Increased Expression Of Gs
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
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ABSTRACT
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Expression of the stimulatory G protein,
Gs
, 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
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
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
under regulation by inducible
metallothionein promoters. Transfectants were exposed to varying
concentrations (0125 µM) of zinc sulfate
that produced a 3-fold range of membrane Gs
expression. The range of Gs
expression
produced was found to mimic a physiologically relevant spectrum of
Gs
expression in membranes derived from
human tissues and rat brain. It was observed that induction of
Gs
expression increased constitutive as well
as stimulated cAMP accumulation. Moreover, induction of
Gs
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
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
expression may also differ
in the expression of certain cAMP-dependent genes.
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INTRODUCTION
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The adenylyl cyclase (AC) signal transduction pathway is a
ubiquitous cascade that modulates numerous membrane, cytosolic, and
genomic events. The stimulatory G protein, Gs
, 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
expression in erythrocyte and lymphocyte
membranes derived from human subjects (1). We also observed a 3-fold
range of Gs
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
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
expression. For example, it has been shown that reconstitution of
Gs
into cyc-S49 cells, a mutant cell line that does not
express Gs
, results in a dose-dependent increase in AC
activity and cAMP accumulation (1, 12, 15, 16). A dose-dependent effect
of Gs
on cAMP accumulation is not only observed within a
low concentration range, but also when Gs
is
overexpressed. In this regard, Montminy and co-workers recently
demonstrated that overexpression of wild type Gs
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
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
content and activation
of the AC system. Would the degree of Gs
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
were induced after
transfection of an expression vector for Gs
under
regulation by inducible promoters that allowed for a physiologically
relevant spectrum of membrane Gs
. The range of G protein
expression was determined based on the spectrum of Gs
expression we have previously measured in membranes from human
erythrocytes and lymphocytes (1). The effects of enhanced
Gs
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
expression has a marked impact on activation
of the AC pathway.
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RESULTS
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Range of Gs
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
content
from these accessible tissues shows that levels of Gs
vary over a 3- to 4-fold range (Fig. 1
). A similar range
of Gs
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. 1
). HAD rats
demonstrate cerebellar membrane Gs
levels that are, on
average, higher than LAD rats (HAD 100 ± 5 vs. LAD
75 ± 7, P < 0.01). A similar range of
Gs
expression was observed in frontal cortex,
hippocampus, and hypothalamus in HAD and LAD rats (data not shown).
Range of Gs
Expression Induced in
Transfected HEK 293 Cells
Wild type HEK 293 cells express both the 52-kDa and 45-kDa forms
of Gs
in approximately equal amounts. The range of
Gs
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 (0125 µM) demonstrated
Gs
expression over a near 3-fold range for the 52-kDa
form of Gs
(Fig. 2A
/2B).
Gs
expression was directly related to zinc concentration
in pMTmGsSV transfectants. The cytosolic Gs
fraction was
negligible by immunoblotting (not shown). Consequently, total cellular
Gs
content closely reflects membrane levels and was
measured in lieu of membrane Gs
content. As expected,
cells transfected with the control plasmids pMTsSV and pMTmSV, followed
by exposure to 0125 µM zinc sulfate, did not
demonstrate any change in level of expression of the 52-kDa form of
Gs
, from either total cellular or membrane extracts,
attesting to the fact that zinc itself does not induce expression of
Gs
in control cells (Fig. 2
). 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
expression (Fig. 2C
). In contrast, cAMP accumulation in control
transfectants (in which zinc did not induce Gs
expression) remained low.
Note that the 45-kDa form of Gs
is not altered by
transfection and thus serves as an internal standard for loading and
transferring of protein during immunoblot process (Fig. 2
). Comparison
of the mouse metallothionen-driven expression vector (pMTsGsSV) and the
sheep metallothionen-driven expression vector (pMTmGsSV) revealed
approximately 50% less expression of Gs
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
than the transfected pMTsGsSV plasmid.
To be certain that the concentration (picograms of
Gs
/µg membrane protein) and the range of
Gs
expression (e.g.
3-fold) induced by the
expression vector were similar to those measured in human erythrocyte
membranes, a standard Gs
curve was constructed. The
standard curve was generated by measuring increasing amounts of
purified, recombinant rGs
(080 ng/lane) on the same
gel with membranes derived from the highest and lowest
Gs
expressors derived from erythrocytes and from the
highest and lowest Gs
expressors derived from HEK cells
transfected with pMTGsSV (Fig. 3
). Gs
levels ranged from 70425 pg/µg membrane protein in membranes
derived from erythrocytes and 90500 pg/µg membrane protein for
transfected HEK cells induced with zinc.
Constitutive Activation of the AC Pathway as a Function of
Gs
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
expression, with no further increase observed at the
30-min sampling time. Accumulation of cAMP was related to levels of
Gs
expression for four levels, at each time point
sampled (Fig. 4
). 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 expression vector) or
pMTmGsSV (mouse metallothionein-driven Gs expression
vector). Transfectants were incubated in either 0 or 100
µM zinc, which produced four levels of Gs
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.
