Introduction

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Opioid dependence is characterized by an enhanced neuronal excitability toward stimulatory input (1). The underlying cellular mechanisms involve up-regulation of the cAMP second messenger system (2), which results from sensitization of AC activity (1, 3, 4). Although the role of cAMP in drug addiction is well recognized, the regulatory mechanism leading to an increase in AC activity is largely unknown.

Opioid receptors belong to the family of seven-transmembrane domain receptors that regulate their appropriate intracellular effector systems via inhibitory G proteins (4, 5). Acute activation of an opioid receptor leads to the inhibition of AC and subsequently to a reduction of intracellular cAMP levels (6). During the course of chronic opioid treatment, however, initially attenuated cAMP levels begin to recover and, in some cell systems (7-9) and brain areas (10), even exceed those originally observed in control cells. The increase in AC activity is generally referred to as "sensitization" of AC (4) and is mediated by an active counter-regulation of stimulatory receptor systems (7, 8, 11). The individual regulatory changes found comprise alterations in the quantity of stimulatory receptors (7, 8, 11) and G proteins (7) as well as an enhanced functional coupling efficiency between both entities (7, 11). However, there also were some cell systems (8) and brain areas (10) in which sensitization of AC develops without any apparent quantitative changes in stimulatory signal transduction components, suggesting the existence of additional functional mechanisms.

Stimulation of AC is mediated by the activated, GTP-bound form of G_s (12). As a variety of other signal transduction proteins (13-15), the G_s subunit undergoes post-translational palmitoylation near the amino terminus (16-18). Palmitoylation of G_s is reversible and turns over rapidly after receptor activation (18-20). Thus, palmitoylation possesses the potential to regulate G_s signaling. Indeed, palmitoylation of G_s is required for intact receptor signaling (17) and has been implicated in the regulation of subcellular localization of the protein (21). However, despite these informations, the role of G_s palmitoylation for intracellular signaling remains unclear (13).

To investigate whether changes in G_s palmitoylation may contribute to the enhancement of stimulatory signal transduction during the state of opioid dependence, we used human mammary epidermoid A431 carcinoma cells (22) stably transfected with the rat µ-opioid receptor cDNA (23). Chronic opioid treatment of clonal A431/µ13 cells largely enhances the capacity of stimulatory AC signaling, which develops without any apparent quantitative changes at the level of stimulatory G proteins and AC (8). Thus, A431/µ13 cells represent a useful model system for studying functional changes in stimulatory AC signaling. Here, we report that chronic opioid treatment of A431/µ13 cells enhances stimulatory AC signaling by reducing the palmitoylation state of G_s. Deacylation of G_s was found (i) to increase intrinsic G_s activity and (ii) to promote G_s/AC interaction. These results support the concept that changes in stimulatory transmembrane signaling contribute to the state of opioid dependence.

	Experimental Procedures

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Materials. [³H]Forskolin (31 Ci/mmol) and [9,10-³H]palmitic acid (30 Ci/mmol) were from NEN DuPont (Dreieich, Germany). ¹²⁵I-cAMP tracer (2000 Ci/mmol) from Amersham International (Braunschweig, Germany). Rabbit anti-cAMP antibody was purchased from BioMakor (Rehovot, Israel). Geneticin (G418) and tissue culture reagents were from GIBCO BRL (Eggenstein, Germany). CNBr-activated Sepharose 4B was from Pharmacia (Freiburg, Germany). (R)-()-Isoproterenol bitartrate and Ro 20-174 (4-[(butoxy-4-methoxyphenyl)-methyl]2-imidazolidinone) were from Research Biochemicals International (Köln, Germany). PGE₁, cAMP, ATP, GTP, hydroxylamine, and Tunicamycin (mixture of isomers A, B, C, and D; catalogue No. T-7765), as well as all standard laboratory reagents, were obtained from Sigma Chemical (Deisenhofen, Germany).

