Increased Expression Of Gs{alpha} Enhances Activation Of The Adenylyl Cyclase Signal Transduction Cascade

Xioaju Yang, Francis Y. G. Lee Sr. and Gary S. Wand

Departments of Medicine and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the stimulatory G protein, Gs{alpha}, can vary over a 3-fold range in human tissues and in rodent central nervous system. In fact, the offspring of alcoholics have higher levels of Gs{alpha} expression in certain tissues compared with the offspring of nonalcoholics. The aim of this research was to test the hypothesis that a causal relationship exists between the level of expression of Gs{alpha} and induction of the adenylyl cyclase (AC) cascade. The methodology employed transient transfection of HEK 293 cells with a cDNA for the 52-kDa form of Gs{alpha} under regulation by inducible metallothionein promoters. Transfectants were exposed to varying concentrations (0–125 µM) of zinc sulfate that produced a 3-fold range of membrane Gs{alpha} expression. The range of Gs{alpha} expression produced was found to mimic a physiologically relevant spectrum of Gs{alpha} expression in membranes derived from human tissues and rat brain. It was observed that induction of Gs{alpha} expression increased constitutive as well as stimulated cAMP accumulation. Moreover, induction of Gs{alpha} expression increased events distal to the accumulation of cAMP including the phosphorylation of the transcription factor, cAMP response element binding protein and transcriptional activation of cAMP-dependent reporter genes. In summary, these studies show that the amount of Gs{alpha} expression has a marked impact on the level of activity of the AC cascade from the membrane through to the nucleus. It is hypothesized that individuals who differ in Gs{alpha} expression may also differ in the expression of certain cAMP-dependent genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The adenylyl cyclase (AC) signal transduction pathway is a ubiquitous cascade that modulates numerous membrane, cytosolic, and genomic events. The stimulatory G protein, Gs{alpha}, serves to couple and amplify ligand-induced signals transmitted from receptor to particular isoforms of AC. In previous work, we identified a 3-fold range of Gs{alpha} expression in erythrocyte and lymphocyte membranes derived from human subjects (1). We also observed a 3-fold range of Gs{alpha} expression throughout the central nervous system (CNS) of various rodent lines (2). Moreover, we have recently shown that the nonalcoholic offspring of alcoholic men have higher levels of Gs{alpha} expression in erythrocyte and lymphocyte membranes compared with the nonalcoholic offspring of nonalcoholic men (1). This information is provocative in view of twin and adoption studies that have demonstrated genetic determinants for the development of alcoholism (3, 4, 5). In fact, a series of studies have identified abnormalities in the AC pathway of alcoholic subjects (6, 7, 8, 9, 10, 11, 12, 13, 14); some of these abnormalities may predate the onset of alcoholism (9, 10, 11).

These observations suggest potential functional differences in the AC system between individuals with high and low Gs{alpha} expression. For example, it has been shown that reconstitution of Gs{alpha} into cyc-S49 cells, a mutant cell line that does not express Gs{alpha}, results in a dose-dependent increase in AC activity and cAMP accumulation (1, 12, 15, 16). A dose-dependent effect of Gs{alpha} on cAMP accumulation is not only observed within a low concentration range, but also when Gs{alpha} is overexpressed. In this regard, Montminy and co-workers recently demonstrated that overexpression of wild type Gs{alpha} constitutively stimulates the phosphorylation of cAMP response element binding protein (CREB) and cAMP-regulated enhancer (CRE)-dependent transcription in somatotrophs (17). Moreover, high expression of Gs{alpha} in the hearts of transgenic mice significantly decreased the lag period necessary for GppNHp to stimulate AC activity (18).

