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Guanosine-5′-O-thiodiphosphate functions as a partial agonist for the receptor-independent stimulation of neural adenylate cyclase
Brain Research, 488 (1989) 105-113 Elsevier
105
BRE 14505
Guanosine-5"-O-thiodiphosphate functions as a partial agonist for the receptor-independent stimulation of neural adenylate cyclase M.M. Rasenick, J.M. Hughes and N. Wang Department of Physiology and Biophysics and the Committee on Neuroscience, University of Illinois College of Medicine, Chicago, IL 60680 (U.S.A.) (Accepted 1 November 1988) Key words: Signal transduction; Guanosine triphosphate-binding protein; fl-Adrenergic receptor; Receptor-effector coupling; Photoaffinity labeling; Cyclic nucleotide; C6 glioma cell
GTP-binding proteins (G proteins) have been implicated as mediators of several aspects of neuronal signal transduction including ion channels, phosphatidyl inositol turnover and the stimulation or inhibition of adenylate cyclase. Several investigators have employed the stable guanosine diphosphate (GDP) analog, guanosine 5"-O-thiodiphosphate (GDPflS) to block putative G protein-mediated processes. Although GDPflS is assumed to block G protein function, some investigators have reported partial activation of G protein-mediated processes by this compound. In this study we demonstrate that GDPflS functions as a partial agonist for the adenylate cyclase system. In rat cerebral cortex membranes, GDPflS activates adenylate cyclase with an ECs0 similar to the hydrolysis resistant GTP analog, guanylylimidodiphosphate (GppNHp), but to a far lower extent. Further, GDP/~S antagonizes the activation of adenylate cyclase by high doses of GppNHp or GTPyS (another stable GTP analog) but potentiates adenylate cyclase activation by low doses of these nucleotides. High doses of GDPflS provoke, only partially, exchange of nucleotides among G proteins, as measured by the transfer of the photoaffinity GTP analog, azidoanilido-GTP, between the inhibitory and stimulatory GTP-binding proteins. In the presence of the/~-adrenergic agonist, isoproterenoi, GDPflS fails to support stimulation of C6 glioma membrane adenylate cyclase and inhibits GppNHp- or GTPyS-mediated stimulation of that enzyme. Inhibition of C6 membrane adenylate cyclase by GTP analogs is also blocked by GDPflS. Finally, in C6 cells made permeable with saponin, where the /~-adrenergic receptor is 'tightly coupled' to the adenylate cyclase system, GDPflS, GppNHp or GTPyS stimulate adenylate cyclase at high concentrations, but only in the absence of isoproterenol. When isoproterenol is added to these cells, GppNHp or GTPyS enhance adenylate cyclase activation, yet GDPflS acts only to block that process. Thus, GDPflS appears to function as partial agonist for the stimulatory adenylate cyclase G protein but only when an activated neuroreceptor is not coupled to that protein. INTRODUCTION GTP-binding proteins (G) represent a class of signal transducing proteins which link receptors to their intracellular effector molecules, as in the adenylate cyclase or rod outer segment phosphodiesterase systems 7'22. Generally, occupation of a specific neuroreceptor by its agonist will allow binding of G T P (or a GTP analog) to a certain G protein. Subsequently, the activated G protein acts as mediator of the intracellular signaling process to be stimulated or inhibited in response to that neurotransmitter. Whereas the identity of G pro-
teins stimulating (Gs) and inhibiting (Oi) adenylate cyclase is well established, the identity of G proteins apparently involved in other signaling processes (phosphatidyl inositol turnover; ion channel modulation) has not been established. Further, while several elegant biochemical and genetic studies have attempted to establish the precise mechanism for the coordinate regulation of r e c e p t o r - G protein-mediated signal transduction processes, it is impossible for these studies to account for the complexities of the intact neuron 15. Some neurophysiologists have attempted to study G protein-regulated processes by injection of G
Correspondence: M. M. Rasenick, Department of Physiology and Biophysics, M/C 901, University of Illinois College of Medicine, P.O. Box 6998, Chicago, IL 60680, U.S.A. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
106 proteins or G protein inhibitors into living cells. The stable GDP analog, guanosine 5"-O-(2-thiodiphosphate) (GDPflS), has been used by several investigators as an inhibitor of GTP-dependent processes 6 and this compound appears to bind to a variety of GTP-binding proteins, fixing them in the inactive state. GDPflS has been thought to inactivate the GTP binding proteins which stimulate (Gs) or inhibit (Gi) adenylate cyclase 5'11. Further, GDPflS has been used in electrophysiological studies to inhibit processes thought to be mediated through the activation of adenylate cyclase ~2. Most recently, GTP-binding proteins have been implicated as direct activators or inhibitors of ion channels, and GDPflS has been used as an inhibitor of G protein effects in some of these studies as well 1"4"1°. Several inconsistencies have appeared in an understanding of GDPflS action in the adenylate cyclase system. Whereas GDPflS has been effective at antagonizing role of GTP or GTP analogs in the hormone- or neurotransmitter-induced stimulation or inhibition of adenylate cyclase, GDPflS has been reported to increase cAMP accumulation in some systems 2°. Further, pressure injection of GDPflS has been observed to block the effects of serotonin (5-HT) in promoting secretion from helix neurons, but GDPflS activated the target cells in the absence of the hormone 12. In this study, we have attempted to answer these apparent contradictions by describing the site of GDPflS action. We demonstrate that GDPflS acts as a partial agonist for G~ only when G~ is not associated with a neuroreceptor. MATERIALS AND METHODS
Tissue preparation Enriched synaptic membrane fractions from 21day-old Sprague-Dawley rats were prepared as per Rasenick et al. 17 and stored under liquid N 2 until use. C6 cells were grown in Dulbecco's MEM; 4.5 g glucose/liter 10% fetal bovine serum, and harvested just prior to confluence. Membranes were prepared as indicated in Rasenick and Kaplan 16 and stored under liquid N 2 until use.
Adenylate cyclase assays Adenylate cyclase was assayed in cerebral cortex
membranes as described by Hatta et al. ~' or in C6 membranes as described in Rasenick and Kaplan ~, Unless otherwise noted, assay incubation mixtures included 15 mM HEPES, pH 7.4.1 mM dithiothreitol (DTI'), 0.3 mM phenylmethylsulfonylfluoride, 5 mM MgC1, 50 ,uM ATE [a-32p]ATP (5 x 1(I5 cpm/tube), 60 mM NaC1, 0.25 mg/ml bovine serum albumin, 0.5 mM isobutylmethylxanthine, 1 unit/ml adenosine deaminase, 0.5 mg/ml creatine phosphate, 0.14 mg/ml creatine phosphokinase and 15 units/ml myokinase. Assays were stopped by the addition of 100,ul of 2% SDS, 1.4 mM cAMP, 40 mM ATP and cAMP accumulated was determined by the method of Salomon 19 using [3H]cAMP (3 x 104 cpm/tube) to monitor recovery.
Permeable C6 cells C6 cells were grown to near confluence on 24-well plates and made permeable with saponin (10 ~g/ml) as per Rasenick and Kaplan 16. Assays were performed as described in Rasenick and Kaplan16; incubations including 0.5 mM ATP, [a-32p]ATP (2 x 10 6 cpm/well), 1 mM MgC1 and 0.5 mM isobutylmethylxanthine in Hanks buffer. Total assay volume was 150 ¢tl/well and reactions were stopped after 15 min by addition of 300/~1 ice-cold HEPES (15 raM, pH 7.4) and freezing on dry-ice. [32p]cAMP produced was determined as above.