Cell culture, chronic opioid treatment, and membrane preparation. Parental human mammary epidermoid carcinoma (A431) cells were stably transfected with plasmid pRC/CMV (InVitrogen, San Diego, CA) containing the rat µ-opioid receptor cDNA (18). Clones resistant to G418 were isolated and screened for µ-opioid receptor expression by [³H]diprenorphine binding (8). All experiments reported here were performed with clone A431/µ13 (B_max = 302.9 ± 46 fmol/mg of membrane protein; K_d = 1.3 ± 0.6 nM; six experiments). A431/µ13 cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 200 µg/ml G418 in a humidified atmosphere of 95% air/5% CO₂ at 37°. At 50% confluency, morphine (10 µM) was added to the medium for 2 days to induce opioid dependence (8). Parallel flasks of the same passage, which were kept in the absence of morphine, served as controls. Cells were harvested after trypsination and membranes were prepared as described previously (24). Murine S49cyc lymphoma cells were grown in DMEM containing 10% heat-inactivated horse serum. Membranes were prepared as described previously (25) and stored in aliquots (10 mg/ml in 5 mM Tris·HCl buffer, pH 7.4, containing 1 mM dithiothreitol and 1 mM EGTA) at 70° until use.

Determination of AC activity. Membrane-bound AC activity was determined in a reaction mixture (100 µl volume) containing 40 mM Tris·HCl, pH 7.4, 0.2 mM EGTA, 0.2 mM dithiothreitol, 100 mM NaCl, 10 mM MgCl₂, 0.5 mM ATP, 5 µg/ml phosphocreatine, 5 IU/ml creatine phosphokinase, 10 µM GTP, and 30 µM Ro 20-1724. Reactions were started by the addition of 10 µg of membrane protein, incubated for 10 min at 28°, and stopped with 500 µl of 0.01 M HCl. In some cases, [AlF₄] (30 µM) or the stable guanine nucleotide Gpp(NH)p (100 µM) was included to determine receptor-independent stimulation of AC activity. Membranes from opioid-dependent cells were measured in the presence of morphine (10 µM) to avoid spontaneous withdrawal. The amount of cAMP generated was determined by radioimmunoassay (26).

S49cyc reconstitution assay. Membranes of A431/µ13 cells were extracted for 1 hr at 4° with sodium cholate (1% w/v) in NMT buffer (50 mM Tris·HCl, pH 7.4, containing 10 mM MgCl₂ and 100 mM NaCl). Insoluble material was removed by centrifugation (10,000 × g; 15 min). G_s-deficient S49cyc membranes (10 µg/tube) were reconstituted on ice for 20 min with 10 µg of detergent-extracted proteins from A431/µ13 cell membranes. In some experiments, 50 ng of purified bovine brain G was added to the tubes. Subsequently, [AlF₄] (30 µM)-stimulated AC activity was determined as described previously (27). All assays were done in triplicate.

Depalmitoylation of G_s. G_s from control or opioid-dependent A431/µ13 cells was chemically depalmitoylated in a cell-free system (28). Sodium cholate extracts (10 µg of protein/µl) were incubated for 30 min on ice in the presence of neutral hydroxylamine (1 M; pH 8.0). Controls received Tris·HCl, pH 8.0. The samples were diluted 10-fold in NMT buffer before AC activity was determined in the S49cyc reconstitution assay. In a second approach, palmitoyl-G_s was depalmitoylated in vivo by blocking a palmitoyl-CoA transferase activity (29). Cells were washed serum free and cultured for an additional 3 hr with DMEM containing tunicamycin (25 µg/ml) and 1% defatted bovine serum albumin. Tunicamycin treatment did not significantly affect cell viability as determined by trypan blue exclusion. Opioid-dependent cells were incubated in the presence of morphine (10 µM). Subsequently, the cells were harvested, sodium cholate (1% w/v) extracts were prepared, and reconstitutive AC activity was determined as above.

Metabolic labeling with [³H]palmitic acid. Steady state levels of G_s palmitoylation were determined by metabolic labeling with [³H]palmitic acid under saturating conditions (30). A431/µ13 cells were plated onto 24-well culture dishes and grown for 2 days in the absence (control) or presence of morphine (10 µM) to induce dependence. Some wells received morphine together with the opioid antagonist naloxone (10 µM). On the day of experimentation, the cells were washed three times with prewarmed DMEH (DMEM plus 25 mM HEPES, pH 7.4) and incubated for 1 hr at 37° with DMEH containing 5% dialyzed fetal calf serum and 5 mM sodium pyruvate in the absence or presence of the drugs given chronically. Metabolic labeling was initiated by the addition of 0.5 mCi/ml [³H]palmitic acid for an additional 3 hr. Incubations were stopped with 2 ml/well of ice-cold phosphate buffered saline. All subsequent steps were performed at 4°. The cells were washed three times with phosphate-buffered saline and lysed in 100 µl buffer A (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 2.5 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin) plus 0.5% sodium dodecyl sulfate. After 1 hr, the solubilate was diluted 5-fold with buffer A and centrifuged for 10,000 × g for 15 min. Immunoprecipitation for 4 hr was performed with Protein A-purified anti-G_s antibodies (11) coupled to CNBr-activated Sepharose 4B beads (10 mg/ml; 20 µl/tube). The pellets were washed three times with buffer A, boiled for 3 min in Laemmli sample buffer without a reducing agent (17) and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were soaked in ENHANCE (Amersham), dried, and fluorographed for 3-6 weeks. Incorporation of the radiolabel into G_s was determined by videodensitometry of the films using the Herolab E.A.S.Y. system (Wiesloch, Germany).