The present study was conducted to test the hypothesis that there is a relationship between cellular Gs{alpha} content and activation of the AC system. Would the degree of Gs{alpha} expression correlate with constitutive activation and/or agonist-activation of the AC cascade? To this end, an in vitro model was developed in which variable amounts of Gs{alpha} were induced after transfection of an expression vector for Gs{alpha} under regulation by inducible promoters that allowed for a physiologically relevant spectrum of membrane Gs{alpha}. The range of G protein expression was determined based on the spectrum of Gs{alpha} expression we have previously measured in membranes from human erythrocytes and lymphocytes (1). The effects of enhanced Gs{alpha} expression were determined by measuring cellular events downstream from the receptor-G protein complex (e.g. cAMP accumulation, phosphorylation of CREB, and reporter gene transcriptional activity). The results of these studies show that the level of Gs{alpha} expression has a marked impact on activation of the AC pathway.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Range of Gs{alpha} Expression in Humans and Rats
Erythrocyte, lymphocyte, lymphoblast, and platelet membranes derived from healthy nonalcoholic human subjects between the ages of 18 and 30 were prepared. Measurement of membrane Gs{alpha} content from these accessible tissues shows that levels of Gs{alpha} vary over a 3- to 4-fold range (Fig. 1Go). A similar range of Gs{alpha} expression was observed in membranes derived from rat cerebellum in two lines of ethanol-sensitive rats, high alcohol drinking (HAD) and low alcohol drinking (LAD) (Fig. 1Go). HAD rats demonstrate cerebellar membrane Gs{alpha} levels that are, on average, higher than LAD rats (HAD 100 ± 5 vs. LAD 75 ± 7, P < 0.01). A similar range of Gs{alpha} expression was observed in frontal cortex, hippocampus, and hypothalamus in HAD and LAD rats (data not shown).



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Figure 1. Range of Gs{alpha} in Accessible Tissues

Immunoblot analyses for membrane Gs{alpha} derived from healthy nonalcoholic subjects between the ages of 18 and 30 and cerebellum of rats are depicted. Each data point is the Gs{alpha} value from each individual sample. Relative autoradiographic densities of total membrane Gs{alpha} of humans vary over a 3- to 4-fold range. Gs{alpha} content in cerebellar membranes of LAD (solid diamonds) and HAD (solid triangles) rats also varies over a 3- to 4-fold range.

 
Range of Gs{alpha} Expression Induced in Transfected HEK 293 Cells
Wild type HEK 293 cells express both the 52-kDa and 45-kDa forms of Gs{alpha} in approximately equal amounts. The range of Gs{alpha} concentrations that is observed in humans and rats was obtained in vitro in HEK 293 cells by transient transfection of pMTmGsSV as described below in Materials and Methods. Cells transfected with pMTmGsSV and induced with variable concentrations of zinc (0–125 µM) demonstrated Gs{alpha} expression over a near 3-fold range for the 52-kDa form of Gs{alpha} (Fig. 2AGo/2B). Gs{alpha} expression was directly related to zinc concentration in pMTmGsSV transfectants. The cytosolic Gs{alpha} fraction was negligible by immunoblotting (not shown). Consequently, total cellular Gs{alpha} content closely reflects membrane levels and was measured in lieu of membrane Gs{alpha} content. As expected, cells transfected with the control plasmids pMTsSV and pMTmSV, followed by exposure to 0–125 µM zinc sulfate, did not demonstrate any change in level of expression of the 52-kDa form of Gs{alpha}, from either total cellular or membrane extracts, attesting to the fact that zinc itself does not induce expression of Gs{alpha} in control cells (Fig. 2Go). In parallel cultures, transfectants incubated for 30 min in serum-free media accumulated cAMP in a manner that was dependent on the level of Gs{alpha} expression (Fig. 2CGo). In contrast, cAMP accumulation in control transfectants (in which zinc did not induce Gs{alpha} expression) remained low.



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Figure 2. Effects of Varying Concentrations of Zinc on Gs{alpha} Expression

A, Each lane represents extracts of 200,000 transfected cells which were incubated in varying concentrations of zinc for 30 h following transfection. The mouse metallothionein promoter was inducible in a dose-dependent manner and is demonstrated by overexpression of the 52 kDa protein as a function of increasing zinc doses for the Gs{alpha} transfectants (right panel). In contrast, incubations of control transfectants in increasing concentrations of zinc had no effect on Gs{alpha} expression, indicating that zinc itself does not increase Gs{alpha} expression (left panel). B, Densitometric analysis of Immunoblot. C, The effects of varying Gs{alpha} expression on constitutive cAMP accumulation. In parallel with analyses of Gs{alpha} content, 200,000 simultaneously transfected cells were treated with a serum-free medium containing 0.5 mM IBMX for 30 min at 20 C, after which wells were prepared for cAMP RIA. Data points are means of four separate samples from each condition and are accompanied by error bars denoting SEM for cAMP RIA. The experiment was repeated twice with similar results.

 
Note that the 45-kDa form of Gs{alpha} is not altered by transfection and thus serves as an internal standard for loading and transferring of protein during immunoblot process (Fig. 2Go). Comparison of the mouse metallothionen-driven expression vector (pMTsGsSV) and the sheep metallothionen-driven expression vector (pMTmGsSV) revealed approximately 50% less expression of Gs{alpha} in pMTsGsSV transfectants compared with pMTmGsSV transfectants, for each concentration of zinc used. In the absence of zinc, the transfected pMTmGsSV plasmid produced more constitutive expression of Gs{alpha} than the transfected pMTsGsSV plasmid.