Photoaffinity experiments p3-azidoanilido-PI-GTP (AAGTP) was synthesized by the method of Pfeuffer 13 with p-azidoaniline provided by Dr. George Wheeler. As described in Hatta et al. 9, membranes were incubated with 1.2 x 10 -7 M [32p]AAGTP for 3 min, washed twice and subjected to a 10 min incubation (23 °C) in the presence of the indicated nucleotide. After this, UV photolysis was performed for 5 min followed by SDS-PAGE and radiofluorographic analysis of the dried gel. Radioactivity incorporated by proteins of interest was determined by excising bands corresponding to those in the autoradiograph and counting in a Beckman LS 5800 liquid scintillation counter s . In tandem experiments, membranes were exposed to 10 -4 M AAGTP, washed and assayed for adenylate cyclase in the presence of the indicated nucleotide.
107
Purification and analysis of guanine nucleotides
All other reagents were of highest purity obtainable.
Guanine nucleotides obtained commercially were dissolved in distilled water to make a 10-2 M solution. 200 pl vols. were applied carefully to the A G 50W-X 4 resin (25 x 1 cm pre-equilibrated with HCI, pH 1.15) and washed in with 2 ml of pH 1.15 HC1 solution. The column was eluted with pH 1.15 HC1 and samples were collected with fraction size of 1 ml and flow rate of 1 ml/min. In the preliminary experiments, a mixture of GTPTS and GDPflS was applied to the column. GTPyS appeared first and totally separated from GDPflS peak. The purified nucleotides were collected and pooled separately, lyophilized and dissolved in water. For thin-layer chromatography (TLC) analysis the nucleotides were spotted on a polyethyleneimine cellulose sheet and developed in 0.5 M NaCI and 0.75 M Tris, pH 8.0. Rf values for nucleotides in this system were: GDPflS, 0.24; GTPflS, 0.09; GTPTS, 0.010; GTP, 0.18; GDP, 0.39; GMP, 0.57.
Materials All nucleotides (except A A G T P ) used in this study were obtained from Boehringer Mannheim (Elkhart, Ind.). p-Azidoaniline (used in the synthesis of A A G T P ) was a gift from G.L. Wheeler and 1-propranolol was a gift from Ayerst laboratories.
400
RESULTS
Activation of adenylate cyclase by various GTP analogs When synaptic membranes were incubated with various GTP analogs and assayed for adenylate cyclase (Fig. 1) it was found that GTPTS, G p p N H p and GDPflS (but not GTP) were capable of activating that enzyme. The ECs0 value for GTPTS was calculated at 2 x 10-8 M and that for G p p N H p or GDPflS was approximately 4 x 10 -7 M. The Vmax of adenylate cyclase activation with GTPTS or G p p N H p was about 300 pmol cAMP/mg protein/ rain; the maximal activation of the enzyme achieved in the presence of GDPflS was only 60% of that seen with the triphosphate analogs.
Antagonism of adenylate cyclase activation by GD PflS Data in the initial experiment raised the possibility that GDPflS was acting as a partial agonist for the activation of adenylate cyclase in synaptic membranes. This was tested by assaying adenylate cyclase with increasing doses of GTPyS in the presence of 10 p M GDPflS (Fig. 2). Under these conditions, GDPflS acted as a partial agonist, contributing to the stimulation of adenylate cyclase by low concentra-
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Fig. 1. Activation of rat cerebral cortex adenylate cyclase by various GTP analogs. Rat cerebral cortex synaptic membranes (12 gg protein/tube) were assayed for adenylate cyclase as described in the methods section. Assays were carried out in the presence of the indicated nucleotide at the indicated concentration and stopped after 10 min. Means of triplicate determinations +_S.E.M. from 1 of 4 identical experiments are depicted. Where error bars are not seen, they are smaller than the symbols.
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Fig. 2. Antagonism of adenylate cyclase activation by GDPflS. Synaptic membrane adenylate cyclase was assayed in the presence of the indicated concentrations of GTPyS with or without GDPflS (1/~M). Means of triplicate determination + S.E.M. from 1 of 3 identical experiments are shown. Where error bars are not seen, they are smaller than the symbols.
108 tions of GTPTS and inhibiting the stimulatory effects of higher GTPTS concentrations. GTPyS concentrations exceeding 10 g M overrode GDPflS effects.
Purity and stability of GDPflS Some studies employing GDPflS have noted effects which were attributed to GTPflS, the phosphorylated product of GDPflS. A variety of synaptic membrane phosphatases hydrolyze GTP rapidly, rendering it ineffective at stimulating (Fig. 1) or inhibiting (not shown) synaptic membrane adenylate cyclase. Thus, it was thought unlikely that GTPflS would regulate the enzyme. Nonetheless, we investigated whether GTPflS was generated during the course of these experiments by the synaptic membranes. Fig. 3 shows that no GTPflS was produced during the course of these incubations. Further, the GDPflS used was free of contamination from GTP~'S. The commercially available GDPflS contained about 2% GDP. When the purified GDPflS was used in experiments as shown in Fig. 1, no differences were seen compared to the commercial preparation.
Photoaffinity studies In previous studies from this laboratory, we demonstrated that G p p N H p or other hydrolysisresistant analogs of GTP induce inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing of these membranes with buffer dilution and centrifugation. A subsequent incubation with GppNHp, GTP~'S or unlabelled A A G T P 'disinhibits' adenylate cyclase in a dosedependent fashion (Fig. 4c). If the hydrolysisresistant photoaffinity GTP analog ([32p]AAGTP) is used in similar experiments, under the conditions where adenylate cyclase is inhibited, A A G T P is bound primarily to the inhibitory GTP-binding protein, G+ and a similar protein, G o. A subsequent incubation with G p p N H p (or other GTP analogs) causes a shift in the incorporation of A A G T P from Gi/o to G s, the sum of the A A G T P binding to these two proteins remaining constant (Figs. 4a and 4b). Under these conditions, A A G T P appears to transfer directly between Gi/o and G s. Fig. 4a compares the ability of G p p N H p and GDPflS to promote the apparent exchange of A A G T P bound from G~ to G s. Whereas G p p N H p caused a 5-fold increase in the
ratio of A A G T P bound between G~ and Gi/,,, GDPflS promoted only a 2-fold increase. Further, G p p N H p was more potent than GDPflS; calculated ECs0s were 2 × 10 6 M for G p p N H p and 2 x 10 5 M for GDPflS. Finally, the data in 4c show the 'distribution' of adenylate cyclase by G p p N H p vs GDPflS. Corollary to data in Fig. 4a and b, GDPflS relieved inhibition of adenylate cyclase only 50% as extensively as G p p N H p and with a 100-fold lower potency; ECs( ~ = 5 × 10-s for G p p N H p vs 5 × 10.6 for GDPflS. GDPflS promoted the apparent transfer of A A G T P from G i to G~ with a similarly lower efficacy and potency (Fig. 4b).
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Fig. 3. Purity and stability of guanosine thiophosphates. GDP/3S and GTP~S were purified on Dowex columns as described in Materials and Methods. GDPflS was contaminated by approximately 2% GDP. To monitor possible breakdown of GDPflS and/or production of GTPflS, samples of GDP/3S (1 raM) were incubated (at the indicated temperature) with synaptic membranes under the conditions (and buffers) used for the adenylate cyclase assay. Thin-layer chromatographic analysis was performed as described in Materials and Methods. No GTPflS was formed. Rf values for nucleotides in this system were: GDPflS, 0.24; GTPflS, 0109; GTP, 0.18; GDP, 0.39; GMP, 0.57.
