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PubMed1990

Biochemical characterization of three stimulatory GTP-binding proteins. The large and small forms of Gs and the olfactory-specific G-protein, Golf.

The biochemical properties of three stimulatory guanine nucleotide-binding protein (G-protein) alpha subunits, the large and small forms of Gs, Gs-l (52 kDa) and Gs-s (45 kDa), and the olfactory specific G-protein, Golf, have been compared. Complementary DNAs (cDNAs) encoding each alpha subunit were independently expressed in a mammalian cell line deficient in endogenous stimulatory G-proteins (S49 cyc-kin-). Gs-l and Gs-s respond similarly to activation by the beta-adrenergic agonist isoproterenol (EC50 = 80 and 60 nM, respectively) and the receptor-independent G-protein activators guanosine 5-O-3-(thio)triphosphate) (GTP gamma S) and AlF-4. The ability of Golf to interact with the beta-adrenergic receptor was also examined. Surprisingly, Golf interacts with beta-adrenergic receptors and is activated by isoproterenol (EC50 = 120 nM). All three G-proteins respond similarly to treatment with different alpha, beta, and gamma thiophosphoryl analogs of GTP. Specifically, (R)-GTP alpha S and GTP gamma S activate each G-protein, whereas (S)-GTP alpha S and (R)- or (S)-GTP beta S are inactive. In addition, similar to Gs alpha, Golf alpha is covalently modified and constitutively activated by cholera toxin. These studies demonstrate that all three stimulatory G-proteins are functionally and structurally similar, however, subtle differences between Golf and the two forms of Gs appear to modulate their interactions with receptors.

Adenylyl Cyclases↗

PubMed1990

A mutation of the beta 2-adrenergic receptor impairs agonist activation of adenylyl cyclase without affecting high affinity agonist binding. Distinct molecular determinants of the receptor are involved in physical coupling to and functional activation of Gs.

Activation of guanyl nucleotide regulatory proteins (G proteins) by hormones and neurotransmitters appears to require the formation of high affinity agonist-receptor-G protein ternary complexes. In the case of the beta 2-adrenergic receptor, multiple regions of the molecule have been implicated in coupling to the stimulatory G protein Gs. This finding raises the possibility that discrete regions of the receptor mediate ternary complex formation, whereas different loci may be involved in other aspects of G protein activation. To date, however, mutagenesis studies with the beta 2-adrenergic receptor have not clarified this question since mutant receptors with impaired abilities to activate Gs have generally possessed a diminished capacity to form the ternary complex as assessed in binding assays. We have expressed in a mammalian cell line a mutant beta 2-adrenergic receptor comprising a seven-amino acid deletion in the carboxyl-terminal region of its third cytoplasmic loop (D267-273), a region proposed to be critically involved in coupling to Gs. When tested with beta-adrenergic agonists, the maximal adenylyl cyclase response mediated by this mutant receptor was less than one-half of that seen with the wild-type receptor. Nevertheless, D267-273 exhibited high affinity agonist binding identical to that of the wild-type receptor. In addition, agonist-induced sequestration of the receptor, a property not mediated by Gs, was also normal. These findings indicate that the formation of high affinity agonist-receptor-Gs complexes is not sufficient to fully activate Gs. Instead, an additional stimulatory signal appears to be required from the receptor. Our data thereby suggest that the molecular determinants of the beta 2-adrenergic receptor involved in formation of the ternary complex are not identical to those that transmit the agonist-induced stimulatory signal to Gs.

Adenylyl Cyclases↗

PubMed1990

Cyclic AMP regulation of Gs protein. Thyrotropin and forskolin increase the quantity of stimulatory guanine nucleotide-binding proteins in cultured thyroid follicles.

