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PubMed1988

Noncoordinate regulation of cardiac Gs protein and beta-adrenergic receptors by a physiological stimulus, chronic dynamic exercise.

We used a physiological stimulus, chronic dynamic exercise, in pigs to examine resultant changes in chronotropic responsiveness to catecholamine and biochemical features of cardiac beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein, GS. Long-term treadmill running resulted in a substantial (44%) down-regulation of right atrial beta-adrenergic receptors, but the dose of isoproterenol yielding a 50% maximal increase in heart rate was decreased by 57% (from 0.07 +/- 0.03 to 0.03 +/- 0.01 microgram/kg; P less than 0.02) despite this decrease in receptor number. This disparity between receptor number and physiological responsiveness suggested altered signal transduction. We therefore quantitated GS in myocardial membranes obtained before and after chronic exercise in a competitive ELISA based on an antipeptide antibody developed to the alpha S portion of GS. We found a 42% increase in the amounts of GS in right atrial membranes (from 11.4 +/- 0.8 to 16.2 +/- 2.0 pmol/mg; P less than 0.05) and a 76% increase in the amounts of GS in left ventricular membranes (from 15.6 +/- 2.6 to 27.4 +/- 5.2 pmol/mg; P = 0.02) after chronic running. These data suggest that in the heart physiological perturbations can result in changes in the levels of GS, that GS and beta-adrenergic receptor number are not coordinately regulated, and that GS may contribute to altered adrenergic responsiveness independently of changes in beta-adrenergic receptor number.

Animals↗

PubMed1988

Subunit dissociation is the mechanism for hormonal activation of the Gs protein in native membranes.

We have recently reported (Ransnäs, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.

Animals↗

PubMed1994

The human thyrotropin receptor activates G-proteins Gs and Gq/11.

The human thyrotropin receptor leads upon activation to the stimulation of phospholipase C and adenylyl cyclase. It is presently not known whether this bifurcating signaling occurs via two different G-proteins (Gq/11 and Gs) or via one G-protein (Gs). Receptor-activated Gs releases beta gamma subunits and alpha s, which then could regulate phospholipase C and adenylyl cyclase, respectively. In order to elucidate the signaling pathways induced by the activated thyroid-stimulating hormone (TSH) receptor, we studied the coupling of the TSH receptor to Gs and Gq/11 in human thyroid membranes. TSH concentration dependently led to the activation of two forms of Gs (Gs short and Gs long) as well as of Gq and G11, demonstrating that signaling pathways induced by TSH already bifurcate in the course of the receptor-G-protein interaction. These data strongly suggest the concept that phospholipase C and adenylyl cyclase activation through the TSH receptor are mediated by Gq/11 and Gs, respectively.

Adenylyl Cyclases↗

PubMed1998

Glutathione metabolism and glutathione S-conjugate export ATPase (MRP1/GS-X pump) activity in cancer. II. Cell-to-cell variability, relation with cellular activation state and functional absence of GS-X pump in lymphocytes.

A severe complication in late-stage cancer patients is host immunosuppression. It is suggested that overproduction of the highly cytostatic and cytotoxic antiproliferative cyclopentenone prostaglandins (CP-PGs) within the plasma of cancer-bearing subjects may contribute to immunosuppression. Lymphoid tissues of Walker 256 tumor-bearing rats accumulate large amounts of CP-PGs while the tumor tissue itself does not. Moreover, tumor cells may present differential sensitivity to CP-PGs due to the expression of the multidrug resistance-associated protein (MRP1) gene product which shows a Mg(2+)-dependent vanadate-sensitive glutathione S-conjugate export ATPase (GS-X pump) activity that extrudes CP-PGs from cells as glutathione S-conjugates. In this study, the possibility that deficient GS-X pump activity in immune cells that may be involved in the accumulation of CP-PGs is investigated. Rat lymph node lymphocytes do not exhibit any notable activity even when mitogen-stimulated. Conversely, although rat peritoneal resident (quiescent) or thioglycollate-stimulated (inflammatory) macrophages exhibit low GS-X pump activity, Bacillus Calmette-Guérin (BCG)-activated macrophages show a notable rise in the activity of the ATPase, suggesting that the cellular activation state may modulate GS-X pump activity/expression and that, under appropriate stimuli (e.g., during immune response) macrophages may provide a self-defense against electrophilic CP-PGs by forming GS-conjugates that can be extruded from cells through the GS-X pump. ras oncogene expression may be linked with MRP1/GS-X pump expression/activity, since C2C12 promyoblasts transformed by v-H-ras transfection doubled GS-X pump activity. These results support the proposition that the accumulation of CP-PGs and the immunosuppression of tumor-bearing subjects may be attributed to a lack of GS-X pump activity/expression in lymphocytes.

