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The Journal of Neuroscience, September 15, 1998, 18(18):7178-7188
Dynamic Regulation of RGS2 Suggests a Novel Mechanism in
G-Protein Signaling and Neuronal Plasticity
Tatsuya
Ingi1,
Andrejs
M.
Krumins3,
Peter
Chidiac3,
Greg M.
Brothers4,
Stephen
Chung4,
Bryan E.
Snow4,
Carol A.
Barnes5,
Anthony A.
Lanahan1,
David P.
Siderovski4,
Elliott M.
Ross3,
Alfred G.
Gilman3, and
Paul F.
Worley1, 2
Departments of 1 Neuroscience and
2 Neurology, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21210, 3 Department of Pharmacology,
University of Texas Southwestern Medical Center, Dallas, Texas 75235, 4 Quantitative Biology Laboratory, Amgen Institute,
Toronto, Ontario M5G 2C1, Canada, and 5 Department of
Psychology and Neurology and Division of Neuronal Systems, Memory, and
Aging, University of Arizona, Tucson, Arizona 84724
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ABSTRACT |
Long-term neuronal plasticity is known to be dependent on rapid
de novo synthesis of mRNA and protein, and recent
studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of
G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons
of the hippocampus, cortex, and striatum in response to stimuli that
evoke plasticity. Although several members of the RGS family are
expressed in brain with discrete neuronal localizations, RGS2 appears
unique in that its expression is dynamically responsive to neuronal
activity. In biochemical assays, RGS2 stimulates the GTPase activity of
the  subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein
in phospholipid vesicles containing M2 muscarinic acetylcholine
receptors. RGS2 also inhibits both Gq- and
Gi-dependent responses in transfected cells. These studies
suggest a novel mechanism linking neuronal activity and signal
transduction.
Key words:
RGS; immediate early genes; seizure; neuronal plasticity; G-protein; GTPase-activating proteins; MAP kinase
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INTRODUCTION |
Neurons respond to extracellular
stimuli using both rapid phosphorylation-dependent and
transcription-dependent mechanisms. These mechanisms collectively
underlie activity-dependent neuronal plasticity (Shatz, 1990 ).
Physiological studies support the notion that phosphorylation evokes
changes in neuronal properties that can last minutes to hours, although
macromolecular synthesis is required to produce changes lasting days to
the life of the organism (Flexner et al., 1963; Agranoff, 1981; Goelet
et al., 1986; Nguyen et al., 1994). To examine molecules involved in
the establishment of transcription-dependent neuronal plasticity, we
have cloned and characterized genes that are rapidly induced in neurons
of the hippocampus by neuronal activity. Currently, we understand this
set of genes to include transcription factors, such as c-fos, c-jun,
and zif268 (Morgan et al., 1987; Saffen et al., 1988), as well as
proteins that may directly modify cellular and synaptic function. The
latter class of proteins are particularly interesting, because their
contribution to plasticity can, in principle, be understood by
examining their direct biochemical and cellular properties. Examples
include the inducible form of cyclooxygenase (Yamagata et al., 1993), a
novel cytoskeleton-associated protein (Arc) (Lyford et al., 1995), and
a protein that selectively binds metabotropic glutamate receptors
(Homer) (Brakeman et al., 1997). These proteins are rapidly induced in
neurons and interact with constitutively expressed cellular and
synaptic proteins to modify neuronal properties.