[³H]Forskolin binding studies. High affinity [³H]forskolin binding to intact cells was performed essentially as described previously (31). Naive, chronically morphine-treated (10 µM; 2 days) or tunicamycin-treated (25 µg/ml; 3 hr) A431/µ13 cells were collected by trypsination, washed three times with ice-cold DMEH, pH 7.4, and equilibrated for 30 min at 4°. Binding reactions (500 µl) were performed for 60 min at 4° in the presence of 40 nM [³H]forskolin and 5 × 10⁶ cells/tube. Receptor-mediated stimulation of G_s/AC interaction was achieved with 10 µM isoproterenol, whereas basal binding of G_s to AC was measured in the absence of a stimulatory ligand. Specific binding was obtained with 10 µM forskolin. In case of opioid-dependent cells, all steps were performed in the presence of morphine (10 µM). Binding reactions were stopped by rapid filtration over Whatman GF/C filters followed by four washes with 5 ml of ice-cold 50 mM Tris·HCl buffer, pH 7.4. Incorporated radioactivity was determined by scintillation counting at 60% efficiency (LS 1801; Beckman Instruments, Columbia, MD). All reactions were done in triplicate.

	Results and Discussion

Top Summary Introduction Procedures Results & Discussion References

Opioid dependence in A431/µ13 cells. Stimulatory AC-coupled receptor systems play an important role in the cellular mechanisms underlying opioid dependence (7, 11). Using human mammary epidermoid carcinoma A431 cells stably expressing the rat µ-opioid receptor (clone A431/µ13) as a model system, we demonstrated previously that chronic opioid-induced sensitization of AC is associated with an increased signaling activity of the endogenous ₂-adrenoceptor system (8). Up-regulation of stimulatory AC signaling in A431/µ13 cells is characterized by significantly elevated levels of both basal (3.5 ± 0.4 versus 4.9 ± 1.1 fmol of cAMP/mg of membrane protein/min) and isoproterenol (10 µM)-stimulated AC activity (41.9 ± 6 versus 55.6 ± 4 fmol of cAMP/mg of membrane protein/min; mean ± standard deviation; four or more experiments). These changes are prevented by pertussis toxin pretreatment (16 ng/ml; 2 days) and by coincubation of the cells with the opioid antagonist naloxone (10 µM; 2 days), indicating a specific opioid receptor-mediated effect. We originally attributed the increase in AC activity to the presence of an increased number of ₂-adrenoceptors because no additional changes were found for both the quantity of G_s and the maximum catalytic activity of AC (8). This conclusion was substantiated by the finding that ICI-118,551 [(±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methyleth-yl)amino]-2-butanol hydrochloride], an inverse agonist at the ₂-adrenoceptor, largely reversed the increase in basal cAMP accumulation. However, as reported here, further experiments revealed that chronic opioid treatment also produced an increase in AC activity after direct activation of G_s by either 30 µM [AlF₄] or 100 µM Gpp(NH)p (Fig. 1), and these effects were not sensitive to ICI-118,551. These observations indicate the existence of an additional postreceptor mechanism involved in sensitization of AC.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chronic opioid-induced sensitization of AC-A431/µ13 cells were chronically treated with (filled bars) or without (open bars) 10 µM morphine for 2 days. Membranes were prepared, and AC activity was determined in the presence of isoproterenol (10 µM), [AlF4] (30 µM), or Gpp(NH)p (100 µM). Opioid-dependent cells were measured in the presence of morphine (10 µM) to prevent spontaneous withdrawal. AC activity is expressed in pmol of cAMP formed/min/mg of membrane protein. Values are mean ± standard deviation from at least four individual experiments. ***, Significantly different from untreated controls (p < 0.001; Student's t test).

