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Graeme Milligan - One of the best experts on this subject based on the ideXlab platform.
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Agonist regulation of cellular Gs Alpha-Subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human beta 2 adrenoceptor.
Biochemical Journal, 1994Co-Authors: Elaine J. Adie, Graeme MilliganAbstract: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.
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Concurrent down-regulation of IP prostanoid receptors and the Alpha-Subunit of the stimulatory guanine-nucleotide-binding protein (Gs) during prolonged exposure of neuroblastoma x glioma cells to prostanoid agonists. Quantification and functional imp
Biochemical Journal, 1992Co-Authors: Elaine J. Adie, Ian Mullaney, Fergus R. Mckenzie, Graeme MilliganAbstract: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.
Yves Audigier - One of the best experts on this subject based on the ideXlab platform.
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Assays for studying functional properties of in vitro translated Gs Alpha Subunit.
Methods in enzymology, 1994Co-Authors: Yves AudigierAbstract:Publisher Summary This chapter discusses different procedure designed to study the functional properties of mutated in vitro translated Gsα Subunits. The relationship between the structure and function of a protein has been widely explored by in vitro mutagenesis of the cDNA encoding the protein and expression of the mutated protein in an eukaryotic cell. If the synthesis of the endogenous protein can be abolished, this approach becomes more fruitful because the functional properties of the mutated protein can be directly assayed on the transfected cell. Isolation of the cyc- variant of the mouse lymphoma S49 cell line, which does not synthesize the α Subunit of the GTP-binding protein Gs has provided this ideal situation for studying the functional domains of Gs α Subunits. An alternative approach, based on the synthesis of large amounts of α Subunit in bacteria and its use for reconstitution of cyc- membranes, is more attractive, but it faces the problem of posttranslational modifications which are lacking in the bacterially expressed proteins.
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Mutagenesis of the amino-terminal glycine to alanine in Gs Alpha Subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation.
The Journal of biological chemistry, 1993Co-Authors: R Van Der Neut, C Pantaloni, I Nebout, J Bockaert, Yves AudigierAbstract:Abstract Proteolytic removal and genetic deletion of the amino-terminal domain of G protein Alpha Subunit have shown that this region is necessary for interaction with beta gamma Subunits. In the Alpha Subunits which undergo myristoylation, myristoylation of the amino-terminal glycine modulates the affinity of Alpha Subunit for the beta gamma complex. To determine the role of the same glycine in nonmyristoylated Alpha Subunits, we substituted it for alanine in Gs Alpha and characterized the properties of the mutated chain G2A Gs Alpha. The mutant could still bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) as revealed by its resistance to trypsin proteolysis and was able to interact with the membrane. However, G2A Gs Alpha was a poor substrate for cholera toxin-catalyzed ADP-ribosylation either in the soluble form or when membrane-associated. Addition of beta gamma Subunits increased the sedimentation rate of G2A Gs Alpha in sucrose gradients. Binding experiments performed on cyc- membranes reconstituted by G2A Gs Alpha showed that the GTP-induced shift of isoproterenol affinity for the beta-adrenergic receptors was reduced. On the same membranes, isoproterenol, GTP gamma S and NaF were 2-fold less effective for activating adenylylcyclase when compared to cyc- membranes reconstituted by Gs Alpha. This differential stimulation of adenylylcyclase was not due to an affinity change for the effector but to a decrease in the maximal activation. Thus the G2A substitution affected beta gamma-dependent properties on reconstituted membranes such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation and we propose that the decreased activation of adenylylcyclase might result from the same defect. Although not essential for association with beta gamma Subunits, the amino-terminal glycine of nonmyristoylated Gs Alpha might play a modulatory role in this interaction.
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Amino acids 367-376 of the Gs Alpha Subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 Alpha Subunit.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Laurent Journot, C Pantaloni, J Bockaert, Marie-alix Poul, H Mazarguil, Yves AudigierAbstract:Abstract Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the Alpha Subunit could be indirect by means of the other beta gamma Subunits or polydisperse multimers. In the activated state, although the Alpha Subunit is dissociated from other Subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins Alpha Subunits, the C-terminal domain of Gs Alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs Alpha C-terminal domain. We first deleted codons 2-6 of Gi1 Alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 Alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 Alpha by different domains of the Gs Alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs Alpha). Our results showed that addition of amino acids 367-376 of Gs Alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 Alpha.
Jean Feunteun - One of the best experts on this subject based on the ideXlab platform.
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Oncogenic potential of guanine nucleotide stimulatory factor Alpha Subunit in thyroid glands of transgenic mice
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: F M Michiels, Bernard Caillou, Monique Talbot, F Dessarps-freichey, M T Maunoury, Martin Schlumberger, L. Mercken, Roger Monier, Jean FeunteunAbstract:Abstract Transgenic mice have been used to address the issue of the oncogenic potential of mutant guanine nucleotide stimulatory factor (Gs) Alpha Subunit in the thyroid gland. The expression of the mutant Arg-201-->His Gs Alpha Subunit transgene has been directed to murine thyroid epithelial cells by bovine thyroglobulin promoter. The transgenic animals develop hyperfunctioning thyroid adenomas with increased intracellular cAMP levels and high uptake of [125I]iodine and produced elevated levels of circulating triiodothyronine and thyroxine. These animals demonstrate that the mutant form of Gs Alpha Subunit carries an oncogenic activity, thus supporting the model that deregulation of cAMP level alters growth control in thyroid epithelium. These animals represent models for humans with autonomously functioning thyroid nodules.