In the present study, we report that a member of the regulators of the
G-protein signaling (RGS) family (Dohlman and Thorner, 1997; Koelle,
1997), RGS2, is rapidly and selectively upregulated in brain neurons in
response to plasticity-inducing synaptic stimuli. Heterotrimeric GTP
binding or G-proteins transduce a vast array of receptor-initiated
signals into appropriate cellular responses (Gilman, 1987; Hamm and
Gilchrist, 1996; Neer and Smith, 1996). The intensity and duration of
the response is known to be regulated at the level of the receptor by
mechanisms that include agonist-dependent phosphorylation of the
receptor and subsequent receptor inactivation and turnover. G-protein
function is also critical to signal regulation. In their inactive
conformation, G-proteins are heterotrimers that contain GDP-bound ,
 , and  subunits. G-protein activation is initiated on agonist
binding to the receptor, which elicits a conformational change that is
transmitted to the G-protein and causes the G subunit to release
GDP. Subsequent binding of GTP results in dissociation of GTP- from
, and each of these separated G-protein components can regulate
downstream effectors. Signaling is terminated when the G subunit,
which possesses intrinsic GTPase activity, hydrolyzes GTP and returns
to the GDP-bound state. G then reassociates with G to reform
inactive heterotrimers.
Recently, a novel gene family has been identified that functions to
accelerate the rate of intrinsic GTP hydrolysis by G and thereby
reduce the duration of G-protein activation (Dohlman and Thorner, 1997;
Koelle, 1997; Berman and Gilman, 1998). Studies in yeast first
identified a gene, termed SST2, that is critical for desensitization of
mating pheromone responses (Chan and Otte, 1982a,b). SST2
loss-of-function mutants respond to concentrations of pheromone that
are 100-fold lower than those required to elicit a response in
wild-type cells (supersensitivity). Moreover, wild-type yeast
desensitizes to pheromone after ~2 hr of continuous exposure, whereas
SST2 mutants do not desensitize. SST2 defined a novel gene and the
first member of the RGS family (Dietzel and Kurjan, 1987). Another RGS
gene, termed EGL-10, was identified in Caenorhabditis elegans and as a regulator of serotonin-stimulated egg laying (Koelle and Horvitz, 1996). Homologs of SST2 and EGL-10 were identified in mammals and comprise a family of RGS proteins (Druey et al., 1996;
Koelle and Horvitz, 1996; Siderovski et al., 1996) that accelerate the
intrinsic GTPase activities of subunits of heterotrimeric G-protein
(Berman et al., 1996; Chen et al., 1996; Hunt et al., 1996; Watson et
al., 1996) and reduce the duration of the activated GTP-bound state of
the subunits. To date, eighteen RGS proteins have been identified
in mammalian tissues; all are highly homologous within an ~130 amino
acid RGS domain (Koelle and Horvitz, 1996; Siderovski et al., 1996).
In vitro assays indicate that several RGS proteins stimulate
the GTPase activity of the Gi subfamily (Berman and Gilman,
1998), whereas RGS4 stimulates both Gi and Gq (Hepler et al., 1997; Huang et al., 1997; Yan et al.,
1997). RGS family members typically do not stimulate the GTPase
activity of Gs; the only exception is a truncated form
of RGS3 (Chatterjee et al., 1997).
Here, we report that mammalian RGS2 mRNA is rapidly and transiently
upregulated in brain neurons by synaptic activity. RGS2 appears to act
selectively to increase the GTPase activity of Gq when
single-turnover assays are performed in solution. However, RGS2 also
activates the GTPase activity of Gi when the G-protein is reconstituted in phospholipid vesicles with M2 muscarinic
acetylcholine receptors (mAChRs). Together, these finding suggest a
novel mechanism that may be important in long-term neuronal responses
to synaptic activity.
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MATERIALS AND METHODS |
Animals and supplies. Adult male rats (Sprague Dawley
or Fischer-344) were used in studies of RGS regulation. Developmental studies used male and female Sprague Dawley pups of the indicated age.
Radiochemicals were obtained from New England Nuclear Life Science
Products. All other reagents were from Fisher Scientific (Houston, TX)
and Sigma (St. Louis, MO), unless specifically noted.
Northern analysis. This procedure was performed as described
previously (Linzer and Nathans, 1983) with 2 µg of
poly(A+) RNA per lane. The cDNA probe used for
Northern analysis of RGS2 was a 1 kb fragment cloned by differential
screening of an oligo-dT primed brain library, as described previously
(Lyford et al., 1995), and included a 3' UTR sequence. The cDNA
fragment was labeled by the random priming technique (Pharmacia,
Piscataway, NJ) using [-32P]dCTP.
