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PubMed1998

Constitutively active Gs alpha is associated with an increased phosphodiesterase activity in human growth hormone-secreting adenomas.

Because phosphodiesterase (PDE) expression and activity are controlled by cAMP, we investigated whether activating mutations of Gs alpha gene that occur in human GH-secreting adenomas are associated with increased PDE activity. We studied 10 adenomas with wild-type Gs alpha (gsp-) and 8 with mutant Gs alpha (gsp+). Although, in the absence of PDE inhibitors, intracellular cAMP levels were similar in gsp+ e gsp- adenomas, the PDE blockade with 3-isobutyl-1-methylxanthine induced a marked increase in cAMP in all but one gsp+ adenoma (% increase: from 77 to 2900) and a slight rise in only 2 gsp-. Similar results were obtained with the PDE4 selective inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl)]-2-pyrrolidinone. In vitro GH release was significantly higher in gsp+ than in gsp- adenomas (315 +/- 158 vs. 82 +/- 53 micrograms/well; P < 0.01), and PDE blockade caused a further increase in 3 of 5 gsp+ adenomas but not in gsp- tumors. By direct measurement, PDE activity was about 7-fold higher in gsp+ than in gsp- adenomas (320 +/- 213 vs. 48 +/- 23 pmol/min.mg protein; P < 0.05) and was largely 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone sensitive. This study first demonstrates that activating mutations of the Gs alpha gene that naturally occur in pituitary adenomas is associated with an increased PDE activity that might, at least partially, counteract the constitutive activation of the cAMP-dependent pathway.

1-Methyl-3-isobutylxanthine↗

PubMed2006

A case of McCune-Albright syndrome associated with Gs alpha mutation in the bone tissue.

The syndrome of McCune-Albright syndrome (MAS) is clasically defined as a triad presentation with the findings of polyostotic fibrous dysplasia, café-au-lait spots, and sexual precocity. However, not all patients present with complete symptoms. A 52-year-old man was diagnosed as having a variant of McCune-Albright syndrome with the following findings: polyostotic fibrous dysplasia, acromegaly due to pituitary tumor and subclinical hyperthyroidism due to toxic multinodular goiter. Sexual precocity and café-au-lait spots were not noted. Acromegaly was confirmed by laboratory examination (IGF-1, glucose suppression test and TRH stimulation test). Long acting somatostatin analogue was used as treatment. Although the pituitary tumor could not be removed due to technical problems, mass lesions on the cranium were removed subtotally. Histopathological evaluation demonstrated that the lesion complied with fibrous dysplasia. Genomic DNAs were isolated from the craniofacial bones and peripheral leucocytes of the patient. After amplifying the related regions, Gs alpha (Gs alpha) gene was analysed by automatic DNA sequence analysis. An activating mutation of the Gs alpha gene (Arg 201 Cys) was found in the genomic DNA isolated from the bone tissue of the patient, but not in the genomic DNA isolated from the blood. We described a case of MAS associated with Gs alpha mutation in the bone tissue, presenting with polyostotic fibrous dysplasia, subclinical hyperthyroidism and acromegaly.

Acromegaly↗

PubMed2002

A randomized, double-masked clinical trial of Optisol-GS vs Chen Medium for human corneal storage.

OBJECTIVE: To determine whether the clinical outcome of corneal transplantation performed with tissue stored in Chen Medium (CM; Chen Laboratories, Phoenix, Md) is superior to that of Optisol-GS (Chiron Ophthalmics, Irvine, Calif). DESIGN: Randomized, double-masked trial. PARTICIPANTS AND SETTING: Ninety patients undergoing corneal transplantation at a tertiary center. INTERVENTION: Donor cornea pairs were stored, one in CM and the other in Optisol-GS, transplanted, and followed up for 3 months. MAIN OUTCOME MEASURES: Endothelial cell loss (ECL) at 3 months. Secondary outcomes were thickness and epithelialization at days 1, 7, 30, and 90, as well as primary and secondary graft failure. RESULTS: Mean percentages of ECL 3 months after surgery were similar for corneas stored in Optisol-GS and corneas stored in CM. The 95% confidence interval (CI) for the difference was -3.4% to 13.2%. Covariate-adjusted 95% CI was -6.1% to 9.1%. Pachymetry values were similar in the 2 treatment groups. Eighty-eight percent of the corneas stored in each medium achieved epithelialization of 75% to 100% of the surface area within 1 week after transplantation. No primary donor failures occurred in either group. One graft that was preserved in CM failed during the study period. CONCLUSION: The clinical outcomes of corneal transplantation with tissue that was preserved in CM were similar to those of grafts preserved in Optisol-GS.