To be certain that the concentration (picograms of Gs{alpha}/µg membrane protein) and the range of Gs{alpha} expression (e.g. ~3-fold) induced by the expression vector were similar to those measured in human erythrocyte membranes, a standard Gs{alpha} curve was constructed. The standard curve was generated by measuring increasing amounts of purified, recombinant rGs{alpha} (0–80 ng/lane) on the same gel with membranes derived from the highest and lowest Gs{alpha} expressors derived from erythrocytes and from the highest and lowest Gs{alpha} expressors derived from HEK cells transfected with pMTGsSV (Fig. 3Go). Gs{alpha} levels ranged from 70–425 pg/µg membrane protein in membranes derived from erythrocytes and 90–500 pg/µg membrane protein for transfected HEK cells induced with zinc.



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Figure 3. Assessing Gs{alpha} Expression with a Gs{alpha} Standard Curve

Increasing amounts of purified, recombinant rGs{alpha} (0–80 ng) were run on the same gel with representative human erythrocyte membranes and membranes derived from transfected HEK 293 cells that were exposed to several concentrations of zinc (0–100 uM) before harvest. Erythrocyte Gs{alpha} levels, as assessed by PhosphorImager, varied by 3.2-fold. HEK 293 cell Gs{alpha} levels, as assessed by PhosphorImager, varied by 3.47-fold. The experiment demonstrates that the expression vector pMTmGsSV can induce the concentration and range of Gs{alpha} expression found in human erythrocyte membranes.

 
Constitutive Activation of the AC Pathway as a Function of Gs{alpha} Expression
To determine the kinetics of cAMP accumulation, transfectants were incubated in serum-free media for 1, 3, 5, 10, and 30 min. Intracellular cAMP accumulation plateaued at 10 min for all levels Gs{alpha} expression, with no further increase observed at the 30-min sampling time. Accumulation of cAMP was related to levels of Gs{alpha} expression for four levels, at each time point sampled (Fig. 4Go). For subsequent experiments, the 10-min incubation time was therefore employed.



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Figure 4. Kinetics of Constitutive cAMP Accumulation

HEK 293 cells were transfected with either pMTsGsSV (sheep metallothionein promoter-driven Gs{alpha} expression vector) or pMTmGsSV (mouse metallothionein-driven Gs{alpha} expression vector). Transfectants were incubated in either 0 or 100 µM zinc, which produced four levels of Gs{alpha} expression: 100 (sheep vector, 0 µM zinc), 132 (sheep vector, 100 µM zinc), 188 (mouse vector, 0 µM zinc), and 361 (mouse vector, 100 µM zinc). Accumulation of cAMP was measured at 1, 3, 5, and 10 min following incubation in serum-free media containing 0.5 mM IBMX. Each point represents the average of four separate measurements for each condition, with associated error bars denoting SEM. The experiment was repeated twice with similar results.

 
To determine whether the degree of Gs{alpha} expression also affects the adenylyl cyclase cascade distal to cAMP accumulation, phospho-CREB levels as well as the transcriptional activity of cAMP-dependent reporter genes were measured in "low" and "high" Gs{alpha}-expressing cells. The transcription factor, CREB, is inactive until it is phosphorylated by the catalytic unit of protein kinase A (PKA-C). The phosphorylation of CREB occurs after cAMP-induced translocation of PKA-C into the nucleus. To this end, HEK-293 cells were transfected with pMTmGsSV (mouse metallothionein-driven Gs{alpha} expression vector) and incubated with or without zinc (75 uM) for 48 h and then harvested for measurements of Gs{alpha} and phospho-CREB. A 3.5-fold induction of Gs{alpha} resulted in almost a 3-fold induction of phospho-CREB (Fig. 5Go).



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Figure 5. The Phosphorylation of CREB as a Function of Gs{alpha} Expression

HEK-293 cells (200,000 cells per well) were transfected with pMTmGsSV (mouse metallothionein-driven Gs{alpha} expression vector) and incubated in the presence or absence of zinc (75 uM) for 48 h and then harvested for measurements of Gs{alpha} and phospho-CREB. Induction of Gs{alpha} by zinc (A) resulted in elevated phospho-CREB levels in nuclear extract (B). Mean densitometric data for Gs{alpha} and for phospho-CREB are the result of three experiments performed in triplicate; *, P < 0.001.