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Fig 4. Photoaffinity studies, a: AAGTP labelling and nucleotide exchange. Synaptic membranes were incubated with [a-~2P] AAGTP as described in methods. After 2 buffer washes, membranes were resuspended in 2 mM HEPES, pH 7.4, with I mM MgC1 and incubated with H20 or the indicated concentrations of GppNHp or GDPflS for 10 min and then UV irradiated. Thirty ~tg samples of each tube were subjected to SDS-PAGE and radiofluorography. Unlabelled nucleotide concentrations are the intermediate value between the two labelled concentrations. Results are from one of two identical experiments, b: transfer of AAGTP from Gi/o to G, evoked by GppNHp and GDPflS. Bands corresponding to G, and G~o were excised from the gel in Fig. 4a and radioactivity in each band was determined by scintillation counting. After background subtraction, the ratio of 32p clam in the 42 kDa (Gs) vs 40 kDa (G~o) bands was determined and is represented above. Note that the total 32p cpm in Gs + G~o averaged 1732 + 109 for each sample lane of the gel in Fig. 4a. c: reversal of adenylate cyclase inhibition by GppNHp and GDPflS. Synaptic membranes were pre-incubated with 100/zM unlabelled AAGTP for 3 rain at 23 °C. Following dilution and washing, membranes were assayed for adenylate cyelase, omitting EGTA and adding 1 mM rather than 5 mM MgCI; temperature = 23 °C. The membranes were not subjected to UV irradiation and the experiment was done under yellow light. The increase in adenylate cyclase activity parallels the increase in AAGTP transferring to G, seen in the labelling experiment in Fig. 4a (see text). Means of triplicate determinations from 2 experiments were normalized and are represented above.
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Fig. 5. Effects of GDP/3S on/3-receptor-mediated stimulation of adenylate cyclase in C6 glioma cell (membranes). Membranes prepared from C6 cells were assayed for adenylate cyclase as indicated in Materials and Methods. a: membranes were assayed in the presence (/k ,&) or absence (r-],l) of isoproterenol (1 #M) and the indicated concentration of GDP/~S ( I , & ) or GTP~,S (rq,A). Means of triplicate determinations for one of 3 experiments are depicted, b: C6 membranes were assayed as above in the presence of 1 MM isoproterenol (C],I,A) and the indicated concentration of GTPTS ([],A) or GDP/3S (I). One group of samples (&) was also assayed in the presence of 10 #M GDPflS. Mean + S.E.M. from 1 of 3 experiments is depicted.
t3-Receptor-mediated stimulation of C6 adenylate cyclase (membranes) Results presented thus far indicate that GDPflS functions as a partial agonist for the activation of adenylate cyclase in the absence of a hormone or neurotransmitter. As a consequence of cell disruption, synaptic membrane adenylate cyclase is insensitive to hormone or neurotransmitter15'24; however, membranes made from C6 glioma cells respond to isoproterenol (as well as to G p p N H p or GTPTS alone) for the activation of that enzyme. Specifically, 10 HM GTP~,S produced a 5-fold activation of C6 m e m b r a n e adenylate cyclase which, in the presence of 1 HM isoproterenol, was increased to 10-fold. U n d e r these same conditions, GDP/3S was ineffective at stimulating adenylate cyclase and isoproterenol did not alter this (Fig. 5a). When GDPflS (10 ktM) was included in the C6 membrane adenylate cyclase assay along with isoproterenol and GTPvS, the GTPTS concentration required for activation of adenylate cyclase was increased by about 30-fold ( - E C s 0 = 0.2 vs 6 # M ) (Fig. 5b). Effects of (1 #M) isoproterenol were blocked completely by 10 MM 1-propranolol (not shown).
Blockade of adenylate cyclase inhibition in C6 cells by GDPI3S When C6 membranes were incubated with NaF
(tO increase adenylate cyclase activity), subsequent assay with G p p N H p evoked a dose-dependent inhibition of adenylate cyclase (Fig. 6). U n d e r the same conditions, GDPflS stimulated the enzyme, although the efficacy of stimulation (34% at 10-5 M GDPflS) was much less than the inhibition (54% at
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Fig. 6. Blockade of adenylate cyclase inhibition in C6 cells by GDPflS. Membranes prepared from C6 cells were assayed for adenylate cyclase under the conditions described in Fig. 4c with the addition of 10 mM NaF and the indicated concentration of GppNHp ([5]) or GDPflS (I). Samples indicated by filled triangles (A) were incubated with 10 #M GDPflS plus the indicated concentration of GppNHp. Means + S.E.M. for 1 of 3 experiments are shown.
111 10-6 M GppNHp). Further, GppNHp-induced inhibition of adenylate cyclase was mitigated by 10 #M GDPflS (Fig. 6), this effect being overriden by excess concentrations of GppNHp.
Effects of GDP~S on adenylate cyclase in permeable C6 cells This laboratory has demonstrated previously, that when the basic structure of the C6 cell is preserved and those cells are made permeable with saponin,
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25
DISCUSSION
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guanine nucleotides activate adenylate cyclase only at very high concentrations. However, in the presence of a fl-adrenergic agonist, GTP analogs enhance adenylate cyclase activity significantly. Thus, unlike the case in C6 membranes, G s appears tightly coupled to the receptor in the permeable C6 cells. This system is analogous to an intact neuron in which GTP analogs might be pressure-injected. In the absence of isoproterenol, GTPyS or GDPflS stimulate adenylate cyclase only at concentrations in excess of 10-5 M (Fig. 7a). Under these conditions, 10-4 M GDPflS increased basal adenylate cyclase by 38.7 + 3.6% (average of 5 triplicate determinations ranging from 29% to 48%. A similar modest increase (range 24-61%) was seen with GTPyS in the absence of isoproterenol. However, in the presence of isoproterenol, GTPyS was a potent stimulator of adenylate cyclase (about 5-fold the basal value) whereas GDPflS (100 #M) increased activity to the same extent that it did in the absence of isoproterenoi. When GDPflS (10 #M) was added to permeable cells in the presence of isoproterenol and increasing concentrations of GTPyS, GDPflS acted to inhibit the stimulatory effects of GTPyS. Under these conditions the dose of GTPyS required to activate adenylate cyclase was increased about 10-fold (Fig. 7b).
•
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Fig. 7. Effects of GDP/~S on adenylate cyclase in permeable C6 cells. C6 cells were grown to near-confluence on 24-well plates, made permeable with saponin, and assayed for adenylate cyclase as described, a: cells were assayed in the
presence of the indicated concentration of GTPyS ([3,/k) or GDPflS (II,A) and the presence (kx,A) or absence (D,II) of 1 pM isoproterenol. Means of triplicate experiments from one of 4 similar experiments are represented. See text for normalized values of 10~ GDPflS or GTPyS in the absence of isoproterenol, b: samples were assayed as in (a), with the indicated concentration of GTPyS without (r-l) or with (11) 10 pM GDPflS. All samples contained isoproterenol (1 #M). Means _+ S.E.M. for 1 of 3 experiments are shown.