Adenosine Triphosphatases↗

PubMed2003

Functional domains essential for Gs activity in prostaglandin EP2 and EP3 receptors.

The interaction of cell surface hormone receptors with heterotrimeric G proteins is crucial for hormonal actions. The domains of the receptor, which interact with and activate G protein, have been extensively studied. However, precise molecular mechanisms underlying regulation of the receptor-induced G protein activation are still poorly understood. Prostaglandin E(2) (PGE(2)) receptors comprise of four subtypes, EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 couple to Gs and EP3 to Gi. To assess the functional domains essential for Gs activation in prostanoid receptors, EP2, EP3beta and each intracellular loop- (IC-) interchanged EP2/EP3 chimeras were tested for agonist binding and functional responses. In EP2 receptor, substitution of IC1 or IC3 resulted in loss of binding activity, while substitution of IC2, N- (IC2N) or C-terminal half region of IC2 (IC2C) had no effects on the binding activity. Wild-type EP2 and IC2C-substituted EP2 showed agonist-induced Gs activity, but IC2- and IC2N-substituted EP2 failed to elicit Gs activity upon agonist stimulation. On the other hand, in EP3 receptor substitution of IC1 resulted in loss of PGE(2) binding, while substitution of IC2, IC3, IC2N or IC2C had no effects on binding activity. Wild-type EP3beta, IC3- or IC2C-substituted EP3 failed to show Gs activity upon agonist stimulation, but IC2- or IC2N-substituted EP3 chimera showed agonist-dependent Gs activity. These results indicated that the second intracellular loop of the EP2 plays an essential role in activation of Gs.

Animals↗

PubMed2003

Coupling to Gs and G(q/11) of histamine H2 receptors heterologously expressed in adult rat atrial myocytes.

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.

Animals↗

PubMed2002

[Effects of clonidine on myocardial Gs alpha mRNA expression after scalds in rats].

AIM: To explore the role of clonidine (Clo) on myocardial Gs alpha mRNA expression after scalds in rats. METHODS: A 30% skin-full-thickness scald was produced by immersing rats in 95 degrees C water for 10 s. The myocardial Gs alpha mRNA expression level, cyclic AMP content and adenylyl cyclase (AC) activity were determined with dot blotting hybridization, in situ hybridization, radioimmunoassay and indirect method. RESULTS: Three hours after scalds, the myocardial Gs alpha mRNA was significantly decreased to (61 +/- 20)% of the control group (P < 0.01). AC activity and cAMP content were also decreased. Clo (0.3, 1.0 and 3.0 mg.kg-1, i.p.) was shown to increase myocardial Gs alpha mRNA expression level (P < 0.01 or P < 0.05) after scalds to (131 +/- 28)%, (142 +/- 51)% and (139 +/- 48)% of the scald group, respectively, which were correlated with the Clo dose (gamma = 0.597, P < 0.05). Clo 1.0 mg.kg-1 and 3.0 mg.kg-1 (i.p.) promoted AC activity and increased cAMP content, but Clo 0.3 and 0.1 mg.kg-1 showed no significant effect (P > 0.05). Selective I1-imidazoline receptor antagonist efaroxan (Efa) (10, 5 mg.kg-1, i.p.) was found to partially reverse the effect of Clo, while Efa 2.5 mg.kg-1 showed no significantly influence. The reduced quantity of Gs alpha mRNA expression level correlated well with the Efa dose (gamma = 0.900, P < 0.05). The change of AC and cAMP was similar to Gs alpha mRNA. CONCLUSION: Clo increased the myocardial Gs alpha mRNA expression, AC activity and cAMP content after scalds in rats.

Adenylyl Cyclases↗

PubMed1997

Effect of activating and inactivating mutations of Gs- and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes.

Previous investigations have demonstrated that both Gs- and the Gi-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of Gs alpha vs. Gi2 alpha, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-Gs alpha or the inactivating G226A(H21a)-Gs alpha point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the Gi2 alpha wild type or the missense mutations R179E-Gi2 alpha, Q205L-Gi2 alpha, and G204A(H21a)-Gi2 alpha. The activating [R201C]Gs alpha-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-L1 adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]Gs alpha-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]Gi2 alpha slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]Gi2 alpha and [Q205L]Gi2 alpha over differentiated controls. The inactivating [H21a]Gi2 alpha-mutant obliterated all signs of preadipocyte differentiation. It is concluded that Gi2 plays a positive and much more important role than Gs in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process.