 
To determine whether enhanced expression of Gs{alpha} also induced transcription of cAMP-dependent genes, cells were either cotransfected with pMTGs{alpha}SV and a reporter gene construct containing a region of the proenkephalin promoter [-2000/+53] ligated to the chloramphenicol acetyltransferase (CAT) gene, or in parallel cultures, cells were cotransfected with the control vector and the CAT reporter construct (see Materials and Methods). The reporter gene contains two CREs that are responsive to the phosphorylated form of CREB. A 2.5-fold increase in Gs{alpha} expression induced a 2-fold increase in proenkephalin-CAT activity (Fig. 6Go).



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Figure 6. Cyclic AMP-Dependent Reporter Gene Expression as a Function of Gs{alpha} Expression

HEK 293 cells were plated at a density of 8 x 106 cells per T75 flask and cotransfected with pREJCAT[-2000/+53] (CAT reporter gene; see Materials and Methods) and MTmGsSV or pREJCAT[-2000/+53] and the control vector. DNA precipitates contained 10 µg of each test plasmid and 2 µg pCH110ßgal plasmid. After 18 h, cells were detached from flask with trypsin-EDTA and replated at a density of 6 x 105 cells per well in the presence or absence of 75 uM zinc for 48 h. Data was normalized to ß-galactosidase activity from an internal ß-galactosidase control plasmid. Induction of Gs{alpha} by zinc (A) resulted in increased CAT activity (B). Mean densitometric data for Gs{alpha} and mean CAT activity data are the result of three experiments performed in triplicate; *, P < 0.001.

 
To determine whether enhanced Gs{alpha} expression would also increase expression of a cAMP-dependent reporter gene endogenous to HEK 293 cells, the effects of increasing Gs{alpha} expression were assessed on ß2-adrenergic receptor mRNA levels. It was observed that a 2.5-fold induction of Gs{alpha} resulted in 3-fold increased steady state levels of ß2-adrenergic receptor mRNA (Fig. 7Go).



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Figure 7. Enhanced Gs{alpha} Expression Induces Expression of an Endogenous cAMP-Dependent Reporter Gene

HEK-293 cells were transfected with pMTmGsSV (mouse metallothionein-driven Gs{alpha} expression vector) and incubated in the presence or absence of zinc (75 uM) for 48 h and then harvested for measurements of Gs{alpha} and ß2-adrenergic receptor mRNA. Induction of Gs{alpha} by zinc (A) resulted in elevated ß2-adrenergic receptor mRNA levels (B). Mean densitometric data are the result of three experiments performed in triplicate; *, P < 0.001. C, Ethidium bromide identification of 28S and 18S RNA in gel (control for loading). D, Methylene blue staining of 28S and 18S RNA on filter (control for transfer).

 
Agonist Activation of the AC Pathway as a Function of Gs{alpha} Expression
Experiments were conducted with PgE1 to determine the influence of Gs{alpha} expression on activation of the AC pathway by an agonist. To this end, HEK 293 cells (75,000 cells per well) were transfected with pMTmGsSV and incubated in 0, 50, or 100 µM zinc for 48 h to induce three levels of Gs{alpha} expression (100 ± 11 vs. 200 ±19 vs. 278 ± 18). Then cells were exposed to a submaximal doses of 0.1 uM PGE1 (19) and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) or only IBMX for 10 min in serum-free media. PGE1-stimulated cAMP accumulation increased as a function of Gs{alpha} expression (Fig. 8Go).



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Figure 8. PgE1-Stimulated cAMP Accumulation as a Function of Gs{alpha} Expression

HEK 293 cells (75,000 cells per well) were transfected with pMTmGsSV and incubated in 0, 50, or 100 µM zinc for 48 h to induce three levels of Gs{alpha} expression. Then cells were exposed to 0.1 uM PGE1 and 0.5 mM IBMX or 0.5 mM IBMX for 10 min in serum-free media. Each point represents the average of three separate experiments with error bars denoting SEM. *, P < 0.01.

 
Effects of PGE1 Exposure on the Phosphorylation of CREB as a Function of Gs{alpha} Expression
Two levels of Gs{alpha} expression were obtained by transfection of HEK 293 cells with pMTsGsSV and pMTmGsSV (100 ± 31 vs. 180 ± 17). Transfectants were then incubated in serum-free media either with or without 0.1 µM PgE1 for 15 min, after which phospho-CREB was measured. In the absence of PgE1, an 80% increase in Gs{alpha} resulted in a 60% increase in phospho-CREB (100 ± 10 vs. 162 ± 18, P < 0.01) (Fig. 9Go). Moreover, in the presence of PgE1, the higher Gs{alpha} condition was associated with increased phospho-CREB levels compared with the lower Gs{alpha} condition (285 ± 28 vs. 410 ± 26, P < 0.01).



