From the Weis Center for Research, Geisinger Clinic,
Danville, Pennsylvania 17822 and the § Department of
Cellular and Molecular Physiology, The Pennsylvania State College of
Medicine, Hershey, Pennsylvania 17033-0850
Received for publication, October 30, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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The G protein The heterotrimeric G proteins control diverse
biological processes by conveying signals from cell-surface receptors
to intracellular effectors. Although function was originally ascribed
to the GTP-bound The G protein Animal Care and Approval--
Mice were segregated by sex and
group-housed in plastic microisolator cages in ventilated racks
(Thoren Caging Systems, Inc., Hazelton, PA). Mice were given ad
libitum access to water and Mouse Diet 9F (Purina Mills, St.
Louis, MO). Environmental factors included temperature and humidity
control and a 12-h light/dark cycle. The animal facility is maintained
as virus antibody-free and parasite-free. Animal research protocols
were approved by the Geisinger Clinic institutional animal care and use committee.
Genotyping--
Southern blot analysis was performed on genomic
tail DNA cut with KpnI. The probe used for this analysis was
an 0.8-kb fragment 5' of the modified Gng7 allele.
Alternatively, PCR analysis was performed using primers
(Invitrogen) shown in Table I. Briefly, primers flanking
the 3' loxP site (JR385 and JR387) were used competitively to amplify
the wild type and Gng7fl alleles, and a third
primer upstream of the 5' loxP site (JR413) was included to amplify
simultaneously the wild type and deleted Gng7 alleles.
Amplification of the bacteriophage P1 Cre transgene was
conducted with JR353 and JR354.
RT-PCR--
RNA was isolated from olfactory bulb, frontal
cortex, striatum, hypothalamus, midbrain, cerebellum, pons, or
whole brain using TRIzol reagent (Invitrogen). First-strand cDNA,
prepared from 2 µg of RNA using Moloney murine leukemia virus
reverse transcriptase (M-MLV RT, Promega Corp., Madison, WI), was used
as a template to amplify the G Immunoblotting--
Powdered brain tissues were homogenized in
HME with proteinase inhibitors (20 mM Hepes, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 1 mM benzamidine, 0.1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µM
leupeptin, 1.4 µM pepstatin, 27 µM
1-chloro-3-tosylamido-7-amino-2-heptanone, 28 µM
L-1-tosylamido-2-phenylethyl chloromethyl ketone).
Membranes were obtained by centrifugation onto a sucrose cushion (see
below), and membrane-associated proteins were extracted with 1%
cholate at 4 °C overnight. Protein concentrations were determined
using an Amido Black assay (15). Equal amounts of proteins were loaded onto 12% Nu-Page gels (Invitrogen) and transferred to NitroPure nitrocellulose (Osmonics, Inc., Westborough, MA) using a high temperature transfer procedure (16). Immunoblotting was performed as
described previously (14) with antisera specific for G Adenylyl Cyclase Assay--
Brain tissues were homogenized in
Buffer A (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.3 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 30 µM
leupeptin, 1 µM pepstatin A) with 10% sucrose using a
Brinkmann homogenizer (Brinkmann Instruments). Membranes were then
isolated by centrifugation (65 min at 100,000 × g)
onto a cushion of Buffer A with 44.5% (w/v) sucrose. The membranes at
the interface were transferred to a new tube, washed twice with Buffer
A, and collected by centrifugation (30 min at 100,000 × g). Protein concentrations were determined with Coomassie
Plus (Pierce). Adenylyl cyclase activity (18) was determined by
incubating membrane protein (20 µg) at 30 °C for 10 min in 0.1 ml
of buffer containing 50 mM Hepes, pH 7.4, 0.2 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, 0.5 mM ATP, 1 mM
isobutylmethylxanthine, 5 mM creatine phosphate, 50 units/ml creatine phosphokinase, and various agonists as indicated in
Fig. 3. For stimulation of striatal membranes with dopamine
agonists, more consistent results were obtained using buffer containing
10 mM imidazole, pH 7.3, 0.2 mM EGTA, 0.5 mM MgCl2, 0.5 mM dithiothreitol,
0.1 mM ATP, and 0.5 mM isobutylmethylxanthine (19). Reactions were terminated by the addition of 1 ml of 0.1 N HCl, 1 mM EDTA. The cAMP concentrations were
determined by automated radioimmunoassay using a Gamma-Flo instrument
(Atto Instruments, Inc., Potomac, MD) as described previously (20).