This study was carried out to clarify the way in which thyrotropin (TSH) and forskolin regulate the adenylylcyclase complex in thyroid follicle cells. We examined the effects of chronic treatment of pig thyroid follicles with TSH or forskolin on the state of G proteins by (a) assaying adenylylcyclase activity, (b) analyzing the ADP-ribosylation of stimulatory G protein (Gs) by cholera toxin, and (c) quantifying the Gs subunits by Western blotting with antipeptide antibodies. Chronic exposure (18 h) of thyroid follicles to a low concentration of TSH (0.01-0.1 milliunit/ml) enhanced the subsequent response of adenylylcyclase to TSH. Higher concentration of TSH (1 milliunit/ml) induced a homologous desensitization of this response. In cells pretreated with forskolin, the TSH-stimulated adenylylcyclase activity was higher than in control cells. The forskolin-or guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p)-stimulated adenylylcyclase activity was always significantly increased after chronic treatment of cells with TSH or forskolin. Treatment of cultured thyroid follicle membranes with [32P]NAD and cholera toxin resulted in labeling of the Gs alpha (45-52-kDa) component. Culturing follicles with TSH (0.001-1 milliunit/ml) or forskolin (0.01-10 microM) greatly affected the cholera toxin-mediated ADP-ribosylation of the Gs alpha subunit. Gs alpha labeling increased progressively to level off at 1 milliunit/ml TSH or 1 microM forskolin (150-200%). Gs alpha immunoreactivity was increased in parallel (200-300%). The immunoreactivity of G beta subunits in cells cultured with TSH or forskolin was also increased compared with control cells. Cycloheximide abolished the effects of TSH and forskolin on the follicles, suggesting that new protein synthesis is required. These results indicate that Gs protein subunits are up-regulated by TSH and forskolin and suggest that their synthesis in thyroid cells is mediated, at least in part, by a cyclic AMP-dependent mechanism.

Adenosine Diphosphate Ribose↗

PubMed1989

Modification of the rat adipocyte A1 adenosine receptor-adenylate cyclase system during chronic exposure to an A1 adenosine receptor agonist: alterations in the quantity of GS alpha and Gi alpha are not associated with changes in their mRNAs.

The A1-adenosine receptor (A1AR) adenylate cyclase system in rat adipocytes undergoes heterologous desensitization following chronic in vivo exposure to an A1AR agonist (+)-N6-(R-phenylisopropyl)adenosine [J. Biol. Chem. 262:841-847 (1987)]. This desensitization involves an absolute increase in adenylate cyclase activity and a refractoriness to receptor ligands that are inhibitory to adenylate cyclase. In this study, receptor changes were characterized using an A1AR antagonist radioligand, [3H]8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropyl xanthine. Saturation binding studies demonstrated a 47% decrease in total A1AR density without a change in KD. Agonist competition studies revealed a decreased percentage of receptors, from 55% to 35%, in the high affinity state following desensitization. An increase in GS alpha of 49% was found by Western blotting using specific GS alpha antibodies. Further, an antibody that recognizes Gi alpha 1 adn Gi alpha 2 was used to quantitate these subtypes of Gi alpha and both were decreased by 59% following desensitization. However, when an antibody that recognizes Gi alpha 3 was used, no change in Gi alpha 3 was found, demonstrating, in this case, differential regulation of Gi alpha subtypes. The mechanisms responsible for changes in GS alpha and Gi alpha were studied by measuring the levels of their mRNAs from normal and desensitized adipocytes. Using either labeled cDNAs (Gi alpha 2, Gi alpha 3) or oligonucleotides (GS alpha, Gi alpha 1), Northern analysis demonstrated that mRNAs for GS alpha and all three isoforms of Gi alpha are present in adipocytes but that there are no changes in the levels of any of these transcripts following desensitization. These data suggest that desensitization of the A1AR-adenylate cyclase system involves a down-regulation of A1ARs and an additional loss of A1AR agonist high affinity sites. Further, an increase in GS alpha, a decrease in Gi alpha 1 and Gi alpha 2, and no change in Gi alpha 3 were found. The regulation of GS alpha and the subtypes of Gi alpha in this system does not occur by altering the levels of their respective transcripts.

Adenylyl Cyclases↗

PubMed1986

Catecholamine-stimulated GTPase cycle. Multiple sites of regulation by beta-adrenergic receptor and Mg2+ studied in reconstituted receptor-Gs vesicles.