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Figure 9. Effects of PGE1 on the Phosphorylation of CREB as a Function of Gs{alpha} Expression

Two levels of Gs{alpha} expression were obtained by transfection of HEK 293 cells with pMTsGsSV and pMTmGsSV, after which cells were incubated in the presence or absence of PgE1, followed by immunoblot analyses. A, Representative Gs{alpha} immunoblot; sMT, sheep promoter; mMT, mouse promoter. B, Densitometric analysis of immunoblots after two experiments performed in triplicate. (a = 100 ± 10; a' = 285 ± 28; b = 162 ± 18; b' = 410 ± 26; a vs. b, P < 0.01; a' vs. b', P < 0.01; a vs. a', P < 0.0125; b vs. b', P < 0.01).

 
Induction of Gs{alpha} did not result in compensatory changes in either Gi2{alpha} (Fig. 10Go) or Gß36 subunit expression (data not shown).



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Figure 10. Gi2{alpha} Levels as a Function of Gs{alpha} Expression

HEK 293 cells were transfected with pMTmGsSV and incubated in 0, 50, or 75 µM zinc for 48 h to induce three levels of Gs{alpha} expression. Cells were harvested, and Gs{alpha} and Gi2{alpha} levels were measured. Induction of Gs{alpha} did not influence Gi2{alpha} expression over the 48-h incubation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study set out to determine whether physiologically relevant differences in Gs{alpha} expression are sufficient to alter the AC cascade. We were surprised to observe that the most pronounced effect of varying Gs{alpha} expression was an effect that occurred independently of agonist stimulation. Namely, increased Gs{alpha} expression increased constitutive accumulation of cAMP. In fact, accumulation of cAMP was proportional to the amount of Gs{alpha} expression. The induction of Gs{alpha} did not induce a compensatory change in the expression of Gi2{alpha} or Gß36. Less marked, but still quite significant, was the observation that accumulation of agonist-stimulated cAMP levels was also proportional to the amount of Gs{alpha} expression. This observation was made using endogenous Gs{alpha}-coupled receptors and also by employing a foreign receptor to this cell line that is known to couple to Gs{alpha} (i.e. PTH receptor; data not shown).

To determine whether variable Gs{alpha} expression could also affect the AC signal cascade distal to cAMP accumulation, the phosphorylation of the cAMP-dependent transcription factor CREB and the amount of phospho-CREB-dependent gene transcription were studied. Hormones and neurotransmitters that stimulate cAMP induce the phosphorylation of CREB. After G protein-coupled activation of AC and the generation of cAMP, cAMP binds to regulatory subunits of protein kinase A, inducing dissociation and production of active monomeric catalytic subunits. The catalytic unit of PKA translocates into the nucleus where it phosphorylates CREB on serine 133. CREB, implicated in the regulation of long- term potentiation and synaptic plasticity, has been posited to play an important role in mechanisms underlying learning and memory (20, 21, 22). Our data show that enhanced expression of Gs{alpha} is associated with an increase in the amount of phospho-CREB, in the presence and in the absence (e.g. constitutive) of an agonist.

CREB is a leucine zipper family member and binds to a palindromic response element (TGACGTCA), known as the cAMP-regulated enhancer (CRE), in cAMP-inducible genes. CREB becomes transcriptionally active after phosphorylation of Ser-133 by cAMP-dependent and cAMP-independent signal cascades (21). Because increased Gs{alpha} expression resulted in both increased cAMP and phospho-CREB levels, it was plausible to assume that enhanced expression of Gs{alpha} would also induce more CRE-dependent gene transcription. Using the proenkephalin-CAT reporter construct, we show that an increase in Gs{alpha} induced an increase in phospho-CREB as well as an increase in proenkephalin-CAT transcriptional activity. Moreover, we also show increased expression of an endogeneous cAMP-dependent gene, the ß2-adrenergic receptor gene, as a function of Gs{alpha} expression. Induction of Gs{alpha} resulted in increased steady state levels of ß2-adrenergic receptor mRNA.