GDPflS has been reported to function as an antagonist of the adenylate cyclase system. Accordingly, investigators have used this compound in order to ascertain whether a G protein might mediate a certain cellular event. Accordingly, GDPflS has been used to block the activation6 and inhibition 11 of adenylate cyclase. Further, GDPflS has been employed to facilitate the opening of a K + channel 1 or to attenuate a voltage-sensitive Ca 2+ channel 4, both of which may be G protein-sensitive. Finally GDP/~S has been used to block receptormediated processes which have been surmised to act via the activation of adenylate cyclase12. Under these conditions, pressure-injection of GDPflS has resulted in the activation of those processes if receptor agonist was omitted from the outside of a cell. Similarly, high concentrations of GDPflS activate adenylate cyclase in permeable C6 cells. Such results
112 have been assumed by some investigators to be due to the presence of contaminating GTPyS in the GDPflS or the formation of GTPflS. In the present study however, GDPflS is uncontaminated and no formation of GTPflS was noted (Fig. 3). Data in Figs. 1 and 2 show that GDPflS behaves as a classic partial agonist 2 with regard to the G~-mediated stimulation of cerebral cortex adenylate cyclase. Data in Fig. 2 help to distinguish between two possible mechanisms of GDPflS action: partial agonism of G Sand antagonism of G i. Clearly, either would result in increased activation of adenylate cyclase. However, if GDPflS acted primarily to inhibit G i, then the adenylate cyclase activity in the presence of GDPflS plus GTPyS would be parallel to that of GTP),S alone at GTPyS concentrations exceeding l 0 -7 M. This is not the case (see Fig. 2). We have demonstrated previously that the photoaffinity GTP analog, AAGTP, can be transferred from Gi/o to Gs by the addition of an hydrolysisresistant GTP analog to the membranes 9. Added GTP analogs appear to cause the formation of G protein complexes (Hatta and Rasenick, unpublished) and AAGTP binding is increased on G S due to cooperative effects~. It appears that G~, G~, Go and a 32 kDa GTP binding protein (G32) are involved in this process. GDPflS is 10-fold less potent than GppNHp in promoting nucleotide exchange and 100-fold less potent in alleviating the stable inhibition of adenylate cyclase (Fig. 4). Furthermore the efficacy of GDPflS is 20-30% that of GppNHp in either of the above parameters. This contrasts with data in Fig. 1, where GDPflS has 60% of the efficacy and equal potency to GppNHp. Further, GDPflS competes with AAGTP for binding tO G s, Gi, G O or G32 with a potency near that of GppNHp 8. A possible explanation for the greater effects of GDPflS on the stimulation of adenylate cyclase (Fig. 1) than on nucleotide exchange (Fig. 4) is that the latter process appears to require activation of G~ and Gi/o. GDPflS does not activate G~ (Fig. 6 and ref. 11). Thus if GDPflS has partial agonist properties for G~ but not G~/o, it would have a greater effect upon a Gs-dependent process (adenylate cyclase stimulation) than on a process requiring activation of multiple G proteins. Although we demonstrate that GDPflS is a partial
agonist for the receptor-independent stimulation of adenylate cyclase, isoproterenot-induced stimulation (Figs. 5 and 7b) is blocked by this compound. These results are somewhat different from those seen in the turkey erythrocyte, where GDPflS antagonized the stimulation of adenylate cyclase5'6. This may be due to the distinction between the coupling of adenylate cyclase to stimulatory receptors in the different systems. In the tightly coupled turkey erythrocyte, isoproterenol and GTP are required to activate the enzyme 3, the occupied receptor allowing the binding of GTP to G~~s. Cerebral cortex synaptic membranes bind receptor agonists normally and display G protein-mediated down-regulation of agonist affinity23. However, a concomitant of cell disruption is the loss of neurotransmitter-activated adenylate cyclase in brain regions where intact-cell preparations show profound neurotransmitter-induced cAMP accumulation ~5,24 It appears that the presence of an activated receptor causes the coupled adenylate cyclase to respond differently to GDPflS. Human fat cells have been noted to respond similarly in this regard 2°. These data suggest the possibility that an agonist occupied receptor causes a coupled G~ to respond differently to GDPflS than an uncoupled G~. C6 glioma cells provided a useful system to test this hypothesis. C6 adenylate cyclase has several properties associated with neuronal adenylate cyclase, except that some degree of receptor coupling is maintained upon cell disruption. Permeable C6 cells maintain tight coupling between the fl receptor and G~ and offer a parallel to a neuron in which GTP analogs are pressure-injected. Consistent with the above hypothesis is the observation that in permeable C6 cells, GDPflS, GppNHp or GTPyS (all at >10ktM) caused a small yet reproducible increase in adenylate cyclase activity (Fig. 7a). However, in the less tightly coupled C6 membranes, GppNHp or GTPyS (without isoproterenol) activated the enzyme to a much greater extent than GDPflS (Fig. 5a). In addition to the receptor-independent activation of G proteins, fluoride appears to uncouple G proteins from receptors 5"21. Consistent with this uncoupling, in the presence of NaF, GDPflS stimulates C6 membrane adenylate cyclase to a greater extent than in the absence of NaF (compare effects of GDPflS in Fig. 6 with those in Fig. 5a).
113
Partial agonist activity of GDPflS is one of a series of observations which suggest that adenylate cyclase in cells of neural origin is regulated in a fashion distinct from that e n z y m e in o t h e r cells and tissues. O t h e r discrepancies b e t w e e n neural and non-neural a d e n y l a t e cyclase systems are the presence of calm o d u l i n activation, loss of r e c e p t o r coupling upon cell disruption and interaction with m e m b r a n e associated cytoskeletal c o m p o n e n t s (see refs. 14,15 for reviews). T h e relevance of these discrepancies to n e u r o t r a n s m i t t e r action and responsiveness have yet to be d e t e r m i n e d .
ACKNOWLEDGEMENTS
REFERENCES
potassium conductance in Aplysia neuron R15, J. Gen. Physiol., 83 (1984) 269-285. 13 Pfeuffer, T., GTP-binding proteins in membranes and the control of adenylate cyclase activity, J. Biol. Chem., 252 (1977) 7224-7234. 14 Rasenick, M.M., O'Callahan, C.M., Moore, C.A. and Kaplan, R.S., GTP-binding proteins which regulate normal adenylate cyclase interact with microtubule proteins. In M. DeBrabander and J. DeMey, (Eds.), Microtubules and Microtubule Inhibitors, Elsevier, Amsterdam, 1985, pp. 313-323. 15 Rasenick, M.M., Marcus, M.M., Hatta, Y., DeLeonJones, E and Hatta, S., Regulation of neuronal adenylate cyclase. In Y. Erlich, R. Lenox, E. Kornecki and W. Berry (Eds.), Molecular Mechanisms of Neuronal Responsiveness, Plenum, New York, 1987, pp. 123-133. 16 Rasenick, M.M. and Kaplan, R., Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells, FEBS Lett., 207 (1986) 296-301. 17 Rasenick, M.M., Stein, P.J. and Bitensky, M.W., Evidence that the regulatory subunit of adenylate cyclase interacts with cytoskeletal components, Nature (Lond.), 294 (1981) 560-562. 18 Rodbell, M., The role of hormone receptors and GTPregulatory proteins in membrane transduction, Nature (Lond.), 284 (1980) 17-22. 19 Salomon, Y., Adenylate cyclase assay, Adv. Cyclic Nuleotide Res., 10 (1979) 35-53. 20 Schreyer, C.R., Pineyro, M.A., Kirkland, J.L. and Gregerman, R.I., Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5"-O-(2-thiodiphosphate), J. Biol. Chem., 259 (1984) 7038-7044. 21 Stein, P.J., Halliday, K.R. and Rasenick, M.M., Photoreceptor GTP binding protein mediates fluoride activation of phosphodiesterase, J. Biol. Chem., 260 (1985) 9081-9084. 22 Stryer, L., Cyclic GMP cascade of vision, Annu. Rev. Neurosci., 9 (1986) 87-119. 23 U'Prichard, D.C. and Snyder, S.H., Guanyl nucleotide influences on [3H]-iigand binding to noradrenergic receptors in calf brain, J. Biol. Chem., 253 (1978) 3444-3452. 24 Von Hungen, K. and Roberts, S., Neurotransmittersensitive adenylate cyclase systems in the brain, Rev. Neurosci., 1 (1974) 231-281.