Reverse Northern analysis. Plasmids containing the indicated
cDNAs were linearized with the appropriate restriction enzymes, and 1 µg of each plasmid was loaded per lane and electrophoresed in 1.5%
agarose gels. Three identical gels were prepared; after denaturation
and neutralization, the cDNAs were transferred to nitrocellulose. Adult
rats were pretreated with cycloheximide (CH) and received a maximal
electroconvulsive seizure (MECS) as described below (Materials
and Methods, Electrophysiology). Control rats were either naive or
treated with CH only. Poly(A+) RNA was isolated
separately from three individual samples using Micro Fastrack
(Invitrogen, San Diego, CA), and nonradioactive double-stranded cDNA
was synthesized using an oligo-dT primer with Superscript reverse
transcriptase (Life Technologies, Gaithersburg, MD) as described
previously (Lanahan et al., 1997). The reaction was terminated and
extracted with phenol, and cDNA was precipitated with ethanol in the
presence of glycogen. The precipitated cDNA mix was then
chromatographed over a NICK column (Pharmacia) and reprecipitated to remove free nucleotides that might hamper the radiolabeling of the cDNA. cDNA was radiolabeled by random priming (Pharmacia) with [-32P]dCTP to a specific activity of
4 × 109 cpm/µg. Identical blots were
prehybridized overnight at 68°C in 5 × SSC, 5 × Denhardt's solution, 10 mM EDTA, 0.2% SDS, 50 mM NaPO4, pH 7.0, and 100 µg/ml boiled
salmon sperm DNA. Three blots were then hybridized overnight in freshly
prepared hybridization solution containing 5 × 106 cpm/ml appropriate 32P-labeled cDNA
probe. Filters were washed at room temperature in 2 × SSC and
0.2% SDS and then washed in 1 × SSC and 0.2% SDS at 68°C.
Levels of hybridization were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Blotted cDNA included the following: c-jun and zif268 (Saffen et al., 1988); c-fos (Morgan et
al., 1987); -tubulin, glucose 6 phosphate dehydrogenase (G6PD), RGS2, RGS3, RGS7, and RGS10 (Koelle and Horvitz, 1996); RGS4
(Berman et al., 1996); and RGS12 and RGS14 (Snow et al., 1997). The
size of the RGS fragments were 1.6 (RGS2), 0.24 (RGS3), 0.7 (RGS4), 1.2 (RGS7), 0.24 (RGS8 and RGS10), 0.6 (RGS12), and 0.9 (RGS14) kb. Because
tissue cDNA is labeled by random priming, it is anticipated that the
sensitivity of reverse Northern analysis may be influenced by the size
of the cDNA insert on the blot. For a given tissue mRNA, only those
labeled cDNAs that include sequence that is represented in the cloned
fragment will hybridize. Thus, the sensitivity of this assay is likely
to be greatest for those family members with the largest inserts.
In situ hybridization. Freshly dissected brain tissue was
rapidly frozen in plastic molds placed on a dry ice-ethanol slurry as
described previously (Cole et al., 1990).