Open Field Study--
A functional observational battery was
used to assess the behavior of mice outside of their home cages. On 5 successive days, mice were placed in the center of a translucent
polypropylene box (45 × 30 × 60 cm). The behavior of each
mouse was observed for 2 min by a researcher who was unaware of the
genotypes of the mice. The latency to first step in seconds was
recorded. The number of times the mouse reared was counted. The
fraction of the box floor explored by the mouse was estimated. The
startle response of the mouse to the sound of a latex glove being
snapped was graded. After the mouse was removed from the box, the fecal boli and urine pools left in the box were counted, and the box was
wiped clean with a contact disinfectant.
Locomotor Activity--
Locomotor activities were quantified in
CLAMS cages (Columbus Instruments, Columbus, OH). The cages were clear
plastic boxes (20 × 10 × 12.5 cm) fitted with three rows of
eight photoelectric sensors (x, y and z directions). The mice were
placed in the CLAMS cages at 11 a.m. and remained in the cages for
3 h. During this time the mice had ad libitum access to
water. Every minute the number of total and consecutive photobeam
breaks for each of the three sensor arrays and the number of contacts
with the sipper tube were recorded.
Acoustic Startle Reflex--
Acoustic startle was measured using
an SR-Lab startle reflex system (San Diego Instruments, San
Diego, CA). A speaker delivered a continuous background noise of 65 db.
During each trial a 40-ms pulse of broadband noise at 110 db was
delivered. In prepulse trials, 20 ms of broadband noise at 70, 80, or
90 db was administered at 100 ms before the pulse. Each mouse received
six consecutive pulse trials followed by six of each prepulse, pulse,
or no stimulation trial, in random order. The interval between trials
varied from 12 to 30 s. Data were collected at 1-ms intervals
starting 50 ms before the first stimulus. The startle magnitude was
calculated by summing the voltage from the startle chamber (minus the
base-line voltage) over the 100 ms after the onset of the pulse. A
single chamber was used for all mice.
Statistical Methods--
Data are presented as the mean ± S.E. Data were analyzed by t test, chi square, or repeated
measures MANOVA using JMP (SAS Institute Inc., Cary, NC).
To investigate the physiologic function of
G-dimer is required for
receptor interaction and effector regulation. However, previous
approaches have not identified the physiologic roles of individual
subtypes in these processes. We used a gene knockout approach to
demonstrate a unique role for the G protein
7-subunit in mice. Notably, deletion of Gng7 caused behavioral changes that were associated with
reductions in the
olf-subunit content and adenylyl
cyclase activity of the striatum. These data demonstrate that an
individual
-subunit contributes to the specificity of a given
signaling pathway and controls the formation or stability of a
particular G protein heterotrimer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-subunit, it is now well established that the
-dimer plays active roles in the signaling process through
upstream recognition of receptors and downstream regulation of
effectors (1). Molecular cloning has identified at least 5
- and 12
-subunit genes in the mouse and human genomes. Structurally,
-subunits are the most diverse, with four subgroups that show less
than 50% identity to each other (2). Moreover,
-subunits exhibit
very different temporal (3, 4) and spatial (5) patterns of expression. These characteristics suggest that
-subunits have heterogeneous functions. However, comparison of their biochemical properties has
revealed only modest differences (6-8), perhaps because of the
inherent limitations of transfection and reconstitution approaches. Gene ablation in mice has proven to be a powerful approach to identifying the functional roles of several G protein
-subunits (9).
We report the first use of a gene targeting strategy to identify a
unique function for a member of the
-subunit family.