1 Andrade, R., Malenka, R.C. and Nicoll, R.A., A Gprotein couples serotonin and GABA receptors to the same channels in hippocampus, Science, 234 (1986) 12611265. 2 Ariens, E.J. and Simonis, A.M., Receptors and receptor mechanisms. In P.R. Saxena and R.P. Forsyth (Eds.), Beta-Adrenoceptor Blocking Agents. Elsevier, Amsterdam, The Netherlands, 1976, pp. 3-27. 3 Cassel, D. and Selinger, Z., Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholaminestimulated GTPase by guanosine 5"-(gamma-thio) triphosphate, Biochem. Biophys. Res. Commun., 77 (1977) 868-873. 4 Dolphin, A.C. and Scott, R.H., Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides, J. Physiol. (Lond.), 386 (1987) 1-7. 5 Downs, R.W., Spiegel, A,M., Singer, M., Reen, S. ahd Aurbach, G.P., Fluoride stimulation of adenylate cyclase is dependent on the guanine nucleotide regulatory protein, J. Biol. Chem., 255 (1980) 949-954. 6 Eckstein, F., Cassel, D., Levkovitz, H., Margalit, L. and Selinger, Z., Guanosine 5"-O-(2-thiodiphosphate), J. Biol. Chem., 254 (1979) 9829-9834. 7 Gilman, A.G., G proteins: transducers of receptor-generated signals, Annu. Rev. Biochem., 56 (1987) 615-649. 8 Gordon, J.H. and Rasenick, M.M., In situ binding of a photoaffinity GTP analog to synaptic membrane G proteins: distribution of bound GTP analog reflects the status of adenylate cyclase, FEBS Len., 235 (1988) 201-206. 9 Hatta, S., Marcus, M.M. and Rasenick, M.M., Exchange of guanine nucleotide between GTP-binding proteins which regulate neuronal adenylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 5439-5443. 10 Hescheler, J., Rosenthal, W., Trautwein, W. and Schultz, G., The GTP-binding protein, Go, regulates neuronal calcium channels, Nature (Lond.), 325 (1987) 445-447. 11 Jakobs, J.J., Determination of the turn-off reaction for the epinephrine-inhibited human platelet adenylate cyclase, Eur. J. Biochem., 132 (1983) 125-130. 12 Lemos, J.R. and Levitan, I.B., Intracellular injection of guanyl nucleotides alters the serotonin-induced increase in
This work was s u p p o r t e d by U.S. Public H e a l t h Service G r a n t M H 39595 and N S F G r a n t BNS 87-19758 and a Biomedical R e s e a r c h S u p p o r t grant to the University of Illinois. M . M . R . is a recipient of a Research Scientist D e v e l o p m e n t A w a r d f r o m the National Institute of M e n t a l H e a l t h ( M H 00699). The authors thank Drs. Mrinalini R a o and Sarah Shefner for advice and criticism as well as Ms. Janice G e n t r y for manuscript p r e p a r a t i o n .
105
BRE 14505
Guanosine-5"-O-thiodiphosphate functions as a partial agonist for the receptor-independent stimulation of neural adenylate cyclase M.M. Rasenick, J.M. Hughes and N. Wang Department of Physiology and Biophysics and the Committee on Neuroscience, University of Illinois College of Medicine, Chicago, IL 60680 (U.S.A.) (Accepted 1 November 1988) Key words: Signal transduction; Guanosine triphosphate-binding protein; fl-Adrenergic receptor; Receptor-effector coupling; Photoaffinity labeling; Cyclic nucleotide; C6 glioma cell
GTP-binding proteins (G proteins) have been implicated as mediators of several aspects of neuronal signal transduction including ion channels, phosphatidyl inositol turnover and the stimulation or inhibition of adenylate cyclase. Several investigators have employed the stable guanosine diphosphate (GDP) analog, guanosine 5"-O-thiodiphosphate (GDPflS) to block putative G protein-mediated processes. Although GDPflS is assumed to block G protein function, some investigators have reported partial activation of G protein-mediated processes by this compound. In this study we demonstrate that GDPflS functions as a partial agonist for the adenylate cyclase system. In rat cerebral cortex membranes, GDPflS activates adenylate cyclase with an ECs0 similar to the hydrolysis resistant GTP analog, guanylylimidodiphosphate (GppNHp), but to a far lower extent. Further, GDP/~S antagonizes the activation of adenylate cyclase by high doses of GppNHp or GTPyS (another stable GTP analog) but potentiates adenylate cyclase activation by low doses of these nucleotides. High doses of GDPflS provoke, only partially, exchange of nucleotides among G proteins, as measured by the transfer of the photoaffinity GTP analog, azidoanilido-GTP, between the inhibitory and stimulatory GTP-binding proteins. In the presence of the/~-adrenergic agonist, isoproterenoi, GDPflS fails to support stimulation of C6 glioma membrane adenylate cyclase and inhibits GppNHp- or GTPyS-mediated stimulation of that enzyme. Inhibition of C6 membrane adenylate cyclase by GTP analogs is also blocked by GDPflS. Finally, in C6 cells made permeable with saponin, where the /~-adrenergic receptor is 'tightly coupled' to the adenylate cyclase system, GDPflS, GppNHp or GTPyS stimulate adenylate cyclase at high concentrations, but only in the absence of isoproterenol. When isoproterenol is added to these cells, GppNHp or GTPyS enhance adenylate cyclase activation, yet GDPflS acts only to block that process. Thus, GDPflS appears to function as partial agonist for the stimulatory adenylate cyclase G protein but only when an activated neuroreceptor is not coupled to that protein. INTRODUCTION GTP-binding proteins (G) represent a class of signal transducing proteins which link receptors to their intracellular effector molecules, as in the adenylate cyclase or rod outer segment phosphodiesterase systems 7'22. Generally, occupation of a specific neuroreceptor by its agonist will allow binding of G T P (or a GTP analog) to a certain G protein. Subsequently, the activated G protein acts as mediator of the intracellular signaling process to be stimulated or inhibited in response to that neurotransmitter. Whereas the identity of G pro-
teins stimulating (Gs) and inhibiting (Oi) adenylate cyclase is well established, the identity of G proteins apparently involved in other signaling processes (phosphatidyl inositol turnover; ion channel modulation) has not been established. Further, while several elegant biochemical and genetic studies have attempted to establish the precise mechanism for the coordinate regulation of r e c e p t o r - G protein-mediated signal transduction processes, it is impossible for these studies to account for the complexities of the intact neuron 15. Some neurophysiologists have attempted to study G protein-regulated processes by injection of G
Correspondence: M. M. Rasenick, Department of Physiology and Biophysics, M/C 901, University of Illinois College of Medicine, P.O. Box 6998, Chicago, IL 60680, U.S.A. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
106 proteins or G protein inhibitors into living cells. The stable GDP analog, guanosine 5"-O-(2-thiodiphosphate) (GDPflS), has been used by several investigators as an inhibitor of GTP-dependent processes 6 and this compound appears to bind to a variety of GTP-binding proteins, fixing them in the inactive state. GDPflS has been thought to inactivate the GTP binding proteins which stimulate (Gs) or inhibit (Gi) adenylate cyclase 5'11. Further, GDPflS has been used in electrophysiological studies to inhibit processes thought to be mediated through the activation of adenylate cyclase ~2. Most recently, GTP-binding proteins have been implicated as direct activators or inhibitors of ion channels, and GDPflS has been used as an inhibitor of G protein effects in some of these studies as well 1"4"1°. Several inconsistencies have appeared in an understanding of GDPflS action in the adenylate cyclase system. Whereas GDPflS has been effective at antagonizing role of GTP or GTP analogs in the hormone- or neurotransmitter-induced stimulation or inhibition of adenylate cyclase, GDPflS has been reported to increase cAMP accumulation in some systems 2°. Further, pressure injection of GDPflS has been observed to block the effects of serotonin (5-HT) in promoting secretion from helix neurons, but GDPflS activated the target cells in the absence of the hormone 12. In this study, we have attempted to answer these apparent contradictions by describing the site of GDPflS action. We demonstrate that GDPflS acts as a partial agonist for G~ only when G~ is not associated with a neuroreceptor. MATERIALS AND METHODS
Tissue preparation Enriched synaptic membrane fractions from 21day-old Sprague-Dawley rats were prepared as per Rasenick et al. 17 and stored under liquid N 2 until use. C6 cells were grown in Dulbecco's MEM; 4.5 g glucose/liter 10% fetal bovine serum, and harvested just prior to confluence. Membranes were prepared as indicated in Rasenick and Kaplan 16 and stored under liquid N 2 until use.