The regulation of the intermediary steps of the catecholamine-stimulated GTPase cycle by beta-adrenergic agonists and Mg2+ was investigated using unilamellar phosphatidylethanolamine-phosphatidylserine vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. The steady-state turnover number of the agonist-stimulated GTPase, normalized according to the receptor-responsive pool of Gs, was 0.8 min-1 for untreated vesicles and 1.7 min-1 for vesicles that had been treated with dithiothreitol to activate the receptors. The binding and release of [alpha-32P]GTP, [3H] GTP, and [gamma-32P]GTP were used to measure the binding and hydrolysis of GTP and the release of GDP. Agonist-liganded receptor stimulated both the binding of GTP and the release of the GDP product, and GDP release per se did not appear to be the mechanism by which receptor stimulated the binding of GTP. Both processes displayed apparent first order rate constants of about 0.5 min-1 for untreated vesicles and both rates increased about 5-fold after dithiothreitol treatment. Both processes were formally catalytic with respect to receptor, in that several (up to 8) molecules of Gs were stimulated per molecule of receptor. The hydrolysis of Gs X GTP to Gs X GDP was unaltered by agonist and occurred with a rate constant of about 4 min-1. The rates of these partial reactions were consistent with the overall rate of steady-state hydrolysis and with the ability of the agonist-liganded receptor to promote the formation of sufficient Gs X GTP to fully stimulate adenylate cyclase in a native membrane. The Mg2+ dependence of agonist-stimulated, steady-state GTPase activity appeared to consist of at least two, distinct Mg2+-requiring processes. Very low concentrations of Mg2+ (approximately 20 nM) were required for hydrolysis of Gs X GTP, and 10 microM Mg2+ was required to maximize the initial rate of agonist-stimulated [alpha-32P] GTP binding.

Adrenergic beta-Agonists↗

PubMed1993

Mutagenesis of the amino-terminal glycine to alanine in Gs alpha subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation.

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.

Adenosine Diphosphate Ribose↗

PubMed1997

Reduced Gs protein function and G alpha s levels in leukocytes of patients with Parkinson's disease.

Early events in signal information transduction beyond dopamine, beta-adrenergic, and muscarinic receptors, involving receptor-coupled G-protein function and G alpha subunit immunoreactive levels were measured in mononuclear leukocytes (MNLs) of 12 never-treated patients with Parkinson's disease in comparison with 10 age- and sex-matched healthy control subjects. Both beta-adrenergic and dopamine receptor-coupled Gs protein function as measured by cholera toxin-sensitive, isoproterenol- and dopamine-induced increases in Gpp(NH)p-binding capacity, in MNLs of patients with Parkinson's disease were found to be significantly reduced in comparison with those in the control group. Muscarinic receptor-coupled non-Gs (Gi or G(o)) protein function: pertussis toxin-sensitive, carbamylcholine-induced increase in Gpp(NH)p-binding capacity, was not found to be significantly different between patients with Parkinson's disease and control subjects. G protein alpha subunits were measured through immunoblotting analyses with specific polyclonal antibodies against G alpha s, G alpha i, and G alpha q subunits. MNL levels of the 45-kDa species of G alpha s were found to be significantly reduced in patients with Parkinson's disease in comparison with control subjects. Other non-Gs proteins (Gi, Gq) did not show any significant quantitative differences between patients with Parkinson's disease and control subjects. The reductions in G alpha s levels in MNLs of patients with Parkinson's disease may explain the beta-adrenergic and dopamine receptor-coupled Gs protein hypofunction detected in MNLs of these patients. As previous studies have failed to observe significant changes in receptor levels in MNLs of patients with Parkinson's disease, our findings of reduced dopaminergic and beta-adrenergic receptor-coupled Gs function and of G alpha s immunoreactive levels in MNLs of Parkinson's patients point to alterations distal to these receptors at the level of the signal-transducing Gs protein.

Adult↗

PubMed1999

Expression of Gi-2 alpha and Gs alpha in myofibroblasts localized to the infarct scar in heart failure due to myocardial infarction.