7-subunit (G
7) was
originally cloned from bovine brain (10). In situ
hybridization of rat brain sections revealed that mRNA for
G
7 is most highly expressed in the striatum (5), where
it is found in 40-50% of medium sized neurons in the caudate putamen
(11). The regional expression of mRNA for G
7 in the
brain mirrors that of the striatum-enriched D1 dopamine receptor (D1R),1
G
olf, and adenylyl cyclase Type V (12), suggesting
involvement of G
7 in the G
olf-mediated
stimulation of adenylyl cyclase by dopamine. Single cell RT-PCR
analysis confirms that D1R and G
7 are expressed in the
same subset of rat neurons (13). Ribozyme suppression studies support a
role for G
7 in the endogenous
-adrenergic receptor
pathway (14) and the heterologously expressed D1R pathway in human
embryonic kidney cells (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
7 transcript with primers
JR384 and JR386 or the elongation factor transcript with primers
EFs and EFas, respectively (Table I).
Primers used in this study
7 (17) at a 1:200 dilution, rat Na+/K+-ATPase
-subunit (Research Diagnostics, Inc., Flanders, NJ) at a 1:1000
dilution, G
s (a generous gift of Dr. Catherine Berlot) used at a 1:500 dilution, G
olf (a generous gift of Dr.
Denis Hervé) used at a 1:1000 dilution, G
o (16)
used at 1:500, G
13 (a generous gift of Dr. N. Dhanasekaran) used at 1:1000, G
q/11 (16) used at 1:200,
and Ras (BD Biosciences) used at a 1:2000 dilution. Immunoblots were
imaged with a PhosphorImager and analyzed with ImageQuant software
(Amersham Biosciences).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
7, we targeted Gng7, the murine gene
encoding G
7, using a Cre/Lox strategy
to provide the potential for conditional inactivation of the gene in
future studies (Fig. 1A). The
complete coding region of Gng7 was contained within an
~10-kb mouse genomic clone (21). This included the two coding exons
split by an ~1.1-kb intron. A targeting vector was designed to flank
the coding region of Gng7 with loxP sites.
Targeted embryonic stem cells, chimeric mice, and F1
heterozygotes with the floxed Gng7 allele
(Gng7fl/+) were produced at Lexicon Genetics,
Inc., The Woodlands, TX. Gng7fl/+ mice were
mated with mice carrying the Cre recombinase driven by the
CMV promoter, BALB/c-TgN(CMV-Cre)#Cgn (Jackson
Laboratories, Bar Harbor, Maine). This resulted in deletion of the
coding region of Gng7 following Cre
recombinase-mediated excision (Fig. 1B). Heterozygous mice
with the deleted Gng7 allele
(Gng7+/
) were back-crossed with C57BL/6J mice
(Jackson Laboratories) for up to five generations, to eliminate the
CMV-Cre transgene and to obtain a more homogeneous
genetic background, and then were intercrossed to obtain the mice used
in the following experiments (Fig. 1C). To confirm the
effectiveness of the gene knockout, expression of Gng7 was
examined in brain, where the gene is most highly expressed (10). RT-PCR
analysis demonstrated that the G
7 mRNA was absent in
brains from Gng7
/
mice (Fig. 1D).
Likewise, immunoblot analysis with G
7 antisera (17)
showed that the G
7 protein was reduced by 50 ± 6%
in cholate-solubilized membrane extracts of brains from
Gng7+/
mice and was not detectable in brains
from Gng7
/
mice (Fig. 1E).
View larger version (31K):
[in a new window]
Fig. 1.
A, targeting strategy. Exons 2 and 3 (black boxes) and the 5'-probe (gray box)
are shown for the wild type allele (Gng7). The ~10-kb
genomic clone is between sites S1 and S3. The
targeted allele (Gng7fl) with LoxP-neoR cassette
in intron 1 and LoxP cassette in the 3'-untranslated sequence
(gray boxes with arrows or
"neo") are shown. The targeting fragment is between
sites S2 and S3. The deleted allele
(Gng7 ) shows the remaining LoxP site following
Cre-mediated deletion of the coding region of
Gng7. PCR primers are located at arrows
a, b, and c. Expected sizes of
KpnI (K) fragments are illustrated. B,
Southern blot of tail biopsy DNA digested with KpnI and
detected with the 5'-probe. Lane 1, molecular weight ladder;
lanes 2-7, progeny of a Gng7+/fl × TgN(CMV-Cre) cross. All lanes show the 14-kb wild
type Gng7 allele, and lanes 4, 5, and
7 also show the 12-kb Gng7
allele.