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Chronic opioid treatment alters the stimulatory activity of G_s. To confirm whether the increase in post-receptor-stimulated AC activity is indeed due to an altered functional activity of G_s, the reconstitutive activity of sodium cholate (1% w/v) extracts prepared from membranes of A431/µ13 cells was determined in the S49cyc assay. Measurements were done in the presence of 30 µM [AlF₄], which constitutively activates G_s. Under these conditions, complementation of G_s-deficient S49cyc membrane AC (25) with detergent extracts from opioid-dependent cells results in ~2-fold higher effector activities compared with control cell extracts (Fig. 2). Western blotting of the detergent extracts was used to verify that identical amounts of G_s were present. Because S49cyc cells contain an AC isoform that is sensitive to stimulation by G subunits (34), we had to exclude the possibility that an altered G content of the sodium cholate extracts could mediate the increase in reconstitutive AC activity. For this, we added a maximal effective amount of purified bovine brain G (50 ng) to detergent extracts from both control and opioid-dependent A431/µ13 cells and determined the effect on reconstitutive AC activity. As expected, the addition of G resulted in a ~2-fold increase in reconstitutive AC activity regardless of whether extracts from control or chronically morphine-treated cells were measured (Fig. 2), indicating that the difference in reconstitutive AC activities observed for sodium cholate extracts from naive and opioid-dependent A431/µ13 cells is not due to an altered G content. Although we cannot rule out entirely any other additional factors present in the detergent extracts, such as inhibitory G protein  subunits, these results suggest that the increased reconstitutive activity of detergent extracts from opioid-dependent cells is due to an increased functional activity of G_s. Thus, besides an increase in ₂-adrenoceptor levels, sensitization of AC in A431/µ13 cells is likely to involve an additional regulatory mechanism at the level of G_s.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Chronic opioid treatment of A431/µ13 cells increases intrinsic Gs activity. Sodium cholate (1% w/v) extracts were prepared from control or chronically morphine-treated (10 µM; 2 days) A431/µ13 cells. Reconstitution of S49cyc membranes with detergent extracts was performed in the absence () or presence (+) of purified bovine brain G subunits (50 ng/tube). Reconstitutive AC activity was determined after activation of Gs with 30 µM [AlF4]. AC activity is expressed in pmol of cAMP generated/min/mg of detergent extract. Values are mean ± standard error from six (G) or three (+G) experiments. ***, Significantly different compared with control cell extract (p < 0.001, Student's t test). Inset, representative immunoblot of detergent extracts used for reconstitution. Staining was performed with a carboxyl-terminal anti-Gs antibody as described previously (9).