OBJECTIVE: Patients surviving large transmural myocardial infarction (MI) are at risk for congestive heart failure with attendant alteration of ventricular geometry and scar remodeling. Altered Gi-2 alpha and Gs alpha protein expression may be involved in cardiac remodeling associated with heart failure, however their expression in scar tissue remains unclear. METHODS: MI was produced in Sprague-Dawley rats by ligation of the left coronary artery. Gi-2 alpha and Gs alpha protein concentration, localization and mRNA abundance were noted in surviving left ventricle remote to the infarct, in border and in scar tissues from 8 week post-MI hearts with moderate heart failure. RESULTS: We observed a 4.5- and 5.0-fold increase in immunoreactive Gi-2 alpha protein concentration occurs in the border and scar regions vs. control values, respectively, in 8-week post-MI rat hearts. Similarly, immunoreactive Gs alpha protein concentration was increased 3.4- and 8.2-fold, respectively, in these tissues vs. controls. Double-fluorescence labeling and phenotyping studies revealed that both Gi-2 alpha and Gs alpha proteins were localized to myofibroblasts in the infarct scar and to viable myocytes bordering the scar. Northern analysis revealed that the Gi-2 alpha/GAPDH ratio was increased in both viable and scar regions (1.24- and 1.85-fold respectively) from experimental hearts when compared to sham-operated control values when compared to noninfarcted left ventricle, the value of this ratio in scar tissue was elevated approximately 1.5 fold. The Gs alpha/GAPDH ratio was significantly increased (1.28-fold) only in the scar region vs. control. CONCLUSION: Our results indicate a marked increase in the expression of Gi-2 alpha and Gs alpha from myofibroblasts of the infarct scar as well as remnant myocytes bordering the scar in 8-week post-MI rat hearts. We suggest that these changes may be associated with ongoing remodeling in the infarct scar in chronic post-MI phase of this experimental model.

Analysis of Variance↗

PubMed1999

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.

Adenylyl Cyclases↗

PubMed2006

Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain.

BACKGROUND AND PURPOSE: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different. EXPERIMENTAL APPROACH: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay. KEY RESULTS: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. CONCLUSIONS AND IMPLICATIONS: The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).

Allosteric Regulation↗

PubMed2002

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling.

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.

Adenylyl Cyclases↗

PubMed2005

Ethanol feeding impairs insulin-stimulated glucose uptake in isolated rat skeletal muscle: role of Gs alpha and cAMP.

BACKGROUND: The mechanism by which chronic alcohol consumption impairs insulin sensitivity is unclear. We investigated the role of the Gs alpha-mediated pathway in decreasing insulin sensitivity in skeletal muscle after ethanol consumption. METHODS: Sixty male Wistar rats, divided into four groups, received either distilled water (controls; group I) or ethanol, which was administered by a gastric tube as a single daily dose of 5 g/kg (group II), 2.5 g/kg (group III), or 0.5 g/kg (group IV). After 20 weeks, fasting plasma glucose and serum insulin levels were measured. The hyperinsulinemic-euglycemic clamp study was performed under anesthesia to estimate whole-body insulin sensitivity. Insulin-stimulated glucose uptake was measured in vitro in dissected gastrocnemius muscle. Expression of glut4, Gs alpha, and Gi alpha was quantified using real-time PCR analysis and western blotting. cAMP levels were measured by ELISA. RESULTS: Compared with controls, the following observations were made: (1) the hyperinsulinemic-euglycemic clamp study revealed impaired insulin action at the whole-body level after ethanol treatment; (2) chronic ethanol feeding at 5 g/kg and 2.5 g/kg significantly decreased both basal and insulin-stimulated glucose uptakes in isolated skeletal muscle (p<0.05), which was accompanied by decreased expression of glut4 (p<0.05); (3) Gs alpha (mRNA and protein) expression in skeletal muscle was significantly increased in all three ethanol groups (p<0.05), and cAMP levels were also increased by ethanol treatment (p<0.05); and (4) there was no significant change in Gi alpha expression in all three ethanol groups. CONCLUSIONS: Chronic ethanol exposure decreased insulin-induced glucose uptake in rat skeletal muscle, which was associated with increased expression of Gs alpha. Because Gs alpha is a negative regulator of insulin sensitivity, the alteration in Gs alpha expression may contribute to the ethanol-induced impairment of insulin signal transduction.