Adenylate cyclase assays Adenylate cyclase was assayed in cerebral cortex
membranes as described by Hatta et al. ~' or in C6 membranes as described in Rasenick and Kaplan ~, Unless otherwise noted, assay incubation mixtures included 15 mM HEPES, pH 7.4.1 mM dithiothreitol (DTI'), 0.3 mM phenylmethylsulfonylfluoride, 5 mM MgC1, 50 ,uM ATE [a-32p]ATP (5 x 1(I5 cpm/tube), 60 mM NaC1, 0.25 mg/ml bovine serum albumin, 0.5 mM isobutylmethylxanthine, 1 unit/ml adenosine deaminase, 0.5 mg/ml creatine phosphate, 0.14 mg/ml creatine phosphokinase and 15 units/ml myokinase. Assays were stopped by the addition of 100,ul of 2% SDS, 1.4 mM cAMP, 40 mM ATP and cAMP accumulated was determined by the method of Salomon 19 using [3H]cAMP (3 x 104 cpm/tube) to monitor recovery.
Permeable C6 cells C6 cells were grown to near confluence on 24-well plates and made permeable with saponin (10 ~g/ml) as per Rasenick and Kaplan 16. Assays were performed as described in Rasenick and Kaplan16; incubations including 0.5 mM ATP, [a-32p]ATP (2 x 10 6 cpm/well), 1 mM MgC1 and 0.5 mM isobutylmethylxanthine in Hanks buffer. Total assay volume was 150 ¢tl/well and reactions were stopped after 15 min by addition of 300/~1 ice-cold HEPES (15 raM, pH 7.4) and freezing on dry-ice. [32p]cAMP produced was determined as above.
Photoaffinity experiments p3-azidoanilido-PI-GTP (AAGTP) was synthesized by the method of Pfeuffer 13 with p-azidoaniline provided by Dr. George Wheeler. As described in Hatta et al. 9, membranes were incubated with 1.2 x 10 -7 M [32p]AAGTP for 3 min, washed twice and subjected to a 10 min incubation (23 °C) in the presence of the indicated nucleotide. After this, UV photolysis was performed for 5 min followed by SDS-PAGE and radiofluorographic analysis of the dried gel. Radioactivity incorporated by proteins of interest was determined by excising bands corresponding to those in the autoradiograph and counting in a Beckman LS 5800 liquid scintillation counter s . In tandem experiments, membranes were exposed to 10 -4 M AAGTP, washed and assayed for adenylate cyclase in the presence of the indicated nucleotide.
107
Purification and analysis of guanine nucleotides
All other reagents were of highest purity obtainable.
Guanine nucleotides obtained commercially were dissolved in distilled water to make a 10-2 M solution. 200 pl vols. were applied carefully to the A G 50W-X 4 resin (25 x 1 cm pre-equilibrated with HCI, pH 1.15) and washed in with 2 ml of pH 1.15 HC1 solution. The column was eluted with pH 1.15 HC1 and samples were collected with fraction size of 1 ml and flow rate of 1 ml/min. In the preliminary experiments, a mixture of GTPTS and GDPflS was applied to the column. GTPyS appeared first and totally separated from GDPflS peak. The purified nucleotides were collected and pooled separately, lyophilized and dissolved in water. For thin-layer chromatography (TLC) analysis the nucleotides were spotted on a polyethyleneimine cellulose sheet and developed in 0.5 M NaCI and 0.75 M Tris, pH 8.0. Rf values for nucleotides in this system were: GDPflS, 0.24; GTPflS, 0.09; GTPTS, 0.010; GTP, 0.18; GDP, 0.39; GMP, 0.57.
Materials All nucleotides (except A A G T P ) used in this study were obtained from Boehringer Mannheim (Elkhart, Ind.). p-Azidoaniline (used in the synthesis of A A G T P ) was a gift from G.L. Wheeler and 1-propranolol was a gift from Ayerst laboratories.
400
RESULTS
Activation of adenylate cyclase by various GTP analogs When synaptic membranes were incubated with various GTP analogs and assayed for adenylate cyclase (Fig. 1) it was found that GTPTS, G p p N H p and GDPflS (but not GTP) were capable of activating that enzyme. The ECs0 value for GTPTS was calculated at 2 x 10-8 M and that for G p p N H p or GDPflS was approximately 4 x 10 -7 M. The Vmax of adenylate cyclase activation with GTPTS or G p p N H p was about 300 pmol cAMP/mg protein/ rain; the maximal activation of the enzyme achieved in the presence of GDPflS was only 60% of that seen with the triphosphate analogs.
Antagonism of adenylate cyclase activation by GD PflS Data in the initial experiment raised the possibility that GDPflS was acting as a partial agonist for the activation of adenylate cyclase in synaptic membranes. This was tested by assaying adenylate cyclase with increasing doses of GTPyS in the presence of 10 p M GDPflS (Fig. 2). Under these conditions, GDPflS acted as a partial agonist, contributing to the stimulation of adenylate cyclase by low concentra-
~Ho GTPTS
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Fig. 1. Activation of rat cerebral cortex adenylate cyclase by various GTP analogs. Rat cerebral cortex synaptic membranes (12 gg protein/tube) were assayed for adenylate cyclase as described in the methods section. Assays were carried out in the presence of the indicated nucleotide at the indicated concentration and stopped after 10 min. Means of triplicate determinations +_S.E.M. from 1 of 4 identical experiments are depicted. Where error bars are not seen, they are smaller than the symbols.
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Fig. 2. Antagonism of adenylate cyclase activation by GDPflS. Synaptic membrane adenylate cyclase was assayed in the presence of the indicated concentrations of GTPyS with or without GDPflS (1/~M). Means of triplicate determination + S.E.M. from 1 of 3 identical experiments are shown. Where error bars are not seen, they are smaller than the symbols.
108 tions of GTPTS and inhibiting the stimulatory effects of higher GTPTS concentrations. GTPyS concentrations exceeding 10 g M overrode GDPflS effects.
Purity and stability of GDPflS Some studies employing GDPflS have noted effects which were attributed to GTPflS, the phosphorylated product of GDPflS. A variety of synaptic membrane phosphatases hydrolyze GTP rapidly, rendering it ineffective at stimulating (Fig. 1) or inhibiting (not shown) synaptic membrane adenylate cyclase. Thus, it was thought unlikely that GTPflS would regulate the enzyme. Nonetheless, we investigated whether GTPflS was generated during the course of these experiments by the synaptic membranes. Fig. 3 shows that no GTPflS was produced during the course of these incubations. Further, the GDPflS used was free of contamination from GTP~'S. The commercially available GDPflS contained about 2% GDP. When the purified GDPflS was used in experiments as shown in Fig. 1, no differences were seen compared to the commercial preparation.