The membrane AC system is a ubiquitous signal transduction pathway that modulates numerous membrane, cytosolic, and genomic events. The stimulatory G protein, Gs{alpha}, serves to couple and amplify ligand-induced signals transmitted from receptor to particular isoforms of AC. In the present study, we extended previous observations (1, 2) and identified a 3-fold range of Gs{alpha} expression in erythrocyte, lymphocyte, transformed lymphocyte, and platelet membranes derived from human subjects. We also identified a 3-fold range of Gs{alpha} expression throughout the CNS of various rodent lines. One reason for conducting these studies was to address our previous observation that the nonalcoholic offspring of alcoholics have higher levels of Gs{alpha} expression in erythrocyte and lymphocyte membranes compared with the nonalcoholic offspring of nonalcoholics (1). In combination, these data suggest potential important differences in the AC system between individuals with high and low Gs{alpha} expression. These observations led us to hypothesize that the amount of membrane Gs{alpha} (within a physiological range) helps modulate the degree of activation of the AC signal transduction system. Our data show a dose-response relationship between the amount of Gs{alpha} a cell expresses and the magnitude to which the AC system can be activated by agonists. It is plausible that a cell population with higher Gs{alpha} expression may be able to transduce a signal of greater magnitude than a cell population with lower Gs{alpha} expression.

How might this information be applied to form a hypothesis vis-a-vis our previous findings of high levels of Gs{alpha} expression in the offspring of alcoholics compared with the offspring of nonalcoholics? In other words, how may the level of Gs{alpha} expression alter the risk for alcoholism? Genetic determinants for alcoholism are supported by both adoption and twin studies (3, 4, 5). The implication of genetic factors in the development of alcoholism and alcohol-related tissue injury has stimulated a search for biological substrates that may confer enhanced risk for alcoholism and for drug-seeking behavior in general (3, 4, 5). In this regard, the AC signal transduction cascade has been examined as one in a series of "candidate" systems that may differ among individuals at high risk and low risk for the future development of this disorder (1, 6, 7, 8, 9, 10, 11).

We speculate that enhanced Gs{alpha} expression could have broad consequences on phenotypes that increase the risk for alcoholism. For example, because high levels of Gs{alpha} expression appear to constitutively activate the AC pathway (e.g. absence of agonist stimulation), it is conceivable that individuals with high Gs{alpha} expression will also have higher levels of expression of many cAMP-dependent gene products (e.g. receptors, hormones, neurotransmitters). Cyclic AMP-dependent genes include many that have been hypothesized to be involved in drug-seeking behaviors [e.g. the opioid precursors proenkephalin and POMC and their receptors, tyrosine hydroxylase, ß-adrenergic receptors (8)].

It is also plausible that the level of Gs{alpha} expression in vivo may modulate tissue responsivity to agonists. Numerous processes, including hormone and neurotransmitter synthesis and release, gene expression, and cell proliferation, are controlled by neurotransmitters and hormones that act through the second messenger, cAMP. Ethanol can stimulate the release as well as augment the action of peripheral hormones and neurotransmitters (6). In the CNS, an important effect of acute ethanol intoxication is the presynaptic release of certain neurotransmitters (e.g. opioids and dopamine) as well as the postsynaptic potentiation of receptors that couple to Gs{alpha}. We speculate that acute intoxication by ethanol initiates a cascade from which differences in membrane Gs{alpha} may play a role in determining differential tissue sensitivity to ethanol. In this model, once ethanol-induced alterations in neurotransmission occur, the magnitude of the postsynaptic response is in part determined by the membrane content of Gs{alpha}. Because the level of expression of Gs{alpha} in the CNS may be an inherited phenotype, viability in Gs{alpha}-mediated signal transduction may be a factor in differences in predisposition to tissue injury induced by ethanol.

In summary, in human tissues and in the CNS of rodents, Gs{alpha} expression can vary as much as 3-fold. In an in vitro model, this range of Gs{alpha} expression increased the accumulation of cAMP, phospho-CREB, and reporter gene transcriptional activity in a dose-dependent manner. We speculate that differences in Gs{alpha} expression in vivo may also alter the AC cascade.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction and Subcloning of the Gs{alpha} Expression Vectors, pMTmGsSV and pMTsGsSV
The inducible Gs{alpha} expression vector pMTsGsSV was constructed by cloning the 0.9-kb XbaI-XhoI sheep metallothionen promoter from pMTCB6 (gift of Dr. Chi Dang), the 1.5-kb XhoI Gs{alpha} fragment encoding for the 52-kDa protein from pHGs8 (gift of Dr. Michael Levine), and the 0.84 kb XhoI-XbaI SV 40 fragment from pMSXND (gift of Dr. Sejin Lee) into Bluescript (Stratagene, La Jolla, CA). The corresponding vector control plasmid, pMTsSV, was constructed by excising the Gs{alpha} insert from pMTsGsSV. The more constitutively active Gs{alpha} expression vector, pMTmGsSV, was constructed by cloning the 2.8-kb EcoRI-BamHI fragment containing the mouse metallothionen promoter and SV 40 from pMSXND into Bluescript, followed by insertion of the 1.5 kb XhoI Gs{alpha} fragment from pHGs8. The corresponding vector control plasmid, pMTmSV, lacks the Gs{alpha} insert.