Alcohol Drinking↗

PubMed2005

Guanine nucleotide exchange-independent activation of Gs protein by beta2-adrenoceptor.

beta2-adrenoceptor-mediated activation of Gs and adenylyl cyclase or other receptor-mediated G protein activations is believed to occur by receptor-catalyzed replacement of GDP with GTP on the alpha-subunit of the G protein. Here we showed that a beta2-adrenoceptor-Gs system, heterologously expressed in cyc- or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). The potency and maximal ability of GDP to activate Gs and adenylyl cyclase were identical to those of GTP. GDP-mediated activation of adenylyl cyclase, similar to that mediated by GTP, was concentration-dependent, required high magnesium concentrations, was inhibited by inverse agonists, and was correlated with the efficacy of receptor ligands used to stimulate the receptor. UDP did not block the GDP-mediated activation, although it completely blocked the formation of a small amount of GTP ( approximately 5% GDP) from GDP. Moreover, the activation of Gs in the presence of GDP was insensitive to cholera toxin treatment of the cells, whereas that observed in the presence of GTP was amplified by the treatment, which showed that the activation observed in the presence of GDP was not mediated by GTP. Therefore, we concluded that GDP itself could mediate beta-adrenoceptor-induced activation of Gs-adenylyl cyclase system as much as GTP. We discuss the results in the context of the current paradigm of receptor-mediated G protein activation and propose an additional mode of activation for beta2-adrenoceptor-G(s) adenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptor-induced Gs protein activation.

Adenylyl Cyclases↗

PubMed1997

Enhanced stimulatory adenylyl cyclase signaling during opioid dependence is associated with a reduction in palmitoylated Gs alpha.

Chronic opioid treatment of stably mu-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/mu 13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 microM; 2 days) A431/mu 13 cells display higher stimulatory AC activities as assessed in the S49cyc- reconstitution assay. This finding is most likely due to an increased functional activity of Gs alpha because the addition of exogenous G beta gamma subunits, which per se stimulate AC in S49cyc- membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment incresed the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of Gs alpha. Indeed, metabolic labelling studies with [3H]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated Gs alpha in the plasma membrane. Furthermore, high affinity [3H]forskolin binding experiments demonstrated that depalmitoylated Gs alpha is able to associated directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of Gs alpha provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of Gs alpha in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.

Adenylyl Cyclases↗

PubMed2006

Determination of Gs alpha protein activity in Albright's hereditary osteodystrophy.

Albright's hereditary osteodystrophy (AHO) is a heterogeneous clinical entity in part associated with pseudohypoparathyroidism (PHP) and other endocrinopathies. It may be caused by diminished Gs alpha protein activity. Heterozygous mutations in the underlying GNAS gene on chromosome 20 have been described. One hundred and six patients with suspected AHO, were investigated, of whom 93 showed a laboratory profile of PHP with low or normal calcium and elevated parathormone with normal vitamin D metabolites, and 13 had no endocrine abnormalities. Gs alpha activity was determined in isolated erythrocyte membranes. Molecular genetic analysis of GNAS exons 2-13 was initiated. Significantly reduced Gs alpha activity was found in 91 patients. In 53 patients with reduced Gs alpha activity, a mutation within GNAS was demonstrated. The mutation detection rate was much lower in AHO patients without endocrinopathies than in those who had PHP. In addition, three of the 15 patients with AHO features but normal Gs alpha activity had genetic variations of GNAS. We conclude that determination of Gs alpha activity can be used as a diagnostic screening procedure in patients with suspected AHO. However, the mutation detection rate in GNAS is highly variable. The genetic heterogeneity of AHO needs further investigation.

Adolescent↗

PubMedDate not supplied

Receptor-mediated activation of the heterotrimeric G-protein Gs results in ATP release from erythrocytes.

BACKGROUND: Adenosine triphosphate (ATP), released from the erythrocyte in response to mechanical deformation, decreased oxygen tension or reduced pH, has been suggested to be an important determinant of vascular resistance in several vascular beds. Mechanical deformation-induced ATP release from rabbit and human erythrocytes was reported to require the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), suggesting that a signal transduction pathway involving CFTR mediates ATP release from erythrocytes. Here we investigate the hypothesis that the heterotrimeric G-protein Gs is also involved in this signal transduction pathway. MATERIALS AND METHODS: The heterotrimeric G-protein Gs was identified in rabbit and human erythrocyte membranes, using gel electrophoresis. The concentration of ATP released into a suspension of erythrocytes, incubated with iloprost or epinephrine, was measured using the luciferin/luciferase assay. RESULTS: The 45 kDa form of the heterotrimeric G-protein Gs was identified in rabbit and human erythrocyte membranes. Incubation of rabbit erythrocytes with iloprost (n=18) or epinephrine (n=6) increased the ATP concentration by 106+/-16% and 156+/-54%, respectively. Epinephrine-induced changes in ATP concentrations were prevented by pretreatment with propranolol. CONCLUSIONS: The heterotrimeric G-protein Gs is present in erythrocyte membranes. Receptor-mediated activation of Gs results in ATP release. These results are consistent with the hypothesis that Gs is a component of a signal transduction pathway for ATP release from erythrocytes.