3T3 Cells↗

PubMed1993

Rare mutations of the Gs alpha subunit gene in human endocrine tumors. Mutation detection by polymerase chain reaction-primer-introduced restriction analysis.

BACKGROUND: The Gs alpha (Gs alpha) gene can be activated to the putative oncogene gsp by specific point mutations at codons 201 or 227. Such mutations have been reported in growth hormone (GH)-secreting pituitary adenomas and thyroid tumors. To clarify the role of Gs alpha gene in human endocrine tumors, 197 tumors were screened for point mutations at codons 201 or 227 of the Gs alpha gene. METHODS: Mutations were detected by primer-introduced restriction analysis (PIRA) of the polymerase chain reaction (PCR) product of genomic DNA. RESULTS: These Gs alpha mutations were present in 4 of 53 pituitary adenomas (4 of 43 GH-secreting adenomas; 1 of these 4 was a GH- and prolactin-secreting adenoma from a patient with familial multiple endocrine neoplasia Type 1), 4 of 66 thyroid tumors (4 of 30 papillary carcinomas), and 1 of 19 adrenocortical adenomas (1 of 6 aldosterone-secreting adenomas). In contrast, none of these Gs alpha mutations were detected in parathyroid tumors, endocrine pancreatic tumors, or pheochromocytomas. CONCLUSIONS: Gs alpha mutations at these two loci may play a role in the pathogenesis of a small population of GH-secreting pituitary adenomas, papillary thyroid carcinomas, and adrenocortical adenomas, but that they are not involved in the pathogenesis of other types of endocrine tumors.

Acromegaly↗

PubMed1994

Modification of cardiac membrane adenylate cyclase activity and Gs alpha by NAD and endogenous ADP-ribosyltransferase.

The mechanism by which NAD stimulates cardiac adenylate cyclase was investigated. In highly purified canine cardiac sarcolemma, NAD stimulated adenylate cyclase activity in the presence of agents which activate Gs (i.e. 5 mM AlF4-, 10 microM GTP gamma S, 10 microM GppNHp or isoproterenol plus 2 nM GTP gamma S). Furthermore, the EC50 of isoproterenol to stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 microM GTP gamma S or isoproterenol plus 2 nM GTP gamma S produced a selective increase in the radiolabeling of a single 45-kDa protein which was identified as Gs alpha by immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from Gs alpha prelabeled in the presence of AlF4- and [32P]-NAD indicating that an arginine residue on Gs alpha was modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1 or 3-aminobenzamide, inhibited AlF4- stimulated ADP-ribosylation of Gs alpha and NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of Gs alpha was not removed under hypotonic or hypertonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess an ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated Gs alpha.

Adenylyl Cyclases↗

PubMed1993

Shedding of Gs protein (a soluble form of the viral glycoprotein) by the rabies virus-infected BHK-21 cells.

We investigated a possible mechanism of G protein shedding occurring in the rabies virus-infected BHK-21 cell cultures. Quantitative analysis showed that about 10% of the newly synthesized G proteins were shed from the cells as Gs protein, a soluble form of G protein which lacked the C-terminal anchoring region of the protein, into the culture medium. Pulse-chase experiments revealed that the release of tritium-labeled Gs proteins began soon after the synthesis, but ceased after a short time (within about 2 hr). On the other hand, the release of the virion-associated G protein began 30 to 60 min behind the start of Gs protein shedding and continued for more than 10 hr. Gs protein could not be detected in the cells by biochemical and immunological methods at the time when large amounts of Gs protein were actively being shed. With an antiserum against the C-terminal of G protein we could detect in the cell a polypeptide having a size that is seemingly the same as that which was lost from Gs protein. These results suggest that Gs proteins are generated by a proteolytic cleavage of newly synthesized G proteins, and the cleavage seems to be stopped at a certain stage of the G protein maturation.

Amino Acid Sequence↗

PubMed1991

Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels.