Aged↗

PubMed2006

Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate.

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.

Animals↗

PubMed2007

Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells.

The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.

Antibodies, Monoclonal↗

PubMed2005

Expression and localization of three G protein alpha subunits, Go, Gq, and Gs, in adult antennae of the silkmoth (Bombyx mori).

In insect olfactory receptor neurons, rapid and transient increases in inositol triphosphate (IP3) and Ca2+ are detected upon stimulation with pheromone or nonpheromonal odorants. This suggests that heterotrimeric guanine nucleotide binding proteins (G proteins) may transduce some odorant responses in insects. We obtained cDNA clones encoding three classes of G protein alpha subunits, Bm Go, Bm Gq, and Bm Gs, from the antennae of the adult male silkmoth (Bombyx mori). RT-PCR experiments showed that the mRNA of these G protein alpha subunits was also present in the various tissues of adult and larval insects. We used immunocytochemistry to localize these G protein alpha subunits in adult male and female antennae. In the adult male antennae, anti-Go antiserum stained the nerve bundles. In contrast, anti-Gq and anti-Gs antisera stained the inner and outer dendritic segments of the putative olfactory receptor neuron. The localizations of Bm Go, Bm Gq, and Bm Gs in the female antennae were the same as in the male antennae. The localizations of Bm Gq and Bm Gs suggest that each subunit mediates a subset of the odorant response.

Amino Acid Sequence↗

PubMed1993

Functional alpha 2-adrenergic receptor-Gs coupling undergoes agonist-promoted desensitization in a subtype-selective manner.

Recently it has become clear that alpha 2-adrenergic receptors (alpha 2AR) functionally couple to Gs as well as Gi, thus inducing a complex modulation of adenylyl cyclase activity. It is unknown whether alpha 2AR-Gs coupling undergoes agonist-promoted desensitization. Therefore, in CHO cells expressing the three cloned human alpha 2AR subtypes (alpha 2C10, alpha 2C4, and alpha 2C2), we assessed the ability of alpha 2AR-mediated stimulation of adenylyl cyclase activity to undergo short-term agonist-promoted desensitization. To isolate alpha 2AR-Gs coupling, cells were pretreated with pertussis toxin, which ablates Gi coupling. Following agonist exposure, both alpha 2C10- and alpha 2C2-mediated stimulation of adenylyl cyclase activity underwent marked desensitization. In distinct contrast, alpha 2C4-mediated stimulation of adenylyl cyclase activity underwent no agonist-promoted desensitization. Thus, alpha 2AR-Gs coupling undergoes agonist-promoted desensitization and does so in a subtype-selective manner.

Adenylyl Cyclase Inhibitors↗

PubMed1993

Abundant Gs alpha mRNA in basket cells of the dentate gyrus in adult rat hippocampus.

In situ hybridization histochemistry (ISHH) has been used to study the differential distribution and relative abundance of mRNAs encoding a stimulatory alpha subunit of the G-protein (Gs alpha) and glutamic acid decarboxylase (GAD) in the dorsal hippocampus in adult rat brain. The present quantitative study shows that GABAergic neurons containing high levels of GAD mRNA, express considerably more Gs alpha message than excitatory principal neurons, the granule cells of the dentate gyrus and the pyramidal cells of CA1 subfield. A subpopulation of basket cells of the dentate gyrus exhibited a uniquely high level of Gs alpha mRNA, in addition to GAD. These findings may indicate a specific functional role for Gs alpha in these GABAergic neurons in the hippocampus.

Animals↗

PubMed1996

Distribution of Gs-alpha activating mutations in human thyroid tumors measured by subcloning.