Lee S Weinstein - One of the best experts on this subject based on the ideXlab platform.
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Gsα Mutations and Imprinting Defects in Human Disease
Annals of the New York Academy of Sciences, 2002Co-Authors: Lee S Weinstein, Min Chen, Jie LiuAbstract:Gs is the ubiquitously expressed heterotrimeric G protein that couples receptors to the effector enzyme adenylyl cyclase and is required for receptor-stimulated intracellular cAMP generation. Activated receptors promote the exchange of GTP for GDP on the Gs Alpha-Subunit (Gs(Alpha)), resulting in Gs activation; an intrinsic GTPase activity of Gs(Alpha) deactivates Gs by hydrolyzing bound GTP to GDP. Mutations of Gs(Alpha) residues involved in the GTPase reaction that lead to constitutive activation are present in endocrine tumors, fibrous dysplasia of bone, and McCune-Albright syndrome. Heterozygous loss-of-function mutations lead to Albright hereditary osteodystrophy (AHO), a disease characterized by short stature, obesity, and skeletal defects, and are sometimes associated with progressive osseous heteroplasia. Maternal transmission of Gs(Alpha) mutations leads to AHO plus resistance to several hormones (e.g., parathyroid hormone) that activate Gs in their target tissues (pseudohypoparathyroidism type IA), while paternal transmission leads only to the AHO phenotype (pseudopseudohypoparathyroidism). Studies in both mice and humans demonstrate that Gs(Alpha) is imprinted in a tissue-specific manner, being expressed primarily from the maternal allele in some tissues and biallelically expressed in most other tissues. This likely explains why multihormone resistance occurs only when Gs(Alpha) mutations are inherited maternally. The Gs(Alpha) gene GNAS1 has at least four alternative promoters and first exons, leading to the production of alternative gene products including Gs(Alpha), XL Alphas (a novel Gs(Alpha) isoform expressed only from the paternal allele), and NESP55 (a chromogranin-like protein expressed only from the maternal allele). The fourth alternative promoter and first exon (exon 1A) located just upstream of the Gs(Alpha) promoter is normally methylated on the maternal allele and is transcriptionally active on the paternal allele. In patients with parathyroid hormone resistance but without AHO (pseudohypoparathyroidism type IB), the exon 1A promoter region is unmethylated and transcriptionally active on both alleles. This GNAS1 imprinting defect is predicted to decrease Gs(Alpha) expression in tissues where Gs(Alpha) is normally imprinted and therefore to lead to renal parathyroid hormone resistance.
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Detection of mutations and polymorphisms of Gs Alpha Subunit gene by denaturing gradient gel electrophoresis.
Methods in enzymology, 1994Co-Authors: Pablo V Gejman, Lee S WeinsteinAbstract:Publisher Summary The genomic structure of the α Subunit of the guanine nucleotide-binding protein G s (G s α ) is composed of 13 exons and 12 introns spanning more than 20 kilobases (kb), whereas the transcribed message of the gene is approximately 1.9 kb in length. Denaturing gradient gel electrophoresis (DGGE) analysis detected several disease-related mutations in individuals affected with Albright hereditary osteodystrophy (AHO) and with the McCune–Albright syndrome (MAS), and it also uncovered a polymorphism that was instrumental to the genetic linkage mapping of the G s α gene. This chapter reviews some basic principles of DNA denaturation and DGGE applications and the specific experimental protocols applied to the analysis of G s α . Many aspects of DGGE are also reviewed in the chapter. In DGGE experiments a temperature gradient is supplanted by a chemical gradient made of a linearly increasing concentration of a urea–formamide mixture in a polyacrylamide gel. Because the denaturation properties of the urea–formamide gradient are reasonably equivalent to those of a thermal gradient at temperatures near the melting temperatures, electrophoresis is performed at 60°.
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mutations of the Gs Alpha Subunit gene in albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis
Proceedings of the National Academy of Sciences of the United States of America, 1990Co-Authors: Lee S Weinstein, Pablo V Gejman, Eitan Friedman, Takashi Kadowaki, Regina M Collins, Elliot S Gershon, Allen M SpiegelAbstract:Abstract Affected members of most kindreds with Albright hereditary osteodystrophy have a partial deficiency of functional Gs, the guanine nucleotide-binding protein that stimulates adenylyl cyclase. By use of the polymerase chain reaction to amplify genomic fragments with the attachment of a high-melting G + C-rich region (GC clamp) and analysis of these fragments by denaturing gradient gel electrophoresis, heterozygous mutations in the Gs Alpha-Subunit gene were found in two kindreds. These included a G----C substitution at the donor splice junction of intron 10 and a coding frameshift created by a single base deletion within exon 10. The findinGs illustrate the heterogeneity of genetic defects in Albright hereditary osteodystrophy and the usefulness of the polymerase chain reaction-denaturing gradient gel electrophoresis method to search rapidly for mutations in a large candidate gene.
Elaine J. Adie - One of the best experts on this subject based on the ideXlab platform.
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Agonist regulation of cellular Gs Alpha-Subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human beta 2 adrenoceptor.
Biochemical Journal, 1994Co-Authors: Elaine J. Adie, Graeme MilliganAbstract: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.
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Concurrent down-regulation of IP prostanoid receptors and the Alpha-Subunit of the stimulatory guanine-nucleotide-binding protein (Gs) during prolonged exposure of neuroblastoma x glioma cells to prostanoid agonists. Quantification and functional imp
Biochemical Journal, 1992Co-Authors: Elaine J. Adie, Ian Mullaney, Fergus R. Mckenzie, Graeme MilliganAbstract: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.