Electrophysiology. MECS stimulation for blot analysis was
performed as follows. Adult rats were pretreated with CH (20 mg/kg, i.p.) and received an MECS using a constant current signal generator (ECT unit, Ugo Basil) as described previously (Cole et al., 1990). As a
negative control, rats were untreated or treated with CH only. For
in situ studies, rats were treated with MECS but not CH. For
long-term potentiation (LTP) studies, Fischer-344 rats were implanted
bilaterally with stimulating and recording electrodes in the perforant
path and hilus of the dentate gyrus as described previously (Worley et
al., 1993). Rats were allowed to recover for at least 2 weeks before
any recordings were performed. Fourteen chronically implanted rats
received high frequency (HF) stimulation in one hemisphere and low
frequency stimulation in the other hemisphere. Electrical stimuli
consisted of 200 msec diphasic constant current pulses given at a
stimulus intensity of 500 mA. The low frequency test stimulation was
delivered at 0.1 Hz, and the HF stimulation parameters consisted of 50 repetitions of a 20 msec train (i.e., eight pulses) delivered at 400 Hz
(400 total pulses). The HF parameters reliably induce LTP (Dragunow et
al., 1989; Jeffery et al., 1990; Worley et al., 1993). After this
treatment, the rats were killed at either 0.5 (n = 2), 1 (n = 2), or 2 hr
(n = 2). Some rats received an intraperitoneal
injection with the NMDA-type glutamate receptor antagonist MK-801 (1 mg/kg) 30 min before the HF stimulus.
Recombinant RGS2 expression. Recombinant RGS2 was expressed
as described previously (Lalumiere and Richardson, 1995). An expressed sequence tag (EST) containing the full open-reading frame of mouse RGS2
was identified within the Amgen (Thousand Oaks, CA) EST Program database and cloned within the NheI site of the baculoviral
expression vector pETL, downstream of the polyhedron promoter
(Lalumiere and Richardson, 1995). Recombinant baculovirus was generated
using BaculoGold baculoviral DNA (PharMingen, San Diego, CA).
Spodoptera frugiperda (Sf9) cells were cultured at 27°C in
spinner vessels to a density of 1 × 106
cells/ml Grace's media (Life Technologies), supplemented with 10% fetal calf serum. Sf9 cells were infected at a multiplicity of
infection of 5.0 with recombinant baculovirus, harvested by centrifugation at 72 hr after infection, swollen in 10 volumes of
hypotonic lysis buffer (in mM: 20 Tris-HCl, pH 8.0, 10 KCl, 2 EDTA, 0.5 EGTA, 5 DTT, and protease inhibitors), and lysed with a
dounce homogenizer. The lysate was clarified by low- and high-speed centrifugations (12,000 × g for 20 min and
100,000 × g for 1 hr), and the supernatant was passed
through a Q Sepharose FF anion exchange column (Pharmacia) equilibrated
with lysis buffer. The column void fraction was dialyzed against SP
Sepharose FF equilibration buffer [in mM: 50 4-mopholinepropanesulfonic acid (MOPS) (Boehringer Mannheim,
Indianapolis, IN), pH 7.4, 10 KCl, 2 EDTA, 5 DTT, 10 KCl, and protease
inhibitors] (Pharmacia) and passed through a SP Sepharose FF cation
exchange column (Pharmacia) equilibrated in the same buffer. The bound
recombinant RGS2 was eluted with a 0-500 mM NaCl
salt gradient. The fractions containing RGS2 were pooled, dialyzed
against storage buffer (in mM: 10 MOPS, pH7.4, 1 EDTA, and
2 DTT), and stored at  80°C.
Preparation of recombinant G-proteins and RGS4. Recombinant
R183C Gq and G-protein subunits were purified
after expression in Sf9 cells (Biddlecome et al., 1996), as were M2
mAChRs (Parker et al., 1991). Gs and myristoylated
Go and Gi1 were synthesized and purified
from Escherichia coli (Linder et al., 1991; Lee et al.,
1994). Recombinant RGS4 was purified from E. coli as
described previously (Berman et al., 1996) and was generously provided
by David Berman (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX).