C, PCR products obtained with primers a,
b, and c of tail biopsy DNA from progeny of a
Gng7+/
× Gng7+/
cross. Lane 1,
X HaeIII marker; lanes
2, 4, 5, and 8, wild type mice
with a single 452-bp band derived from primers a and
b; lanes 3 and 6,
Gng7
/
mice with a single 574-bp band derived
from primers b and c; lanes 7 and
9, heterozygous Gng7+/
mice with
both bands. D, top, RT-PCR products obtained
after 25 cycles of amplification with primers for Gng7 from
cDNA prepared from whole brains of three wild type, three
Gng7+/
, and three
Gng7
/
mice or without added cDNA
(W). Bottom, RT-PCR products obtained after 25 cycles of amplification with primers for elongation factor 1
2
(Eef1a2) from identical aliquots of the cDNAs.
E, Western blot analysis of proteins prepared from brains of
three wild type, three Gng7+/
, and three
Gng7
/
mice or SF9 cells expressing
G
7 (Std). The lower panel shows
G
7 is reduced in membranes from
Gng7+/
mice and absent in membranes from
Gng7
/
mice. The upper panel shows
that rat sodium/potassium ATPase
-subunit (ATPase) is
equal in all lanes.
Genotype analysis of offspring of heterozygous
(Gng7+/) intercrosses revealed the expected
numbers of wild type, heterozygous, and homozygous mice, indicating
that disruption of Gng7 did not affect survival to weaning.
There were no significant differences in the weights of
Gng7
/
mice and their wild type littermates.
Moreover, no increased mortality was seen in
Gng7
/
mice over a 6-month median observation
time. Finally, in two homozygous crosses,
Gng7
/
mice were fertile and weaned litters
of apparently normal size. These observations are in stark contrast to
mice with a deficiency of other components of the D1R signal
transduction pathway. Mice with a homozygous deletion of
Gnas, the gene encoding G
s, die in
utero before implantation (22). Most mice with a homozygous deletion of Gnal (23), the gene encoding
G
olf, fail to thrive and die in the neonatal period.
Moreover, although Gnal
/
mice are fertile,
dams lack fostering skills and none of their litters survive. Similar
failure to thrive is observed in mice with a homozygous deletion of
Drd1a (24, 25), the gene encoding the D1R. These feeding and
fostering deficits may be related to loss of the sense of smell in
Gnal
/
mice (23) and impaired motivated
behavior in Drd1a
/
mice (26), respectively.
Thus, Gng7
/
mice display a more
circumscribed phenotype than mice with a deficiency of Gnas,
Gnal, or Drd1a; and offer a more robust animal model for behavioral studies.
Behavior reflects the underlying function of the brain, making it
a sensitive indicator of alterations induced by genetic manipulation
(27). Anecdotal reports of increased handling reactivity from the
animal care technicians provided the first evidence for a behavioral
phenotype. More systematic screens of their behavior were carried out
using a functional observational battery (28). For this purpose, mice
were observed outside of their home cages; measurements of
neuromuscular function (gait abnormalities), sensorimotor defects
(auditory startle), autonomic responses (fecal boli and urine pools),
and activity levels (latency to first step, rears, and exploration)
were scored on predefined rating scales by a trained observer who was
unaware of the genotypes of the animals. Using this test battery, the
most striking observation was that the Gng7/
mice exhibited an increased startle response as compared with their
wild type littermates. To quantify this effect, a Startle Reflex System
(San Diego Instruments) was used to measure the startle reactivity of
Gng7
/
mice and their wild type littermates.
Notably, the Gng7
/
mice displayed a greater
startle amplitude than their wild type littermates for each stimulus
tested (Fig. 2A). Prepulse
inhibition refers to the reduction in startle response that occurs when
the startling stimulus is preceded by a stimulus of lower intensity and
is often used as a measure of sensorimotor gating. Despite the enhanced
startle response, the Gng7
/
mice showed a
similar degree of prepulse inhibition compared with their wild type
littermates for each stimulus tested (Fig. 2A,
inset).