Photoaffinity studies In previous studies from this laboratory, we demonstrated that G p p N H p or other hydrolysisresistant analogs of GTP induce inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing of these membranes with buffer dilution and centrifugation. A subsequent incubation with GppNHp, GTP~'S or unlabelled A A G T P 'disinhibits' adenylate cyclase in a dosedependent fashion (Fig. 4c). If the hydrolysisresistant photoaffinity GTP analog ([32p]AAGTP) is used in similar experiments, under the conditions where adenylate cyclase is inhibited, A A G T P is bound primarily to the inhibitory GTP-binding protein, G+ and a similar protein, G o. A subsequent incubation with G p p N H p (or other GTP analogs) causes a shift in the incorporation of A A G T P from Gi/o to G s, the sum of the A A G T P binding to these two proteins remaining constant (Figs. 4a and 4b). Under these conditions, A A G T P appears to transfer directly between Gi/o and G s. Fig. 4a compares the ability of G p p N H p and GDPflS to promote the apparent exchange of A A G T P bound from G~ to G s. Whereas G p p N H p caused a 5-fold increase in the
ratio of A A G T P bound between G~ and Gi/,,, GDPflS promoted only a 2-fold increase. Further, G p p N H p was more potent than GDPflS; calculated ECs0s were 2 × 10 6 M for G p p N H p and 2 x 10 5 M for GDPflS. Finally, the data in 4c show the 'distribution' of adenylate cyclase by G p p N H p vs GDPflS. Corollary to data in Fig. 4a and b, GDPflS relieved inhibition of adenylate cyclase only 50% as extensively as G p p N H p and with a 100-fold lower potency; ECs( ~ = 5 × 10-s for G p p N H p vs 5 × 10.6 for GDPflS. GDPflS promoted the apparent transfer of A A G T P from G i to G~ with a similarly lower efficacy and potency (Fig. 4b).
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Fig. 3. Purity and stability of guanosine thiophosphates. GDP/3S and GTP~S were purified on Dowex columns as described in Materials and Methods. GDPflS was contaminated by approximately 2% GDP. To monitor possible breakdown of GDPflS and/or production of GTPflS, samples of GDP/3S (1 raM) were incubated (at the indicated temperature) with synaptic membranes under the conditions (and buffers) used for the adenylate cyclase assay. Thin-layer chromatographic analysis was performed as described in Materials and Methods. No GTPflS was formed. Rf values for nucleotides in this system were: GDPflS, 0.24; GTPflS, 0109; GTP, 0.18; GDP, 0.39; GMP, 0.57.
109
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Fig 4. Photoaffinity studies, a: AAGTP labelling and nucleotide exchange. Synaptic membranes were incubated with [a-~2P] AAGTP as described in methods. After 2 buffer washes, membranes were resuspended in 2 mM HEPES, pH 7.4, with I mM MgC1 and incubated with H20 or the indicated concentrations of GppNHp or GDPflS for 10 min and then UV irradiated. Thirty ~tg samples of each tube were subjected to SDS-PAGE and radiofluorography. Unlabelled nucleotide concentrations are the intermediate value between the two labelled concentrations. Results are from one of two identical experiments, b: transfer of AAGTP from Gi/o to G, evoked by GppNHp and GDPflS. Bands corresponding to G, and G~o were excised from the gel in Fig. 4a and radioactivity in each band was determined by scintillation counting. After background subtraction, the ratio of 32p clam in the 42 kDa (Gs) vs 40 kDa (G~o) bands was determined and is represented above. Note that the total 32p cpm in Gs + G~o averaged 1732 + 109 for each sample lane of the gel in Fig. 4a. c: reversal of adenylate cyclase inhibition by GppNHp and GDPflS. Synaptic membranes were pre-incubated with 100/zM unlabelled AAGTP for 3 rain at 23 °C. Following dilution and washing, membranes were assayed for adenylate cyelase, omitting EGTA and adding 1 mM rather than 5 mM MgCI; temperature = 23 °C. The membranes were not subjected to UV irradiation and the experiment was done under yellow light. The increase in adenylate cyclase activity parallels the increase in AAGTP transferring to G, seen in the labelling experiment in Fig. 4a (see text). Means of triplicate determinations from 2 experiments were normalized and are represented above.
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Fig. 5. Effects of GDP/3S on/3-receptor-mediated stimulation of adenylate cyclase in C6 glioma cell (membranes). Membranes prepared from C6 cells were assayed for adenylate cyclase as indicated in Materials and Methods. a: membranes were assayed in the presence (/k ,&) or absence (r-],l) of isoproterenol (1 #M) and the indicated concentration of GDP/~S ( I , & ) or GTP~,S (rq,A). Means of triplicate determinations for one of 3 experiments are depicted, b: C6 membranes were assayed as above in the presence of 1 MM isoproterenol (C],I,A) and the indicated concentration of GTPTS ([],A) or GDP/3S (I). One group of samples (&) was also assayed in the presence of 10 #M GDPflS. Mean + S.E.M. from 1 of 3 experiments is depicted.
t3-Receptor-mediated stimulation of C6 adenylate cyclase (membranes) Results presented thus far indicate that GDPflS functions as a partial agonist for the activation of adenylate cyclase in the absence of a hormone or neurotransmitter. As a consequence of cell disruption, synaptic membrane adenylate cyclase is insensitive to hormone or neurotransmitter15'24; however, membranes made from C6 glioma cells respond to isoproterenol (as well as to G p p N H p or GTPTS alone) for the activation of that enzyme. Specifically, 10 HM GTP~,S produced a 5-fold activation of C6 m e m b r a n e adenylate cyclase which, in the presence of 1 HM isoproterenol, was increased to 10-fold. U n d e r these same conditions, GDP/3S was ineffective at stimulating adenylate cyclase and isoproterenol did not alter this (Fig. 5a). When GDPflS (10 ktM) was included in the C6 membrane adenylate cyclase assay along with isoproterenol and GTPvS, the GTPTS concentration required for activation of adenylate cyclase was increased by about 30-fold ( - E C s 0 = 0.2 vs 6 # M ) (Fig. 5b). Effects of (1 #M) isoproterenol were blocked completely by 10 MM 1-propranolol (not shown).
Blockade of adenylate cyclase inhibition in C6 cells by GDPI3S When C6 membranes were incubated with NaF
(tO increase adenylate cyclase activity), subsequent assay with G p p N H p evoked a dose-dependent inhibition of adenylate cyclase (Fig. 6). U n d e r the same conditions, GDPflS stimulated the enzyme, although the efficacy of stimulation (34% at 10-5 M GDPflS) was much less than the inhibition (54% at
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Fig. 6. Blockade of adenylate cyclase inhibition in C6 cells by GDPflS. Membranes prepared from C6 cells were assayed for adenylate cyclase under the conditions described in Fig. 4c with the addition of 10 mM NaF and the indicated concentration of GppNHp ([5]) or GDPflS (I). Samples indicated by filled triangles (A) were incubated with 10 #M GDPflS plus the indicated concentration of GppNHp. Means + S.E.M. for 1 of 3 experiments are shown.
111 10-6 M GppNHp). Further, GppNHp-induced inhibition of adenylate cyclase was mitigated by 10 #M GDPflS (Fig. 6), this effect being overriden by excess concentrations of GppNHp.
Effects of GDP~S on adenylate cyclase in permeable C6 cells This laboratory has demonstrated previously, that when the basic structure of the C6 cell is preserved and those cells are made permeable with saponin,
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25
DISCUSSION
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guanine nucleotides activate adenylate cyclase only at very high concentrations. However, in the presence of a fl-adrenergic agonist, GTP analogs enhance adenylate cyclase activity significantly. Thus, unlike the case in C6 membranes, G s appears tightly coupled to the receptor in the permeable C6 cells. This system is analogous to an intact neuron in which GTP analogs might be pressure-injected. In the absence of isoproterenol, GTPyS or GDPflS stimulate adenylate cyclase only at concentrations in excess of 10-5 M (Fig. 7a). Under these conditions, 10-4 M GDPflS increased basal adenylate cyclase by 38.7 + 3.6% (average of 5 triplicate determinations ranging from 29% to 48%. A similar modest increase (range 24-61%) was seen with GTPyS in the absence of isoproterenol. However, in the presence of isoproterenol, GTPyS was a potent stimulator of adenylate cyclase (about 5-fold the basal value) whereas GDPflS (100 #M) increased activity to the same extent that it did in the absence of isoproterenoi. When GDPflS (10 #M) was added to permeable cells in the presence of isoproterenol and increasing concentrations of GTPyS, GDPflS acted to inhibit the stimulatory effects of GTPyS. Under these conditions the dose of GTPyS required to activate adenylate cyclase was increased about 10-fold (Fig. 7b).