Preparation of [-32P]GTP-G substrates for GAP
assays. Purified R183C Gq (~1 nM
final concentration) was incubated with
[-32P]GTP (specific activity, ~500 cpm/pmol) for
2-3 hr at 20°C in a solution containing: 10 µM GTP,
5.5 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS) (Sigma), 50 mM Na HEPES, pH 7.5, 1 mM DTT, 1 mM
EDTA, 0.9 mM MgSO4, 0.1 mg/ml BSA, 30 mM
(NH2)2SO4, and 4%
glycerol. Other G proteins were incubated with
[-32P]GTP in the absence of MgSO4 as
described previously (Berman et al., 1996). The mixture containing
GTP-bound G proteins was gel filtered on G-25 Sephadex, equilibrated
with 1 mM CHAPS buffer (1 mM CHAPS, 50 mM Na HEPES, pH 7.5, 1 mM EDTA, 0.9 mM MgSO4, 1 mM DTT, and
0.018 mg/ml BSA). The flow-through was diluted fourfold into
octylglucoside (OG) buffer (0.1% octyl glucopyranoside, 20 mM Na HEPES, pH 7.5, 80 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.9 mM
MgSO4, 0.01 mg/ml BSA, and 1 mM GTP) and
kept on ice until needed. All of the manipulations above were conducted
in the absence of MgSO4 for G proteins other than
Gq.
In vitro GAP activity. GAP activity was measured by adding
G-GTP substrate to RGS sample and OG buffer (supplemented with 9 mM MgSO4 when Gs,
myr-Go, or myr-Gi were used). One hundred microliter aliquots were withdrawn at the indicated times and were
quenched in 900 µl of 5% (wt/vol) Norit A charcoal in 50 mM NaH2PO4. The charcoal was
pelleted, and 600 µl of supernatant containing 32Pi was
counted. GTPase assays conducted in a buffer containing 0.5%
Lubrol (Berman et al., 1996) yielded results similar to
those obtained in 0.1% OG.
M2 mAChR(Gi) reconstitution assay. The
steady-state GTPase activity of Gi1 was measured in
reconstituted vesicles containing M2 mAChRs and heterotrimeric
Gi1 (Parker et al., 1991; Wong and Ross, 1994) (a generous
gift from Dr. Yaping Tu, University of Texas Southwestern Medical
Center). The vesicles were equilibrated for 5 min at 20°C with
carbachol (1 mM) or atropine (20 µM) in the
presence of RGS2 or RGS4 (500 nM final
concentration) in buffer containing: 4 µM GTP, 20 mM Na HEPES, pH 8.0, 50 mM NaCl, 2 mM MgCl2, and 1 mM EDTA. The
assay was initiated by the addition of an equal volume (30 µl) of the
same buffer without unlabeled GTP but containing 106
cpm of [-32P]GTP. Reactions were terminated after 15 min at 20°C by the addition of 900 µl of 5% Norit A charcoal in 50 mM NaH2PO4. The samples were
centrifuged, and the supernatant was counted to determine the content
of 32Pi.
Cell culture. SV-40 transformed African Green Monkey Kidney
(COS) cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with fetal bovine serum (10%).
Approximately 2 × 106 cells in a plate were
transfected by the calcium phosphate method with various combinations
of pRK5-mAChR (6 µg), pMT2-HA-ERK1 (6 µg), and pRK5-RGS (10 µg).
The total amount of plasmid DNA was adjusted to 22 µg/plate with
vector DNA when necessary. After 40 hr, cells were left untreated or
were stimulated with the agonist and lysed at 4°C in a buffer
containing: 20 mM HEPES, pH 7.5, 10 mM EGTA, 40 mM -glycerophosphate, 1% NP-40, 2.5 mM
MgCl2, 1 mM dithiothreitol, 2 mM sodium vanadate, 1 mM
phenylmethylsulfonylfluoride, 20 µg/ml aprotinin, and 20 µg/ml
leupeptin. The lysate was centrifuged at 14,000 × g
for 20 min at 4°C, and proteins were immunoprecipitated and assayed
for kinase activity.