Adenosine Triphosphate↗

PubMed2003

Presynaptic impairment of synaptic transmission in Drosophila embryos lacking Gs(alpha).

Gs(alpha) is a subunit of the heterotrimeric G-protein complex, expressed ubiquitously in all types of cells, including neurons. Drosophila larvae, which have mutations in the Gs(alpha) gene, are lethargic, suggesting an impairment of neuronal functions. In this study, we examined synaptic transmission at the neuromuscular synapse in Gs(alpha)-null (dgsR60) embryos shortly before they hatched. At low-frequency nerve stimulation, synaptic transmission in mutant embryos was not very different from that in controls. In contrast, facilitation during tetanic stimulation was minimal in dgsR60, and no post-tetanic potentiation was observed. Miniature synaptic currents (mSCs) were slightly smaller in amplitude and less frequent in dgsR60 embryos in normal-K+ saline. In high-K+ saline, mSCs with distinctly large amplitude occurred frequently in controls at late embryonic stages, whereas those mSCs were rarely observed in dgsR60 embryos, suggesting a developmental defect in the mutant. Using the Gal4-UAS expression system, we found that these phenotypes in dgsR60 were caused predominantly by lack of Gs(alpha) in presynaptic neurons and not in postsynaptic muscles. To test whether Gs(alpha) couples presynaptic modulator receptors to adenylyl cyclase (AC), we examined the responses of two known G-protein-coupled receptors in dgsR60 embryos. Both metabotropic glutamate and octopamine receptor responses were indistinguishable from those of controls, indicating that these receptors are not linked to AC by Gs(alpha). We therefore suggest that synaptic transmission is compromised in dgsR60 embryos because of presynaptic defects in two distinct processes; one is uncoupling between the yet-to-be-known modulator receptor and AC activation, and the other is a defect in synapse formation.

Adenylyl Cyclases↗

PubMed2005

Rabbit erythrocytes possess adenylyl cyclase type II that is activated by the heterotrimeric G proteins Gs and Gi.

Adenosine triphosphate (ATP) release from rabbit erythrocytes occurs in response to deformation or reduced oxygen tension. A signal transduction pathway that relates these stimuli to ATP release has been proposed. This pathway includes the heterotrimeric G proteins, Gs and Gi, adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator. Importantly, adenylyl cyclase types II, IV and VII have been reported to be activated by both Gs and Gi. Here, we demonstrate that rabbit erythrocytes possess an adenylyl cyclase subtype that is activated both by the alpha subunit and the betagamma subunit of Gs and Gi, respectively. Washed rabbit erythrocytes released ATP when exposed to the beta adrenergic receptor-mediated activator of Gs, isoproterenol (ISO, 10 microM, n = 8, p < 0.05) as well as in response to incubation with a direct activator of Gi, mastoparan 7 (MAS7, 10 microM, n = 12, p < 0.05). In contrast, an inactive mastoparan derivative, mastoparan 17 (MAS 17, 10 microM, n = 6) did not stimulate ATP release. Importantly, incubation of washed rabbit erythrocytes with either isoprotenerol (ISO) (10 microM, n = 7) or MAS7 (10 microM, n = 11) resulted in increases in cyclic adenosine monophosphate (cAMP) (p < 0.01).Western analysis was used to determine if an adenylyl cyclase capable of being activated by both Gs and Gi was a component of rabbit erythrocyte membranes. We identified adenylyl cyclase type II with two antibodies generated against different epitopes of the protein. These results provide support for the hypothesis that, in rabbit erythrocytes, activation of either Gs or Gi results in the stimulation of adenylyl cyclase resulting in increases in cAMP leading, ultimately, to the release of ATP.

Adenosine Triphosphate↗