•
, -8
, -7
, -6
, -5
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, -3
log G T P 3 . S [M]
Fig. 7. Effects of GDP/~S on adenylate cyclase in permeable C6 cells. C6 cells were grown to near-confluence on 24-well plates, made permeable with saponin, and assayed for adenylate cyclase as described, a: cells were assayed in the
presence of the indicated concentration of GTPyS ([3,/k) or GDPflS (II,A) and the presence (kx,A) or absence (D,II) of 1 pM isoproterenol. Means of triplicate experiments from one of 4 similar experiments are represented. See text for normalized values of 10~ GDPflS or GTPyS in the absence of isoproterenol, b: samples were assayed as in (a), with the indicated concentration of GTPyS without (r-l) or with (11) 10 pM GDPflS. All samples contained isoproterenol (1 #M). Means _+ S.E.M. for 1 of 3 experiments are shown.
GDPflS has been reported to function as an antagonist of the adenylate cyclase system. Accordingly, investigators have used this compound in order to ascertain whether a G protein might mediate a certain cellular event. Accordingly, GDPflS has been used to block the activation6 and inhibition 11 of adenylate cyclase. Further, GDPflS has been employed to facilitate the opening of a K + channel 1 or to attenuate a voltage-sensitive Ca 2+ channel 4, both of which may be G protein-sensitive. Finally GDP/~S has been used to block receptormediated processes which have been surmised to act via the activation of adenylate cyclase12. Under these conditions, pressure-injection of GDPflS has resulted in the activation of those processes if receptor agonist was omitted from the outside of a cell. Similarly, high concentrations of GDPflS activate adenylate cyclase in permeable C6 cells. Such results
112 have been assumed by some investigators to be due to the presence of contaminating GTPyS in the GDPflS or the formation of GTPflS. In the present study however, GDPflS is uncontaminated and no formation of GTPflS was noted (Fig. 3). Data in Figs. 1 and 2 show that GDPflS behaves as a classic partial agonist 2 with regard to the G~-mediated stimulation of cerebral cortex adenylate cyclase. Data in Fig. 2 help to distinguish between two possible mechanisms of GDPflS action: partial agonism of G Sand antagonism of G i. Clearly, either would result in increased activation of adenylate cyclase. However, if GDPflS acted primarily to inhibit G i, then the adenylate cyclase activity in the presence of GDPflS plus GTPyS would be parallel to that of GTP),S alone at GTPyS concentrations exceeding l 0 -7 M. This is not the case (see Fig. 2). We have demonstrated previously that the photoaffinity GTP analog, AAGTP, can be transferred from Gi/o to Gs by the addition of an hydrolysisresistant GTP analog to the membranes 9. Added GTP analogs appear to cause the formation of G protein complexes (Hatta and Rasenick, unpublished) and AAGTP binding is increased on G S due to cooperative effects~. It appears that G~, G~, Go and a 32 kDa GTP binding protein (G32) are involved in this process. GDPflS is 10-fold less potent than GppNHp in promoting nucleotide exchange and 100-fold less potent in alleviating the stable inhibition of adenylate cyclase (Fig. 4). Furthermore the efficacy of GDPflS is 20-30% that of GppNHp in either of the above parameters. This contrasts with data in Fig. 1, where GDPflS has 60% of the efficacy and equal potency to GppNHp. Further, GDPflS competes with AAGTP for binding tO G s, Gi, G O or G32 with a potency near that of GppNHp 8. A possible explanation for the greater effects of GDPflS on the stimulation of adenylate cyclase (Fig. 1) than on nucleotide exchange (Fig. 4) is that the latter process appears to require activation of G~ and Gi/o. GDPflS does not activate G~ (Fig. 6 and ref. 11). Thus if GDPflS has partial agonist properties for G~ but not G~/o, it would have a greater effect upon a Gs-dependent process (adenylate cyclase stimulation) than on a process requiring activation of multiple G proteins. Although we demonstrate that GDPflS is a partial
agonist for the receptor-independent stimulation of adenylate cyclase, isoproterenot-induced stimulation (Figs. 5 and 7b) is blocked by this compound. These results are somewhat different from those seen in the turkey erythrocyte, where GDPflS antagonized the stimulation of adenylate cyclase5'6. This may be due to the distinction between the coupling of adenylate cyclase to stimulatory receptors in the different systems. In the tightly coupled turkey erythrocyte, isoproterenol and GTP are required to activate the enzyme 3, the occupied receptor allowing the binding of GTP to G~~s. Cerebral cortex synaptic membranes bind receptor agonists normally and display G protein-mediated down-regulation of agonist affinity23. However, a concomitant of cell disruption is the loss of neurotransmitter-activated adenylate cyclase in brain regions where intact-cell preparations show profound neurotransmitter-induced cAMP accumulation ~5,24 It appears that the presence of an activated receptor causes the coupled adenylate cyclase to respond differently to GDPflS. Human fat cells have been noted to respond similarly in this regard 2°. These data suggest the possibility that an agonist occupied receptor causes a coupled G~ to respond differently to GDPflS than an uncoupled G~. C6 glioma cells provided a useful system to test this hypothesis. C6 adenylate cyclase has several properties associated with neuronal adenylate cyclase, except that some degree of receptor coupling is maintained upon cell disruption. Permeable C6 cells maintain tight coupling between the fl receptor and G~ and offer a parallel to a neuron in which GTP analogs are pressure-injected. Consistent with the above hypothesis is the observation that in permeable C6 cells, GDPflS, GppNHp or GTPyS (all at >10ktM) caused a small yet reproducible increase in adenylate cyclase activity (Fig. 7a). However, in the less tightly coupled C6 membranes, GppNHp or GTPyS (without isoproterenol) activated the enzyme to a much greater extent than GDPflS (Fig. 5a). In addition to the receptor-independent activation of G proteins, fluoride appears to uncouple G proteins from receptors 5"21. Consistent with this uncoupling, in the presence of NaF, GDPflS stimulates C6 membrane adenylate cyclase to a greater extent than in the absence of NaF (compare effects of GDPflS in Fig. 6 with those in Fig. 5a).
113
Partial agonist activity of GDPflS is one of a series of observations which suggest that adenylate cyclase in cells of neural origin is regulated in a fashion distinct from that e n z y m e in o t h e r cells and tissues. O t h e r discrepancies b e t w e e n neural and non-neural a d e n y l a t e cyclase systems are the presence of calm o d u l i n activation, loss of r e c e p t o r coupling upon cell disruption and interaction with m e m b r a n e associated cytoskeletal c o m p o n e n t s (see refs. 14,15 for reviews). T h e relevance of these discrepancies to n e u r o t r a n s m i t t e r action and responsiveness have yet to be d e t e r m i n e d .
ACKNOWLEDGEMENTS
REFERENCES
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This work was s u p p o r t e d by U.S. Public H e a l t h Service G r a n t M H 39595 and N S F G r a n t BNS 87-19758 and a Biomedical R e s e a r c h S u p p o r t grant to the University of Illinois. M . M . R . is a recipient of a Research Scientist D e v e l o p m e n t A w a r d f r o m the National Institute of M e n t a l H e a l t h ( M H 00699). The authors thank Drs. Mrinalini R a o and Sarah Shefner for advice and criticism as well as Ms. Janice G e n t r y for manuscript p r e p a r a t i o n .