Immunoprecipitation and MAP kinase assay. For the MAP kinase
assay, proteins from clarified supernatants were immunoprecipitated with monoclonal antibody to hemagglutinin (HA) 12CA5 (Boehringer Mannheim) for 2 hr at 4°C, and immunocomplexes were recovered with
Immobilized Protein G (Pierce, Rockford, IL). Bound proteins were
washed three times with lysis buffer supplemented with 2 mM
sodium vanadate, once with 0.5 M LiCl in 100 mM
Tris, pH 7.5, and once with kinase reaction buffer (in mM:
10 MOPS, pH 7.5, 12.5 -glycerophosphate, 7.5 MgCl2, 0.5 EGTA, 0.5 sodium fluoride, and 0.5 sodium
vanadate). Reactions were done in 60 µl volumes of kinase reaction
buffer [10 µCi/reaction [-32P]ATP, 50 µM unlabeled ATP, and 0.25 mg/ml myelin basic protein (MBP) (Sigma)] at 30°C for 30 min. Reactions were terminated by the
addition of a 2 × SDS buffer. Samples were boiled, and proteins were separated by SDS-PAGE (12% gel). Phosphorylated MBP was
visualized by autoradiography and was quantified by liquid
scintillation counting. Parallel samples were immunoprecipitated with
antibody to HA and processed for protein immunoblot analysis with a
ERK1-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
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RESULTS |
Selective and robust induction of RGS2 mRNA in response to
neuronal activation
In previous studies, we developed differential and subtractive
cloning strategies to identify mRNAs that are rapidly upregulated in
the hippocampus by excitatory activity (Yamagata et al., 1994; Brakeman
et al., 1997; Lanahan et al., 1997). In the present study, a phage cDNA
library was prepared from rat hippocampus 4 hr after systemic
administration of CH. CH blocks protein synthesis and stabilizes many
mRNAs that normally turn over rapidly. Rats additionally received MECS
at 30 min intervals for six treatments before being killed. MECS is a
simple and reliable means to induce the expression of immediate early
genes (IEGs) in the hippocampus and causes long-term enhancement of
synaptic contacts in the hippocampus (Barnes et al., 1994; Lanahan et
al., 1997). The cDNA library was then subtracted twice using RNA
prepared from naive rat hippocampus and once with RNA from liver. The
subtracted library was then differentially screened using radiolabeled
cDNA prepared from naive control and MECS-CH rat hippocampus.
Nucleotide sequence analysis of one of the differentially expressed
cDNAs identified it as RGS2.
To explore the dynamic regulation of RGS mRNA expression, we first
performed Northern blot analysis of poly(A+) RNA
from hippocampus of naive and MECS-CH-stimulated rat hippocampus (Fig.
1A). The RGS2 cDNA
probe detected a 1.8 kb message that was strongly induced by MECS-CH.
The size of the mRNA was identical to that reported for the RGS2
transcript (Wu et al., 1995). The rapid induction of RGS2 mRNA in the
presence of CH suggests that it is regulated as an IEG in brain.
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Figure 1.
Selective induction of RGS2 mRNA in hippocampus by
seizure. A, Northern blot analysis of 2 µg/lane
poly(A+) RNA prepared from hippocampus under basal
conditions and after 4 hr MECS-CH induction. The blot was probed with
a 1 kb cDNA corresponding to the 3' nt sequence of RGS2. Note robust
induction of an ~1.8 kb transcript. B, Analysis of RGS
family mRNA levels after seizure by reverse Northern analysis. Southern
blots were prepared using 1 µg of each RGS cDNA, as well as positive
and negative control genes. Duplicated Southern blots were probed with
radiolabeled cDNA prepared from hippocampus of naive control
(top) and MECS-stimulated (bottom) rats.
Comparison of top and bottom blots
demonstrates that MECS-CH increases the intensity of hybridization to
RGS2, as well as representative IEGs (c-jun,
c-fos, and zif268). -tubulin and G6PD
are constitutively expressed and provide a comparison for genes that
are induced by MECS. C, Quantitation of RGS family mRNA.
Levels of hybridization signal from reverse Northern blots were
quantitated using a PhosphorImager (Molecular Dynamics) and are
presented as percentage of G6PD level on each blot. Data are mean ± SEM of triplicate determinations from a representative experiment.
