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 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 Density-dependent nerve growth factor regulation of Gs-alpha RNA in pheochromocytoma 12 cells.

1990 ◽  
Vol 10 (6) ◽  
pp. 3277-3279 ◽  
Author(s):  
G Tjaden ◽  
A Aguanno ◽  
R Kumar ◽  
D Benincasa ◽  
R M Gubits ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
G Protein ◽  
Alpha Subunit ◽  
Low Density ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Density Dependent ◽  
Stimulatory G Protein ◽  
Gs Alpha

Nerve growth factor (NGF) affects levels of the alpha subunit of the stimulatory G protein (Gs-alpha) in pheochromocytoma 12 cells in a bidirectional, density-dependent manner. Cells grown at high density responded to NGF treatment with increased levels of Gs-alpha mRNA and protein. Conversely, in cells grown in low-density cultures, levels of this mRNA were lowered by NGF treatment.

Altered myocardial Gs protein and adenylyl cyclase signaling in rats exposed to chronic hypoxia and normoxic recovery

2003 ◽  
Vol 94 (6) ◽  
pp. 2423-2432 ◽  
Author(s):  
Markéta Hrbasová ◽  
Jiri Novotny ◽  
Lucie Hejnová ◽  
František Kolář ◽  
Jan Neckář ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Contractile Function ◽  
Left Ventricular ◽  
Adaptation To Hypoxia ◽  
Adrenergic Stimulation ◽  
Α Subunit ◽  
Stimulatory G Protein ◽  
Membrane Bound ◽  
The Right

The present work has analyzed the consequences of chronic intermittent high-altitude hypoxia for functioning of the G protein-mediated adenylyl cyclase (AC) signaling system in the right (RV) and left ventricular (LV) myocardium in rats. Adaptation to hypoxia did not appreciably affect the number of β-adrenoceptors and the content of predominantly membrane-bound α-subunit (Gsα) of the stimulatory G protein, but it raised the amount of cytosolic Gsα in RV. The levels of myocardial inhibitory Gα protein were not altered. Activity of AC stimulated by GTP, fluoride, forskolin, or isoprotertenol was reduced by ∼50% in RV from chronically hypoxic rats, and a weaker depression was also found in LV. In addition, hypoxia significantly diminished a functional activity of membrane-bound Gsα in both RV and LV. The RV baseline contractile function was markedly increased in chronically hypoxic animals, and its sensitivity to β-adrenergic stimulation was decreased. Animals recovering from hypoxia for 5 wk still exhibited markedly elevated levels of cytosolic Gsα and significantly lower activity of AC in RV than did age-matched controls, but contractile responsiveness to β-agonists was normal.

Molecular Scanning of the α Subunit of the Stimulatory G Protein Gene in Essential Hypertension

1995 ◽  
Vol 88 (s32) ◽  
pp. 24P-24P
Author(s):  
H Jia ◽  
AD Hingorani ◽  
PA Stevens ◽  
S Monteith ◽  
MJ Brown
Keyword(s):  
Essential Hypertension ◽  
G Protein ◽  
Protein Gene ◽  
Α Subunit ◽  
Stimulatory G Protein

scholarly journals Receptor-Mediated Adenylyl Cyclase Activation Through XLαs, the Extra-Large Variant of the Stimulatory G Protein α-Subunit

2002 ◽  
Vol 16 (8) ◽  
pp. 1912-1919 ◽  
Author(s):  
Murat Bastepe ◽  
Yasemin Gunes ◽  
Beatriz Perez-Villamil ◽  
Joy Hunzelman ◽  
Lee S. Weinstein ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Adrenergic Receptor ◽  
Α Subunit ◽  
Exon 2 ◽  
Amino Terminal ◽  
Stimulatory G Protein ◽  
Β2 Adrenergic Receptor ◽  
Carboxyl Terminal ◽  
G Protein Α Subunit

Abstract XLαs, the large variant of the stimulatory G protein α subunit (Gsα), is derived from GNAS1 through the use of an alternative first exon and promoter. Gsα and XLαs have distinct amino-terminal domains, but are identical over the carboxyl-terminal portion encoded by exons 2–13. XLαs can mimic some functions of Gsα, including βγ interaction and adenylyl cyclase stimulation. However, previous attempts to demonstrate coupling of XLαs to typically Gs-coupled receptors have not been successful. We now report the generation of murine cell lines that carry homozygous disruption of Gnas exon 2, and are therefore null for endogenous XLαs and Gsα (GnasE2−/E2−). GnasE2−/E2− cells transfected with plasmids encoding XLαs and different heptahelical receptors, including the β2-adrenergic receptor and receptors for PTH, TSH, and CRF, showed agonist-mediated cAMP accumulation that was indistinguishable from that observed with cells transiently coexpressing Gsα and these receptors. Our findings thus indicate that XLαs is capable of functionally coupling to receptors that normally act via Gsα.

Sign in / Sign up

Export Citation Format

Share Document