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To determine whether the degree of Gs
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
-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
expression vector) and incubated with or without zinc
(75 uM) for 48 h and then harvested for measurements
of Gs
and phospho-CREB. A 3.5-fold induction of
Gs
resulted in almost a 3-fold induction of phospho-CREB
(Fig. 5
).
To determine whether enhanced expression of Gs
also
induced transcription of cAMP-dependent genes, cells were either
cotransfected with pMTGs
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
expression induced a
2-fold increase in proenkephalin-CAT activity (Fig. 6
).
To determine whether enhanced Gs
expression would also
increase expression of a cAMP-dependent reporter gene endogenous to HEK
293 cells, the effects of increasing Gs
expression were
assessed on ß2-adrenergic receptor mRNA levels. It was
observed that a 2.5-fold induction of Gs
resulted in
3-fold increased steady state levels of ß2-adrenergic
receptor mRNA (Fig. 7
).
Agonist Activation of the AC Pathway as a Function of
Gs
Expression
Experiments were conducted with PgE1 to determine the influence of
Gs
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
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
expression (Fig. 8
).

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Figure 8. PgE1-Stimulated cAMP Accumulation as a Function of
Gs 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 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.
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Effects of PGE1 Exposure on the Phosphorylation of CREB as a
Function of Gs
Expression
Two levels of Gs
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
resulted in a 60% increase in
phospho-CREB (100 ± 10 vs. 162 ± 18,
P < 0.01) (Fig. 9
). Moreover, in the
presence of PgE1, the higher Gs
condition was associated
with increased phospho-CREB levels compared with the lower
Gs
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 Expression
Two levels of Gs 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 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).
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Induction of Gs
did not result in compensatory changes
in either Gi2
(Fig. 10
) or
Gß36 subunit expression (data not shown).
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DISCUSSION
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This study set out to determine whether physiologically relevant
differences in Gs
expression are sufficient to alter the
AC cascade. We were surprised to observe that the most pronounced
effect of varying Gs
expression was an effect that
occurred independently of agonist stimulation. Namely, increased
Gs
expression increased constitutive accumulation of
cAMP. In fact, accumulation of cAMP was proportional to the amount of
Gs
expression. The induction of Gs
did
not induce a compensatory change in the expression of Gi2
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
expression. This
observation was made using endogenous Gs
-coupled
receptors and also by employing a foreign receptor to this cell line
that is known to couple to Gs
(i.e. PTH
receptor; data not shown).
To determine whether variable Gs
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
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
expression
resulted in both increased cAMP and phospho-CREB levels, it was
plausible to assume that enhanced expression of Gs
would
also induce more CRE-dependent gene transcription. Using the
proenkephalin-CAT reporter construct, we show that an increase in
Gs
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
expression. Induction of Gs
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
, 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
expression in
erythrocyte, lymphocyte, transformed lymphocyte, and platelet membranes
derived from human subjects. We also identified a 3-fold range of
Gs
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
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
expression. These observations led us to
hypothesize that the amount of membrane Gs
(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
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
expression may be able to transduce a signal of greater magnitude than
a cell population with lower Gs
expression.
How might this information be applied to form a hypothesis vis-a-vis
our previous findings of high levels of Gs
expression in
the offspring of alcoholics compared with the offspring of
nonalcoholics? In other words, how may the level of Gs
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
expression could have
broad consequences on phenotypes that increase the risk for alcoholism.
For example, because high levels of Gs
expression appear
to constitutively activate the AC pathway (e.g. absence of
agonist stimulation), it is conceivable that individuals with high
Gs
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
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
. We speculate that acute intoxication by
ethanol initiates a cascade from which differences in membrane
Gs
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
.
Because the level of expression of Gs
in the CNS may be
an inherited phenotype, viability in Gs
-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
expression can vary as much as 3-fold. In an in
vitro model, this range of Gs
expression increased
the accumulation of cAMP, phospho-CREB, and reporter gene
transcriptional activity in a dose-dependent manner. We speculate that
differences in Gs
expression in vivo may also
alter the AC cascade.
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MATERIALS AND METHODS
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Construction and Subcloning of the Gs
Expression Vectors, pMTmGsSV and pMTsGsSV
The inducible Gs
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
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
insert from pMTsGsSV. The more
constitutively active Gs
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
fragment from pHGs8. The
corresponding vector control plasmid, pMTmSV, lacks the
Gs
insert.
Expression of Gs
by Transient
Transfection and Zinc Induction
Human embryonic kidney cells (HEK 293) were selected because of
their endogenously low level of membrane Gs
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
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
, 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
(
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
standard curve
was constructed using varying amounts (580 ng) of purified,
recombinant Gs
(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 123136 of CREB (06245; 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
-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
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 Students t-test and ANOVA
where appropriate.
 |
ACKNOWLEDGMENTS
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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.
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FOOTNOTES
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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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