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The finding of increased startle response but normal prepulse
inhibition of the startle response is a phenotype that has been observed previously in mice with mutations in the glycine binding site
of the N1-subunit of the NMDA receptor,
Grin1D481N/D481N (29) and
Grin1D481N/K483Q (30). This is intriguing
because earlier studies had shown that dopamine acting through cAMP to
stimulate protein kinase A, and through DARPP-32 (32-kDa
dopamine- and cAMP-responsive phosphoprotein) to inhibit protein
phosphatase 1, increases the phosphorylation state of the N1-subunit of
the N-methyl-D-aspartic acid (NMDA) receptor and
potentiates NMDA responses (31). This suggests that G7
may be involved in the signal transduction pathway that regulates the
response of striatal
-aminobutyric acid-producing (GABAergic)
neurons to glutamate, a pathway that has been implicated in the
pathogenesis of schizophrenia (32). Alternate mechanisms by which a
deficiency of G
7 may increase the startle response are
suggested by other mouse models. Mice with a deficiency of adrenergic
2c-receptors demonstrate an increased startle response; however, these mice have diminished prepulse inhibition (33). Transgenic mice expressing a dominant mutant of the human inhibitory glycine receptor
1-subunit, TgN(GLRA1R271Q),
as well as mice with the recessive mutation spasmodic
(Glraspd/spd), display a complex neuromuscular
phenotype that includes increased startle response and mimics the human
disease of hyperekplexia (34).
The abundant expression of Gng7 in the striatum suggests a
possible role in control of locomotor activity. As a quantitative test
of this activity, Gng7/
mice and their wild
type littermates were placed in CLAMS cages (Columbus Instruments),
which are equipped with photobeams to measure movement in the
x, y, and z directions. Both wild type and Gng7
/
mice exhibited elevated horizontal
and vertical locomotor activity when introduced into this new
environment. Gng7
/
mice showed less
horizontal (Fig. 2B) and vertical activity (not shown) than
their wild type littermates at all time points, but these differences
were not statistically significant. Habituation refers to the tendency
for the increased locomotor activity to decline upon repeated or
sustained exposure to a new environment, which is often used as a
measure of a learned response. Importantly, both groups of mice
displayed comparable levels of habituation between and within sessions.
On the basis of pharmacologic studies showing
D1 dopamine agonists produce a strong stimulatory effect on
locomotor activity (35), one might have expected a more marked decrease
in locomotor activity in Gng7/
mice. In this
regard, however, other gene knockout studies have not provided strong
support for the pharmacologic studies. For example,
Drd1a
/
mice have been variously reported as
being either hypoactive (24, 26), hyperactive (25), or neither but
showing an altered pattern of activity (36). Moreover,
Gnal
/
mice have increased locomotor activity
in an open field (12), which is the opposite of the result expected
based on pharmacologic studies. This variability may reflect the
complex nature of locomotor activity (37), the confounding effects of
olfactory deficits in Gnal
/
mice (23), or
unidentified compensatory changes that may occur in knockout mice (38).
Further study will be needed to clarify this issue, including more a
restricted disruption of Gng7 within specific regions of the
brain that is made possible by our floxed mouse model.
Finally, the expression of Gng7 along with
Drd1a in a subset of neurons within the striatum suggests a
possible role in regulation of adenylyl cyclase activity (13). To
evaluate this possibility, adenylyl cyclase activity was compared
between those regions of the brain that normally express
Gng7, such as striatum, and other regions that do not
normally express Gng7, such as cerebellum (Fig.
3A). Intriguingly, dopamine
and the D1-specific agonist 6-chloro-PB had
stimulatory effects on adenylyl cyclase activity in the striatum from
wild type mice, but the responses were virtually abolished in the
striatum from Gng7/
mice (Fig.
3B). By contrast, dopamine had no effect on adenylyl cyclase
activity in the cerebellum, but the response to isoproterenol was
comparable in both wild type and Gng7
/
mice
(Fig. 3C). These results establish a functional link between the expression of G
7 and adenylyl cyclase activity.