In the heart, the guanosine 5'-triphosphate (GTP)-binding protein Gs is activated by hormone binding to beta-adrenergic receptors and stimulates the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A. Additionally, Gs can modulate cardiac Ca channels directly in cell-free systems. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (ICa) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the ICa density increased from 12 to 40 microA/cm2 following application of 1 microM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1 microM ISP enhanced ICa only from 9 to 17 microA/cm2 after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPs (the Rp-isomer of adenosine 3',5'-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on ICa. Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of ICa, which probably reflects the direct stimulation of Ca channels by Gs under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 microM cAMP, bath application of 20-40 microM 3-isobutyl-1-methylxanthine (IBMX) enhanced the ICa density to about 41 microA/cm2 indicating saturation of the cAMP pathway. Under this condition, 1 microM ISP was without significant effect on ICa. This result may suggest that direct Gs stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the Gs pathway in intact myocytes. Third, simultaneous application of 1 microM ISP and 40 microM IBMX enhanced ICa up to densities of around 75 microA/cm2 during cell dialysis with 100 microM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that Gs is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of Gs may act to prime L-type Ca channels for cAMP-dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Inhibition of ADP-ribosyltransferase increases synthesis of Gs alpha in neuroblastoma x glioma hybrid cells and reverses iloprost-dependent heterologous loss of fluoride-sensitive adenylate cyclase.

Exposure of NG108-15 cells to 50 mM nicotinamide [an inhibitor of mono(ADP-ribosyl)transferase] for 18 hr led to an increase in membrane associated Gs alpha measured either as cholera toxin substrate or by immunoblotting with a specific antiserum. Prolonged exposure of NG108-15 cells to iloprost is followed by homologous loss of iloprost sensitivity, and heterologous loss of fluoride-dependent activation of adenylate cyclase. Nicotinamide reversed the loss of fluoride sensitivity, but failed to restore iloprost-dependent activation of adenylate cyclase. These results with nicotinamide in NG108-15 cells contrasted with those from platelets, which also exhibit heterologous desensitization of fluoride sensitivity following prolonged exposure to iloprost. Treatment of platelets with 50 mM nicotinamide for 18 hr led to an increase of 75.0 +/- 19.4% in the amount of membrane associated cholera toxin substrate. However, there was no associated increase in the abundance of Gs alpha as determined by immunoblotting. Furthermore, in platelets there was no restoration by nicotinamide of the iloprost-dependent loss of fluoride-sensitive adenylate cyclase activity. It follows that heterologous desensitization in platelets is accompanied by inactivation of Gs alpha, which is retained within the plasma membrane in its inactive state. The nicotinamide-dependent increase in the abundance of membrane associated cholera toxin substrate and immunoreactive Gs alpha in NG108-15 cells is associated with an increase of 72.0 +/- 20.3% in the levels of mRNA encoding Gs alpha. The capacity of nicotinamide to increase the abundance of membrane associated Gs alpha was reversed when the cells were cultured in the presence of 20 micrograms/mL cycloheximide. These results suggest that the ability of nicotinamide to increase the abundance of Gs alpha in NG108-15 cells is mediated by de novo protein synthesis.

ADP Ribose Transferases↗

PubMed1993

Pharmacological characterization of effects of verapamil and GS 283 on isolated guinea pig and rat trachealis.

The pharmacological effects of verapamil and GS 283, 1-(4'-methoxybenzyl)-6,7-dihydroxy-3,4-dihydroxyisoquinoline, were investigated using isolated rat and guinea pig trachealis. Both verapamil and GS 283 inhibited carbachol-, histamine (only guinea pig)-, and high-K(+)-induced contraction in a dose-dependent manner. GS 283 acted as a weak histamine H1 and muscarinic receptor antagonist in guinea pig and rat trachealis with respective pKB values in the range of 5.60 approximately 6.12 and 5.17 approximately 5.83. On the other hand, pyrilamine and atropine showed a typical competitive antagonism on histamine (guinea pig) and on muscarinic receptors (rat trachea) with pKB values of 9.25 +/- 0.21 and 9.37 +/- 0.32, respectively. GS 283 inhibited Ca(2+)-induced contraction on guinea pig trachealis in Ca(2+)-free media. Furthermore, very high concentrations of GS 283 and verapamil completely abolished a phasic contraction induced by carbachol in Ca(2+)-free media, suggesting that verapamil and GS 283 can enter into the cytoplasm, where they may exert secondary actions on internal sites of the muscle, such as the sarcoplasmic reticulum. It is concluded that GS 283 has a Ca2+ antagonistic action along with weak histamine and muscarinic receptor blocking activity in isolated rat and guinea pig tracheal smooth muscle and its mode of action is likely inhibition of Ca2+ influx from plasma membrane and also release from the sarcoplasmic reticulum.

