◽  
Author(s):  
L F Brass ◽  
D R Manning ◽  
M J Woolkalis
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Protein Band ◽  
2D Electrophoresis ◽  
Guanine Nucleotide Binding Protein ◽  
Pi Hydrolysis ◽  
Camp Formation

The hydrolysis of polyphosphoinositides (PI) by phospholipase C during platelet activation produces two key intracellular messengers, inositol triphosphate and diacylglycerol. This process is thought to be regulated by a guanine nucleotide binding protein referred to as Gp. Although the evidence that Gp exists is compelling, to date it has not been isolated. Uncertainty about its identity has been compounded by variations between tissues in the susceptibility of Gp to pertussis toxin and by reconstitution studies which show that pertussis toxin-inhibited PI hydrolysis can be restored by purified Gi, the pertussis toxin-sensitive G protein which inhibits adenylate cyclase. Therefore, it remains unclear whether Gp represents a new G protein or a second role for Gj. When platelets permeabilized with saponin were incubated with pertussis toxin and 32P-NAD, a single 42 kDa protein was 32P-ADP-ribosylated which co-migrated with the purified a subunit of Gi. Preincubating the platelets with an agonist inhibited labeling of this protein by dissociating the G protein into subunits. The extent of inhibition correlated with the number of toxin-sensitive functions caused by the agonist. Labeling was abolished by thrombin, which inhibited cAMP formation and caused toxin-inhibitable PI hydrolysis. Labeling was partially inhibited by vasopressin and platelet activating factor, which caused toxin-inhibitable PI hydrolysis, but had no effect on cAMP formation and by epinephrine, which inhibited cAMP formation, but did not cause PI hydrolysis. Labeling was unaffected by the TxA2 analog U46619, which neither caused toxin-sensitive PI hydrolysis nor inhibited cAMP formation. These observations suggest that the 42 kDa band may contain a subunits from both Gp and Gi and, in fact, 2D electrophoresis resolved the 42 kDa protein band into two proteins with distinct pi. However, those agonists linked functionally only to Gp or only to Gi decreased the labeling of both proteins. Therefore, our data suggest (1) that Gj and Gp are the same protein and (2) that whether a aiven platelet agonist affects adenylate cyclase or phospholipase C or both depends upon factors extrinsic to the G protein.

scholarly journals Primary structure of the α-subunit of bovine adenylate cyclase-inhibiting G-protein deduced from the cDNA sequence

FEBS Letters ◽  
1986 ◽  
Vol 197 (1-2) ◽  
pp. 305-310 ◽  
Author(s):  
Toshihide Nukada ◽  
Tsutomu Tanabe ◽  
Hideo Takahashi ◽  
Masaharu Noda ◽  
Kazuko Haga ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Primary Structure ◽  
Cdna Sequence ◽  
Α Subunit

Visualizing Signal Transduction: Receptors, G-Proteins, and Adenylate Cyclases

1996 ◽  
Vol 91 (5) ◽  
pp. 527-537 ◽  
Author(s):  
Carmen W. Dessauer ◽  
Bruce A. Posner ◽  
Alfred G. Gilman
Keyword(s):  
Signal Transduction ◽  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Crystallographic Analysis ◽  
Α Subunit ◽  
Signalling Network ◽  
Great Insight ◽  
Guanine Nucleotide Binding ◽  
Retinal Cgmp Phosphodiesterase

1. The first glimpses of heterotrimeric G-proteins came with the discoveries of the ubiquitous adenylate cyclase activator, Gs, and the specialized retinal cGMP phosphodiesterase activator, Gt or transducin. The model that evolved for regulation of adenylate cyclase activity by G-proteins soon proved to be a general paradigm for a large number of signalling pathways. Although many different G-proteins interact with a diverse array of receptors and effectors, each is composed of a guanine-nucleotide-binding α-subunit and a tightly associated complex of a β- and a γ-subunit. 2. Receptors catalyse the activation of G-proteins by promoting exchange of GDP for GTP, while G-proteins catalyse their own deactivation as a result of their intrinsic GTPase activity. Crystallographic analysis has described several of the various conformational states that G-proteins undergo as they are activated and deactivated and has provided great insight into the kinetic models of G-protein-mediated signal transduction. 3. The regulation of adenylate cyclase has proven to be intriguing and complex. Gsα activates all forms of mammalian adenylate cyclase; other G-proteins (Gi, Go and Gz) inhibit certain isoforms of the enzyme. The discovery of new isoforms of adenylate cyclase has revealed synergistic and conditional mechanisms of regulation. These include activation or inhibition by the G-protein βγ-subunit complex, activation by Ca2+-calmodulin, and phosphorylation by protein kinases. The large number of receptors, G-proteins and adenylate cyclases provides a complex signalling network that integrates and interprets a multitude of convergent inputs.

scholarly journals Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by βγ-subunits and somatostatin in S49 lymphoma cell membranes

1991 ◽  
Vol 280 (2) ◽  
pp. 303-307 ◽  
Author(s):  
L A Ransnäs ◽  
D Leiber ◽  
P A Insel
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Cell Membranes ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Subunit Dissociation ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.

scholarly journals The effect of iloprost on the ADP-ribosylation of Gsα (the α-subunit of Gs)

1989 ◽  
Vol 261 (3) ◽  
pp. 841-845 ◽  
Author(s):  
L Molina y Vedia ◽  
R D Nolan ◽  
E G Lapetina
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Present Evidence ◽  
Adp Ribosylation ◽  
Specific Antisera ◽  
Membrane Bound ◽  
Gs Alpha ◽  
Adp Ribosyltransferase

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

scholarly journals Glucose Monitoring in Fission Yeast via the gpa2 Gα, the git5 Gβ and the git3 Putative Glucose Receptor

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 513-521
Author(s):  
Robert M Welton ◽  
Charles S Hoffman
Keyword(s):  
Adenylate Cyclase ◽  
Fission Yeast ◽  
G Protein ◽  
Glucose Repression ◽  
Glucose Monitoring ◽  
Coding Region ◽  
Guanine Nucleotide Binding Protein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
G Protein Coupled

Abstract The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six “upstream” genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Gα) while git5 encodes a Gβ subunit. Multicopy suppression studies with gpa2+ previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Gα followed by βγ. We show here that an activated allele of gpa2 (gpa2R176H, carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2R176H allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe.

scholarly journals Quantification of signalling components and amplification in the β-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes

1995 ◽  
Vol 311 (1) ◽  
pp. 75-80 ◽  
Author(s):  
S R Post ◽  
R Hilal-Dandan ◽  
K Urasawa ◽  
L L Brunton ◽  
P A Insel
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Cardiac Myocytes ◽  
Signal Amplification ◽  
Adrenergic Receptor ◽  
Ventricular Myocytes ◽  
Binding Assays ◽  
Beta Adrenergic ◽  
Adult Rat ◽  
Gs Alpha

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5′-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

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