Forskolin is a potent activator of adenylyl cyclase activity.
Remarkably, forskolin had a potent stimulatory effect on adenylyl
cyclase activity in the striatum from wild type mice, but the response was reduced by 50% in the striatum from
Gng7
/
mice (Fig. 3D). By
contrast, response to forskolin was comparable in the cerebellum from
both groups of mice (Fig. 3E). Taken together, these data
demonstrate that G
7 has an important but regionalized role in the regulation of adenylyl cyclase activity in the brain.
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Because the striatal adenylyl cyclase Type V isoform (39)
is synergistically activated by a combination of forskolin and Gs (40), one mechanism that could account for defects in
both receptor-mediated and forskolin-stimulated adenylyl cyclase
activity is a reduced level of the stimulatory G protein
-subunit in
the striatum of Gng7
/
mice. To test this
possibility, the levels of G
s and G
olf, the two known activators of adenylyl cyclase activity, were determined. Notably, the levels of G
s were comparable in the
striatum from both groups of mice level, but the amount of
G
olf was reduced by 82 ± 3% in the striatum
(n = 8) from Gng7
/
mice
(Fig. 4A). To provide
additional evidence for a specific reduction in G
olf, we
examined the levels of representative members of other G protein
-subunit subunit families. Notably, the levels of G
o,
G
13, and G
q/11 were not significantly
reduced in the striatum of Gng7
/
mice (Fig.
4B). These results provide the first evidence that loss of a
-subunit can result in loss of its
-subunit partner.
|
In summary, this paper provides several novel insights. First,
G7 plays a unique role in regulation of adenylyl cyclase
signaling in certain regions of the brain. This is demonstrated by the
finding that loss of G
7 produces both a behavioral and a
biochemical phenotype, indicating that other types of
-subunit are
not able to substitute for this function. Thus, members of the
-subunit family are not functionally interchangeable in the context
of the whole animal. Second, G
7 plays a role in the
stabilization or formation of a G protein heterotrimer
(
olf
7) that is required for
stimulation of adenylyl cyclase activity in the striatum. This is
substantiated by the finding that loss of G
7 reduces the
level of G
olf in the striatum in a specific and
coordinate fashion. Further studies are needed to address the
underlying mechanisms and to determine whether this process is
applicable to other
-subunits. If widespread, these results will
reveal an important new signaling paradigm, namely, the level of a
specific
-subunit controls the stability or assembly of a particular
G protein heterotrimer. This, in turn, provides a probable basis for
the selectivity of the multitude of G protein-coupled receptor signaling pathways that are now known to exist. Finally, the production of mice lacking G
7 provides a unique mouse model for the
study of numerous diseases in which dysfunction of the adenylyl cyclase signaling pathway in the striatum has been implicated, such as Parkinson's disease, Huntington's chorea, Tourette's syndrome, and schizophrenia.
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ACKNOWLEDGEMENTS |
---|
We thank the outstanding technicians in our
animal care facility, Cynthia J. Rhone, Gail L. Gregory, and Shannon
Wescott. We are grateful to Dr. Denis Hervé for providing the
olf antibody, to Dr. Catherine Berlot for supplying the
s antibody and providing helpful advice, and to Dr. N. Dhanasekaran for supplying the
13 antibody. We are
indebted to Drs. Michael C. Nehls and Jean-Pierre Revelli (Lexicon
Genetics Inc.) for producing the Gng7+/fl mice.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM39867 (to J. D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Weis Center for Research, Geisinger Clinic, 100 North Academy Ave., Danville, PA 17822. Tel.: 570-271-6684; Fax: 570-271-6701; E-mail: jrobishaw@geisinger.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211132200
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ABBREVIATIONS |
---|
The abbreviations used are:
D1R, dopamine
D1 receptor;
G7, G protein
7-subunit;
MANOVA, multivariate analysis of
variance;
RT, reverse transcriptase;
CLAMS, comprehensive
laboratory animal monitoring system;
CMV, cytomegalovirus;
olf, olfactory;
NMDA, N-methyl-D-aspartic acid.
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