Constitutive activation of the Gs alpha protein-adenylate cyclase pathway may not be sufficient to generate toxic thyroid adenomas.

These studies examine the regulation of adenylate cyclase in renal cortical membranes from phosphate-deprived and phosphate-deprived acidotic dogs. Enzyme stimulation by parathyroid hormone (PTH) was decreased in phosphate deprivation [Vmax 1,578 +/- 169 vs. 2,581 +/- 219 pmol adenosine 3',5'-cyclic monophosphate (cAMP).mg protein-1 x 30 min-1 in controls, P less than 0.01]. Metabolic acidosis further decreased PTH-stimulated activity. Membranes from phosphate-deprived dogs showed a decrease in Gs alpha-content by cholera toxin-dependent ADP-ribosylation (174 +/- 18 arbitrary units vs. 266.4 +/- 13.6 in controls, P less than 0.01). Metabolic acidosis further decreased Gs alpha-content, P less than 0.01. Gi content by pertussis-dependent ADP-ribosylation was also lower in phosphate-deprived and phosphate-deprived acidotic animals. Gs function was examined by its property to protect the catalytic unit from inactivation by N-ethylmaleimide when preincubated with GTP gamma S. In controls, protection of inactivation was 80% of the maximal activity, whereas in phosphate deprivation protection was less than 50%. In conclusion, metabolic acidosis enhances adenylate cyclase resistance to PTH in phosphate deprivation. These alterations are associated with a decrease in the content and function of Gs alpha, suggesting a role of Gs in the renal adaptation to phosphate depletion and acidosis.

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The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.

The Journal of Cell Biology ◽

10.1083/jcb.111.4.1427 ◽

1990 ◽

Vol 111 (4) ◽

pp. 1427-1435 ◽

Cited By ~ 17

Author(s):

Y Audigier ◽

L Journot ◽

C Pantaloni ◽

J Bockaert

Keyword(s):

Adenylate Cyclase ◽

Binding Proteins ◽

Alpha Subunit ◽

Amino Terminus ◽

Gtp Binding Proteins ◽

Amino Terminal ◽

Alpha Subunits ◽

Gtp Binding ◽

Carboxy Terminal ◽

Gs Alpha

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

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Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways

Biochemical Journal ◽

10.1042/bj2970233 ◽

1994 ◽

Vol 297 (1) ◽

pp. 233-239 ◽

Cited By ~ 23

Author(s):

P A Stevens ◽

S Pyne ◽

M Grady ◽

N J Pyne

Keyword(s):

Smooth Muscle ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Cholera Toxin ◽

Phospholipase D ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Tracheal Smooth Muscle ◽

Constitutive Activation ◽

Cyclic Amp Accumulation

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5′-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

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Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.4.g686 ◽

1993 ◽

Vol 265 (4) ◽

pp. G686-G698 ◽

Cited By ~ 1

Author(s):

B. S. Dixon ◽

E. Sutherland ◽

A. Alexander ◽

D. Nibel ◽

F. R. Simon

Keyword(s):

Adenylate Cyclase ◽

Apical Membrane ◽

Bile Duct Ligation ◽

Basolateral Membrane ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Gtp Binding ◽

Duct Ligation ◽

Apical Membranes ◽

Gs Alpha

Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

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Thyrotropin (TSH) stimulates cell growth and DNA synthesis in monolayer cultures of human thyrocytes independent of the adenylate-cyclase system

Acta Endocrinologica ◽

10.1530/acta.0.114s273 ◽

1987 ◽

Vol 116 (1_Suppl) ◽

pp. S273-S280 ◽

Cited By ~ 7

Author(s):

Peter E. Goretzki ◽

Rainer Koob ◽

Christine Koller ◽

Hans-Dietrich Röher

Keyword(s):