In the search for the prevalence and distribution pattern of Gs-alpha gene mutations in differentiated thyroid tumors we examined 66 tumor tissue samples for the presence of mutations at "hot-spot" codons 201 and 227 using methods based on the polymerase chain reaction, subcloning and sequencing. The prevailing type of single-base substitution at codon 201 (71.4%) corresponded to the replacement of the wild-type sequence CGT (Arg) with TGT (Cys). The fragments of the Gs-alpha gene, including codon 201 or 227 from five follicular carcinomas and one follicular adenoma, were subcloned in Escherichia coli and it was found that the proportion of alleles with mutated codon 201 varied from 3.2% to 43%. Sequencing of the corresponding region has confirmed preliminary data indicating that the single-base changes CGT (Arg) to TGT (Cys) or CGT to CAT (His) occurred. There was only a weak correlation between the prevalence of cells bearing a mutation in the Gs-alpha gene and the level of Gs-alpha protein expression in the corresponding thyroid tumors.

Adult↗

PubMed1988

Direct interaction between membrane-associated nucleoside diphosphate kinase and GTP-binding protein(Gs), and its regulation by hormones and guanine nucleotides.

In previous studies we have proposed that the membrane-associated nucleoside diphosphate kinase (m-NDP kinase) may play a role as a GTP channeling machinery for adenylate cyclase regulation by hormones. In this study, whether the m-NDP kinase has a direct interaction with the component (GTP-binding protein (Gs)) of the glucagon- and beta-adrenergic agonist-sensitive adenylate cyclase systems in rat liver membranes was examined by extraction with octylglucoside, followed by immunoprecipitation by affinity-purified monospecific anti-NDP kinase antibodies. The results demonstrated that the m-NDP kinase and the Gs were extractable as a complexed form and that the complex formation was reversibly regulated, through cell surface receptors, by hormones which had an ability to cause activation of the rat liver adenylate cyclase. Also, it was suggested that guanine nucleotides rather than hormones were primary regulators of the m-NDP kinase-Gs interaction. These results were discussed in relation to the regulatory cycle of the Gs of adenylate cyclase system.

Adenylyl Cyclases↗

PubMed1992

Prevalence of three mutations in the Gs alpha gene among 24 families with pseudohypoparathyroidism type Ia.

Pseudohypoparathyroidism type Ia (PHP-Ia), an inherited multi-hormone resistance syndrome, is associated with deficient cellular activity of the alpha-subunit of the guanine nucleotide-binding protein (Gs alpha) that stimulates adenylyl cyclase. We determined prevalence of three recently described mutations in exons 1 and 10 of the Gs alpha gene among 24 unrelated patients with PHP-Ia. Restriction analysis was used to detect two mutations that produce unique RFLPs, and allele-specific oligonucleotide hybridization was used to detect the other mutation. As none of these mutations were not found, genomic DNA was analyzed with denaturing gradient gel electrophoresis to screen for other mutations in exon 10. Mutations of the initiation codon and exon 10 in the Gs alpha gene thus rarely (< or = 4% each) cause PHP-Ia and the Gs alpha gene mutations causing PHP-Ia are heterogeneous and unique to each pedigree.

Adenylyl Cyclases↗

PubMed1989

Coupling of D1 dopamine receptors to the guanine nucleotide binding protein Gs is deficient in Huntington's disease.

Human brain contains two subtypes of D1 dopamine receptors, which both exist under high- (RH) and low-agonist affinity (RL) sites, but can be distinguished on the basis of the ability of GTP to convert RH into RL. The amygdala contains exclusively GTP-sensitive (GS) D1 receptors, frontal cortex exclusively GTP-insensitive (GI) D1 receptors, and putamen both GS and GI receptors. In contrast with controls, we were unable to detect RH sites in amygdala from patients with Huntington disease (HD). The amount of RH sites in normal and HD frontal cortex were similar. In putamen, the GTP-induced partial conversion of RH into RL, observed in controls, was absent in HD. The results suggest that coupling of GS-D1 receptors with the guanine nucleotide binding protein Gs may be deficient in HD.

Adult↗

PubMed1991

Postmortem cerebral cortex Gs alpha-subunit levels are elevated in bipolar affective disorder.

We examined the relative abundance of G-protein subunits in postmortem brain obtained from 7 patients with bipolar affective disorder (BAD) compared with 7 age- and sex-matched controls. G-protein subunit immunoreactivities were determined in membranes prepared from postmortem prefrontal cortex using SDS-PAGE and immunoblotting with specific polyclonal antisera against selected G-protein subunits: Gsa, Gi(1&2) alpha, Go alpha and G beta(1&2). Of these G-protein subunits, only Gs alpha immunoreactivity was found to be significantly elevated in frontal (+ 34%), and occipital (+ 80%) cortex (P less than 0.05) in BAD compared with control subjects. Smaller increments (+ 22%) in cerebellar Gs alpha immunoreactivity were also found but were not statistically significant. On the basis that increased Gs alpha immunoreactivity may reflect enhanced functional responsiveness of the receptor-effector units to which this coupling protein is integral, the present findings suggest that disturbances in Gs-mediated signal transduction may be involved in the pathophysiology of BAD.