Expression of Gs{alpha} by Transient Transfection and Zinc Induction
Human embryonic kidney cells (HEK 293) were selected because of their endogenously low level of membrane Gs{alpha} expression and membrane AC activity. Cells were plated to semiconfluency at a density of 10 million cells per T75 tissue culture flask. Transient transfection was accomplished by calcium phosphate precipitation in a HEPES buffer (5 Prime-3 Prime Inc., Boulder, CO) with 20 µg plasmid DNA/flask for 18 h at 37 C, 5% CO2. Precipitates were washed with sterile PBS. Cells were detached from flasks with trypsin-EDTA and plated onto 24-well tissue culture plates (200,000 cells per well). Zinc sulfate was included in the complete media at final concentrations of 0, 25, 50, 75, 100, and 125 uM. Cell viability was determined by staining with Trypan blue and was 100% when zinc concentrations did not exceed 125 uM, appreciable with zinc concentrations greater than 150 µM, and 100% lethal at concentrations greater than 200 µM (after a 30-h incubation). Forty-eight hours after the start of transfection, cells were extracted with 1 x Laemli in lysis buffer (1 mM Tris-HCl, 1 mM EDTA, 5 µg/ml leupeptin, 0.3 mg/ml phenylmethylsulfonylfluoride, pH 7.4), boiled for 5 min, and analyzed for Gs{alpha} content by immunoblotting. Alternatively, after overnight transfection, cells were washed free of precipitates with PBS, incubated in original flasks with varying concentrations of zinc, and harvested for membranes by the methods described below. Efficiency of transfection was determined by transfecting separate plates in parallel with the eukaryotic assay vector, pCH110, followed by X-gal (GIBCO BRL, Gaithersburg, MD) staining, and was found to be 53.3 ± 4.1% (n = 12).

Immunoblot Analysis
G protein content from accessible tissues from humans and from the CNS of rodents was determined as previously described (1, 14). Antiserum RM/1 (Dupont NEN, Boston, MA), which recognizes C-terminal regions of both the 45- and 52-kDa forms of Gs{alpha}, was used. G protein signal intensities were determined with a PhosphorImager and analyzed with ImageQuant computer software (Molecular Dynamics, Sunnyvale, CA). Data were expressed as ratios of 52- to 45-kDa forms of Gs{alpha} (~ 1.0 for wild type HEK 293 cells). The lowest level of expression for each set of transfections was assigned an arbitrary value of 100, with higher levels of expression assigned values relative to 100, thereby correcting for differences in sample loading and [125I]protein A (ICN, Costa Mesa, CA) specific activity. The Gs{alpha} standard curve was constructed using varying amounts (5–80 ng) of purified, recombinant Gs{alpha} (CytoSignal Research Product, Irvine, CA).

Phospho-CREB Analysis
To obtain nuclear extract, cells were harvested in lysis buffer (10 mM HEPES, 0.5 mM spermidine·3 HCL, 0.15 mM spermine·4 HCL, 5 mM EDTA, 0.25 mM EGTA, 50 mM NaF, 7.0 mM2-MeOH) with 2 M sucrose. The cellular extracts were spun at 1000 x g for 5 min at 4 C, the nuclear pellets were resupended in 1 ml lysis buffer containing 0.35 M sucrose, followed by centrifugation at maximal speed at 4 C for 30 min. The pellet containing nuclear protein was resuspended in NSB buffer containing 20 mM Tris (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM dithiothreitol, 0.125 mM phenylmethylsulfonyl fluoride, 50% glycerol, and 2 x NUN solution was added (25 mM HEPES, 300 mM NaCL, 1 M urea, 1% NP-40, and 1 mM dithiothreitol). After vigorous vortexing, the samples were shaken at 4 C for 1 h and centrifuged at maximal speed at 4 C for 10 min. The supernatant was stored at -70 C. Final protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL). Immunoblot analysis was performed to detect phosphorylated CREB using an anti-phospho-CREB antisera raised against phosphorylated amino acid residues 123–136 of CREB (06–245; Upstate Biotechnology, Lake Placid, NY). The antisera is specific for pSer133-CREB as described by Ginty et al. (23). Signal intensities were determined as described above.