Palmitoylation attenuates the stimulatory activity of G_s. The G_s subunit is subject to post-translational palmitoylation (16-18), a covalent lipid modification that has been shown to regulate the function of a series of membrane proteins participating in signal transduction, such as G protein-coupled receptors (15), G protein  subunits (13, 17, 18), effector molecules (14), and tyrosine protein kinases (35). Palmitoylation is reversible due to the lability of the thioester bond (13) and thus provides a potential mechanism that could regulate the activity of G_s. In a first step to investigate whether alterations in G_s palmitoylation may account for the increase in the stimulatory activity observed for G_s from opioid-dependent cells, two functional approaches were used to modulate the palmitoylation state of G_s: (i) chemical depalmitoylation in vitro by hydroxylamine treatment (28), and (ii) inhibition of palmitoyl-CoA transferase activity in vivo by tunicamycin (29). Because neither approach involves specific modulation of G_s palmitoylation (tunicamycin also inhibits N-linked glycosylation; hydroxylamine cleaves every thioester bond) and would also affect the functional properties of other signal transduction components, such as receptors, inhibitory G protein  subunits, and AC (14, 15, 18), specific effects of these treatments on the activity of G_s were determined in the S49cyc reconstitution assay after solubilization and persistent activation of G_s by [AlF₄]. Neutral hydroxylamine has been used frequently to remove the palmitate residue from G_s by cleaving the thioester bond (14, 16-18). Depalmitoylation of detergent-solubilized G_s from control cells with hydroxylamine (1 M; 30 min; 4°) was found to considerably enhance its reconstitutive AC activity by ~3.5-fold. Although native G_s from opioid-dependent cells per se exhibits a ~2-fold higher reconstitutive activity compared with control cell G_s, depalmitoylation by hydroxylamine treatment further increased its stimulatory activity, reaching values almost identical to those obtained for depalmitoylated control cell G_s (Fig. 3). Similar results were obtained after depalmitoylation in vivo by tunicamycin treatment (25 µg/ml; 3 hr). Again, depalmitoylation was found to largely increase the stimulatory activity of G_s. In addition, the difference in the stimulatory activity of G_s between control and opioid-dependent cells disappears after depalmitoylation (Fig. 3). These data not only confirm that depalmitoylated G_s is active in vitro (13) but also demonstrate that palmitoylation of G_s attenuates its ability to activate AC. This finding is somewhat unexpected because a previous study showed that removal of the palmitoylation site by mutagenesis reduced the stimulatory activity of a constitutively activated form of G_s (17). However, the same laboratory also reported that a depalmitoylated form of G_z, the G protein  subunit mediating pertussis toxin-insensitive inhibition of AC, possesses an increased functional capacity to inhibit AC (30). Although it is not possible currently to determine the actual intrinsic activities of palmitoylated and deacylated G_s because of the inability to provide stably palmitoylated G_s, our results indicate that post-translational palmitoylation seems to affect the stimulatory activity of G_s.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Depalmitoylation increases the intrinsic activity of Gs. Depalmitoylation of Gs was performed either in vitro by treatment of detergent extracts with neutral hydroxylamine (hatched bars) or in vivo by exposure of the cells to tunicamycin before membrane preparation and solubilization with 1% (w/v) sodium cholate (filled bars). Intrinsic activity of depalmitoylated Gs was determined by the S49 cyc reconstitution assay in the presence of 30 µM [AlF4]. Native detergent extracts from control and opioid-dependent cells served as controls (open bars). Values are mean ± standard error from three independent experiments.

Chronic opioid treatment reduces the palmitoylation state of G_s. Palmitoylated G_s is located exclusively in the plasma membrane, whereas depalmitoylated G_s is found in both the membrane and cytosol (18-20). Based on the observation that chronic opioid treatment does not affect the abundance of membrane-bound G_s (8), the finding of an increased stimulatory activity of G_s may suggest that opioid dependence is associated with a reduced fraction of palmitoylated G_s in the plasma membrane. To test this prediction, we performed metabolic labeling studies with [³H]palmitic acid. Because palmitoylation of G_s is dynamic (18, 20), we first investigated the time course of incorporation of [³H]palmitate over 15 min to 4 hr to determine the time required to reach equilibrium labeling conditions. As shown in Fig. 4A, the greatest incorporation of the radiolabel was achieved within 2 hr of exposure to [³H]palmitic acid. Thus, all subsequent experiments were performed for 3 hr to ensure reliable examination of the steady state levels of G_s palmitoylation.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Regulation of steady state palmitoylation of Gs by chronic opioid treatment. A, Time course of [3H]palmitate incorporation into Gs from naive A431/µ13 cells. Cells were incubated with 0.5 mCi/ml [3H]palmitate for 15 min to 4 hr before cells were lysed, and Gs was immunoprecipitated using a carboxyl-terminal anti-Gs antibody. Immunocomplexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was fluorographed for 3 weeks. The relative intensity of the respective Gs bands was determined by videodensitometry using the Herolab E.A.S.Y. system and is expressed in percent of maximum labeling, which was set at 100%. Values are mean ± variation from one experiment performed in duplicate. B, Effect of acute and chronic opioid treatment on steady state Gs palmitoylation. Naive or chronically morphine pretreated cells were metabolically labeled with 0.5 mCi/ml [3H]palmitic acid for 3 hr. After radiolabeling, Gs was immunoprecipitated and fluorographed as above. Lane 1, naive cells. Lane 2, acute morphine (10 µM; 30 min). Lane 3, chronic morphine pretreatment (10 µM; 2 days). Lane 4, chronic morphine and naloxone pretreatment (10 µM each; 2 days). Values are mean ± standard deviation from one representative experiment performed in triplicate. Similar results were obtained in three separate experiments.