Thyroid Cancer ◽

Cell Growth ◽

Adenylate Cyclase ◽

Dna Synthesis ◽

Thymidine Incorporation ◽

Human Thyroid ◽

Adenylate Cyclase System ◽

Monolayer Cultures ◽

Cancer Metastases ◽

Thyroid Adenomas

Abstract. Monolayer cultures of human thyrocytes from normal thyroids (n = 13), thyroid adenomas (n = 8), differentiated (n = 7), poorly and undifferentiated (n = 5) thyroid cancers as well as thyroid cancer metastases (n = 2) were established to assess the significance of TSH and cAMP on cell growth and DNA synthesis. Cell growth was stimulated by 0.1 IU bTSH/ml and inhibited by 1.0 IU bTSH/ml (P < 0.01), while dibutyryl-cAMP (dbcAMP) failed to show any effect on cell growth at the concentrations (10–5; 10–3 mol/l), tested. Neither did the adenylate-cyclase inhibitor dideoxy-adenosine (ddA) (2 × 10–5 mol/l) stimulate thyrocyte growth. DNA synthesis, however, measured indirectly by [3H]thymidine incorporation, was stimulated not only by TSH 2–12-fold, but also by ddA 1.3–7-fold (P <0.01), and was not affected by dbcAMP. TSH had no effect on [3H]thymidine incorporation in fibroblasts and c-cells from c-cell carcinomas. The stimulatory effect of TSH on thyrocyte growth and DNA synthesis was unpredictable in thyrocytes from cancerous tissues (n = 14) with no obvious correlation to tumour differentiation or stage. Thus, we showed that TSH is a promotor for cell growth and DNA synthesis in monolayer cultures of human thyrocytes from normal and adenomatous human thyroid tissues with no obvious correlation to the AC system. This TSH effect is unpredictable, however, in thyrocytes from human thyroid cancer.

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Attenuation of Gs α coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters

Biochemical Journal ◽

10.1042/bj2950655 ◽

1993 ◽

Vol 295 (3) ◽

pp. 655-661 ◽

Cited By ~ 16

Author(s):

P Svoboda ◽

L Unelius ◽

B Cannon ◽

J Nedergaard

Keyword(s):

Adipose Tissue ◽

Adenylate Cyclase ◽

Plasma Membranes ◽

Coupling Efficiency ◽

Ouabain Binding ◽

Beta Adrenergic ◽

Brown Adipose ◽

Density Band ◽

Gs Protein ◽

Gs Alpha

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [3H]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [3H]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after Gs-protein-mediated activation (with guanosine 5′-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the Gs protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute Gs-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional Gs proteins and adenylate cyclase units. The total content of Gs alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent Gs alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in Gs-protein-mediated coupling efficiency, owing to a decrease in the amount of Gs alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta 3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.

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The effect of iloprost on the ADP-ribosylation of Gsα (the α-subunit of Gs)

Biochemical Journal ◽

10.1042/bj2610841 ◽

1989 ◽

Vol 261 (3) ◽

pp. 841-845 ◽

Cited By ~ 29

Author(s):

L Molina y Vedia ◽

R D Nolan ◽

E G Lapetina

Keyword(s):

Adenylate Cyclase ◽

Cholera Toxin ◽

Alpha Subunit ◽

Α Subunit ◽

Present Evidence ◽

Adp Ribosylation ◽

Specific Antisera ◽

Membrane Bound ◽

Gs Alpha ◽

Adp Ribosyltransferase

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

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Quantification of signalling components and amplification in the β-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes

Biochemical Journal ◽

10.1042/bj3110075 ◽

1995 ◽

Vol 311 (1) ◽

pp. 75-80 ◽

Cited By ~ 95

Author(s):

S R Post ◽

R Hilal-Dandan ◽

K Urasawa ◽

L L Brunton ◽

P A Insel

Keyword(s):

Adenylate Cyclase ◽

G Protein ◽

Cardiac Myocytes ◽

Signal Amplification ◽

Adrenergic Receptor ◽

Ventricular Myocytes ◽

Binding Assays ◽

Beta Adrenergic ◽

Adult Rat ◽

Gs Alpha

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5′-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

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