Bipolar Disorder↗

PubMed1994

Cholera toxin-induced Gs alpha down-regulation in neural tissue: studies on the pineal gland.

Cholera toxin (CT) treatment (50 micrograms/ml) was used to down regulate the alpha subunit of the stimulatory guanine nucleotide binding protein (Gs alpha) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces Gs alpha by approximately 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of G beta, Gi alpha or Go alpha. This effect on Gs alpha was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of Gs alpha. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP (N6,2'-O-dibutyryl adenosine 3',5' monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced alpha- or beta-adrenergic stimulation. alpha 1-Adrenergic activation of the pineal gland elevates [Ca2+]i, which potentiates effects of cyclic AMP; in these studies the response to alpha-adrenergic activation was markedly increased in 48 h CT-treated glands, reflecting Ca2+ potentiation of the effects of elevated levels of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Tubulin stimulates adenylyl cyclase activity in rat striatal membranes via transfer of guanine nucleotide to Gs protein.

Previous studies of rat cerebral cortex and rat C6 glioma cells have demonstrated that dimeric tubulin is capable of activating the G proteins Gs and Gil via transfer of guanine nucleotide from tubulin to Gs alpha and Gil alpha. To provide further information regarding cytoskeletal modulation of adenylyl cyclase, the present study examined effects of tubulin on the activation of the enzyme in rat striatal membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analog 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of adenylyl cyclase by approximately 130%. Furthermore, tubulin-GppNHp activated SKF 38393-sensitive adenylyl cyclase and potentiated forskolin-stimulated activity of the enzyme. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analog [32p]p3 (4-azidoanilido)-p1-5'-GTP ([32P]AAGTP), was incubated with striatal membranes, AAGTP was transferred from tubulin to Gs alpha as well as Gi alpha with the extents of nucleotide transfers being 7.6 +/- 0.8% and 17.8 +/- 1.4% of AAGTP originally bound to tubulin, respectively. These results indicate that, in rat striatum, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gs alpha, supporting the hypothesis that tubulin contributes to the regulation of neuronal signal transduction.

Adenylyl Cyclases↗

PubMed1983

Differential mitogenic effects of methyl isobutyl xanthine and a tumor growth factor on G1-arrested 3T3 T proadipocytes at the predifferentiation GD state and the growth-factor deficiency GS state.

Two growth-states exist in the G1 phase of the 3T3 T proadipocyte cell cycle. GD is the arrest state at which proadipocytes must growth-arrest prior to differentiation. GS is the arrest state at which proadipocytes growth-arrest following deprivation of serum or growth factors. In an attempt to further distinguish these arrest states, we have compared the relative ability of a variety of mitogens to induce GD- and GS-arrested cells to initiate DNA synthesis. The data show that GD-arrested cells at both high and low densities can be induced to proliferate by methyl-isobutyl-xanthine (MIX), whereas high and low density GS-arrested cells are not. The data also show that a tumor growth factor can stimulate the proliferation of both high and low density GD- and GS-arrested cells, whereas other agents are poor mitogens for high density GD-arrested cells. We conclude that MIX and a tumor growth factor (TUGF) can serve as probes to study the characteristics of the GD arrest state.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Carbamylcholine inhibits beta-adrenergic receptor-coupled Gs protein function proximal to adenylate cyclase.

The specific mechanism by which the inhibitory guanine nucleotide binding protein (Gi) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the beta gamma subunit, which is dissociated with activation of Gi, inhibits the function of the stimulatory guanine nucleotide binding protein (Gs) by reducing the concentration of the free alpha s subunit. In the present study, Gs protein function is determined by measuring cholera toxin-blockable, isoproterenol-induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this beta-adrenergic receptor-coupled Gs protein function. Pretreatment of the cardiac ventricle membrane with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist-receptor coupled effect through Gi on Gs protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.

Adenosine Diphosphate Ribose↗

PubMed1990

Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells. Long-term down-regulation of Gs alpha subunits by cholera toxin treatment.

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.

Adenosine Diphosphate Ribose↗