cAMP Accumulation
After incubations, media were aspirated and replaced with 0.3 ml 0.1 N HCl. Acid extracts were boiled for 2 min, frozen at -80 C, centrifuged 14,000 x g, and supernatant was saved for assays. For cAMP RIA, standard solutions were mixed from a 5000 pM cAMP standard; tubes for total counts and nonspecific binding were prepared from buffer (0.05 M NaOAc, pH 6.2). Samples (either 2.5 or 5.0 µl) were taken in duplicate and 100 µl buffer were added to each. Tubes were acetylated with 5 µl of a solution containing 1 part acetic anhydride and 2 parts triethylamine. After addition of 100 µl trace solution [[125I]cAMP (ICN) (10,000 cpm per tube) and 100 µl IgG (Biotek, Lenexa, KA) in 5 cc buffer] and of 100 µl primary antibody solution [0.025 g BSA, 25 µl specific rabbit antibody (ICN) (diluted to 1:200), in 5 cc buffer], samples were incubated overnight at 4 C. Tubes for nonspecific binding did not receive primary antibody solution. Second antibody solution 100 µl [10 ml goat anti-rabbit antibody (Biotek), 250 µl phenylmethylsulfonyl fluoride (30 mg/ml EtOH), 1.25 ml 2% NaN3 in 115 ml 50 mM NaPO4, pH 7.5] was added to each tube and incubated at 20 C for 2 h. One milliliter of buffer was added to each tube, and tubes were spun at 2000 x g at 4 C for 10 min. Pellets were counted on a {gamma}-counter, and counts were analyzed using a RIA program. Differences in sample concentration were corrected by expressing values as picomoles per mg protein.

CAT Assays
To measure transcriptional activity, a plasmid containing the chloramphenicol acetyltransferase (CAT) gene under the control of the rat proenkephalin sequences from bases --2700 to +53 (pREJCAT [-2000/+53]; gift of Dr. Steven Sabol) was employed (24). The proenkephalin sequences contain two cAMP-inducible enhancers of the proenkephalin gene. For cotransfection experiments, HEK 293 cells were plated at a density of 8 x 106 cells per T75 flask and cotransfected with pREJCAT[-2000/+53] and MTmGsSV or pREJCAT[-2000/+53] and the control vector, MtmSV, as described above. DNA precipitates generally contained 10 µg of each test plasmid and 2 µg pCH110ßgal plasmid. After 18 h, cells were detached from the flask with trypsin-EDTA and replated at a density of 6 x 105 cells per well in the presence of 75 uM zinc. Cells were harvested after 48 h for either nuclear protein (phospho-CREB determination) or whole-cell extract (CAT activity, cAMP, and Gs{alpha} levels). CAT activity was determined by a phase-extraction assay as previously described (25). Data were normalized to ß-galactosidase activity from an internal ß-galactosidase control plasmid.

Northern Analysis
Total RNA was isolated (Perfect Total RNA Isolation Kit; 5 Prime-3 Prime). The human ß2-adrenergic receptor cDNA was excised from pBC12B1 [gift of Dr. Charles Emela (34)] by NCOI/SALI digestion to obtain a 2.2-kb insert. The insert was 32P-labeled by the random prime method (Random Prime, Amersham). Northern analyses were performed as previously described (14). Loading and transfer efficiency was assessed by ethidium bromide staining of gel and trypan blue staining of nitrocellulose.

Statistical Analysis
Data were evaluated by Student’s t-test and ANOVA where appropriate.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Sejin Lee, M.D., Ph.D., for assistance in constructing the vectors employed in this study. We also thank June Dameron for secretarial assistance in the preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Gary S. Wand, M.D., Associate Professor of Medicine and Psychiatry, The Johns Hopkins University School of Medicine, Ross Research Building, Room 850, 720 Rutland Avenue, Baltimore, Maryland 21205.

This work was supported by NIH Grant RO1-AA09000 and a generous grant from Alexander and Norma Lattman and Rochelle and Elliot Abramson.

Received for publication October 4, 1996. Revision received April 11, 1997. Accepted for publication April 15, 1997.


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