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View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of tunicamycin and hydroxylamine treatment on Gs palmitoylation. Inhibition of Gs palmitoylation by tunicamycin was investigated by coincubation of A431/µ13 cells for 3 hr with 25 µg/ml tunicamycin and 0.5 mCi/ml [3H]palmitic acid. Cells were lysed and incorporation of the radiolabel into Gs was analyzed as in Fig. 4. Sensitivity of [3H]palmitoyl-Gs to hydroxylamine treatment was analyzed in membranes from A431/µ13 cells that were prelabeled with [3H]palmitic acid. Membrane aliquots were incubated for 30 min at 4° either in the presence of 1 M NH2OH, pH 8.0, or 1 M Tris·HCl, pH 8.0 (control). Membranes were washed and solubilized, Gs was immunoprecipitated, and fluorography was performed as described. Films were exposed for 3 weeks at 70°.

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Depalmitoylation promotes G_s/AC interaction. The regulatory cycle of acylation and deacylation of G_s is well established and closely linked to the activation state of the G protein. On activation, the GTP-bound form of G_s dissociates from G and becomes rapidly depalmitoylated (20). After hydrolysis of GTP, the depalmitoylated and GDP-bound form of G_s may either reassociate with G in the plasma membrane and become rapidly repalmitoylated (36) or redistribute into the cytosol (17, 21). On this basis, it could be anticipated that the reduction in G_s palmitoylation observed after chronic morphine treatment would result in a loss of G_s from the plasma membrane. However, in a previous study (8), we failed to detect any change in the abundance of membrane-bound G_s, indicating that depalmitoylated G_s in opioid-dependent A431/µ13 cells does not redistribute into the cytosol but instead redistributes laterally in the plasma membrane. Because depalmitoylated G_s displays higher stimulatory activity than palmitoyl-G_s and chronic morphine treatment increases basal cAMP accumulation in A431/µ13 cells, we investigated whether depalmitoylated G_s might bind directly to AC. For this, we performed high affinity [³H]forskolin binding studies, which provide a measure for the number of complexes formed between G_s and AC (31). In untreated A431/µ13 cells, specific binding of [³H]forskolin is detectable only after activation of ₂-adrenoceptors by isoproterenol. In contrast, in chronically opioid treated cells, there is substantial [³H]forskolin binding in the absence of any stimulatory ligand, whereas the maximum number of ₂-adrenoceptor-stimulated G_s/AC complexes remains unchanged. Depalmitoylation of intracellular G_s in control cells by tunicamycin treatment (25 µg/ml; 3 hr) mimics the increase in basal [³H]forskolin binding (Fig. 6). The lack of ₂-adrenoceptor-stimulated high affinity [³H]forskolin binding after tunicamycin treatment may reflect receptor depalmitoylation, which has been reported recently to attenuate receptor signaling (15). However, these results also demonstrate that depalmitoylated G_s, which accumulates in the plasma membrane during the state of opioid dependence or after tunicamycin treatment, is able to associate directly with AC, even in the absence of preceding receptor activation. This observation is the first example of the regulation of G protein activity by modulation of its palmitoylation state. The most plausible explanation for this altered protein/protein interaction of depalmitoylated and presumably GDP-bound G_s would be a change in its affinity for G subunits and/or AC. Indeed, depalmitoylated G_s has been shown recently to possess ~5-fold lower affinity for G subunits than palmitoyl-G_s (37). However, inactivated and depalmitoylated G_s is still able to associate with G subunits in the plasma membrane, a critical step in the forward reaction of the palmitoylation cycle that enhances susceptibility of G_s for repalmitoylation by membrane-bound palmitoyl-CoA transferases (36, 37). Binding of depalmitoylated G_s to other membrane proteins, such as AC, could be affected by limiting the availability of free G subunits. Such a mechanism seems likely because G subunits have been shown to contribute to sensitization of AC by an unidentified indirect mechanism (38). Alternatively, because signal transduction molecules are organized in functional compartments within the plasma membrane (39), depalmitoylation of G_s could simply increase its mobility and allow access to additional AC molecules (13).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Chronic opioid and tunicamycin treatment promotes association of Gs with AC. Formation of Gs/AC complexes was determined by high affinity [3H]forskolin binding. Naive, chronically morphine-treated (10 µM; 2 days) or tunicamycin-treated (25 µg/ml; 3 hr) A431/µ13 cells were analyzed in the absence (filled bars) or presence of the -adrenoceptor agonist isoproterenol (10 µM; open bars). Data are given in dpm of specific [3H]forskolin binding/106 cells. Values are mean ± standard error from four experiments.

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