Animals↗

PubMed1994

Ethanol-induced reduction of the amount of stimulatory GTP-binding regulatory protein, Gs, in wild-type S49 murine lymphoma cells: effect of cycloheximide and puromycin.

The activity and the amount of Gs in wild-type S49 murine lymphoma cells decreased by 16-18% and by 29%, respectively, after 24 h treatment of the cells with 50 mM ethanol, through a transient increase of the Gs activity with no alteration in its content after 4 and 8 h treatment. No decrease of the Gs activity was noted when membranes from S49 cells were treated with 50-500 mM ethanol. To study the role of protein metabolism in the ethanol-induced reduction in the amount of Gs, S49 cells were treated with ethanol for 24 h in the presence of inhibitor of protein synthesis. Cycloheximide at concentrations of 0.05-0.15 micrograms/ml and puromycin at 0.15-0.60 micrograms/ml caused a dose-dependent inhibition of cell division in S49 cells to a similar degree. The ethanol-induced reduction of the activity and the amount of Gs was completely abolished by those concentrations of cycloheximide, but not by puromycin. Cycloheximide itself caused no alteration in the Gs activity of S49 cell membranes. These results indicate that the process of the ethanol-induced event of Gs involves cycloheximide-sensitive protein synthesis.

Animals↗

PubMed1991

Alzheimer's disease: specific increases in a G protein subunit (Gs alpha) mRNA in hippocampal and cortical neurons.

The GTP binding protein, Gs, activates adenyl cyclase in direct response to stimulation of several neurotransmitter receptors. In situ hybridization histochemistry (ISHH) with a 35S-labelled oligonucleotide has been used to detect the mRNA encoding the alpha subunit of Gs (Gs alpha) in human hippocampus, temporal and visual cortices and cerebellum, and its level has been compared between Alzheimer's disease (AD) and control brains. A marked regional increase was found in the hippocampus of AD cases. Analysis of levels of Gs alpha mRNA in individual constituent pyramidal cells confirmed this increase (3 to 4-fold in densitometric units) in hippocampal fields CA1, CA3 and CA4, as well as in temporal cortex. Levels of Gs alpha mRNA were also determined relative to total poly(A)+ mRNA in the same cell populations in each case. Gene-specific elevation of Gs alpha mRNA was thereby confirmed in hippocampal fields, and also in temporal cortex. No changes were seen in visual cortex. The increase in Gs alpha mRNA may represent a response by AD neurons in affected areas to receptor alterations, or to an abnormality in receptor-G protein coupling. Alternatively, altered G protein gene expression might be a pathogenic event underlying changes in linked receptor populations.

Aged↗

PubMed1991

Human choriogonadotropin-induced coupling of receptor and Gs protein and the effect of hormone deglycosylation.

The detergent-soluble extract of rat ovary plasma membranes contained a Gs protein of about 100 kDa as shown by its elution behavior on a Bio Gel A-1.5m column. However, the cell membranes exposed to hCG (37 degrees C, 15 min) contained in addition a higher molecular weight Gs protein complex of 300 kDa comprised of human chorionic gonadotropin (hCG) receptor (hCGR) and Gs. The complex bound with an affinity column of GTP-Sepharose and could be released with Gpp(NH)p and GTP inhibited this binding. The presence of the hCGR in the complex was shown by its binding to 125I-hCG. Furthermore, GTP inhibited the binding of hCG to the complex. These results indicate the presence of hCGR and Gs protein complex in the hCG-treated membranes. hCGR and Gs protein were individually purified and reconstituted into phospholipid vesicles. The protein-phospholipid vesicles showed saturation kinetics of binding of 125I-hCG and 3H-Gpp(NH)p. Incubation of phospholipid vesicles with hCG resulted in a 2-3-fold increase in the binding of 3H-Gpp(NH)p and GTPase activity. Activation of Gs protein was dependent on the length of incubation and the hormone concentration. Deglycosylated hCG was about 10 times less potent than hCG suggesting a role of carbohydrates of hCG in inducing hCG-Gs protein interactions. The data with the in vitro reconstitution system rule out the involvement of a carbohydrate-binding lectin in the function of the hormone.

Animals↗

PubMedDate not supplied

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas.

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.

Adenoma↗