Of the eight RGS subtypes surveyed, only RGS2 showed robust induction
(7.3-fold). Increases for other RGS genes were calculated to be 1.7- (RGS4), 1.2- (RGS7), 0.58- (RGS12), and 0.65-fold
(RGS14). Similar results were obtained in two
independent experiments.
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To determine whether other members of the RGS family are regulated by
MECS-CH in the hippocampus, we used the reverse Northern strategy
(Fig. 1B). With this technique (Lanahan et al.,
1997), levels of tissue mRNA are assessed by monitoring the intensity of the hybridization of radiolabeled cDNA prepared from tissue RNA to
Southern blots containing cloned cDNAs of multiple candidate IEGs. We
prepared poly(A+) RNA individually from the
hippocampus of a control rat and from an MECS-CH rat and
converted them to double-strand cDNAs. cDNA was labeled by random
priming and used as a hybridization probe. Southern blots were prepared
using a panel of eight RGS cDNAs, as well as representative known IEGs
and constitutively expressed genes (Fig. 1B).
Duplicate blots were probed with cDNA from the hippocampus of naive
control (Fig. 1B, top) and MECS-CH (Fig. 1B, bottom) rats. In Figure
1B, comparison of the top and
bottom blots confirms that MECS stimulation increases mRNA
levels of representative IEGs (c-jun, c-fos, and zif268), whereas
-tubulin and G6PD were essentially unchanged. Consistent with the
Northern blot analysis, RGS2 mRNA was strongly increased by MECS-CH.
In contrast, hybridization to RGS3, RGS4, RGS7, RGS8, RGS10, RGS12, and
RGS14 was not increased by MECS-CH. To permit quantitative comparisons
between blots, the hybridization signal was determined using a
PhosphorImager (Molecular Dynamics) and normalized to G6PD (Fig.
1C). Of the eight RGS subtypes surveyed, only RGS2 showed a
significant induction with MECS-CH. In this analysis, RGS2 mRNA was
induced more than sevenfold. Reverse Northern analysis provides a
comparison of the relative levels of expression; however, because of limitations in sensitivity of the reverse Northern technique
(see Materials and Methods), we cannot exclude the possibility that
these RGS genes might also be regulated by activity at lower levels.
These results indicate that several RGS family members are expressed in
the hippocampus and that RGS2 mRNA is selectively induced by MECS-CH.
Although RGS2 mRNA is induced by CH alone in blood mononuclear cells
(Siderovski et al., 1994), our reverse Northern analyses indicate that
RGS2 is not induced by CH alone in the rat brain (data not shown).
Expression and activity-dependent induction of RGS2 mRNA in
cerebral cortex and hippocampus
To examine the cellular distribution of natural (control) and
induced expression of RGS2, we performed in situ
hybridization (Fig. 2). Expression was
examined in the forebrain of a naive rat and compared with expression
in rats that received a single MECS and were killed 0.5, 1, or 2 hr
afterward (Fig. 2A). RGS2 mRNA is naturally expressed
in cortex and hippocampus, with prominent cellular localization to
granule and pyramidal neurons of the hippocampus and layer 2 neurons of
the pyriform cortex. Within 30 min after MECS, RGS2 mRNA is seen to be
induced in neuronal cell populations of the hippocampus and throughout
the cortex, amygdala regions, and striatum. These results indicate that
a single seizure is sufficient to induce RGS2. Additionally, because rats did not receive CH in these experiments, we conclude that RGS2
induction does not depend on concomitant CH treatment. No induction is
observed in the thalamus. Expression in cortex and hippocampus remains
elevated at both the 1 and 2 hr time points after MECS. To obtain a
more complete time course of induction, we repeated in situ
analysis with individual hippocampi harvested from rats as long as 8 hr
after MECS (Fig. 2B). In the pyramidal cell layer of
hippocampus and the granule cell layer of dentate gyrus, RGS2 mRNA
levels remain elevated for 2 hr after stimulation and return to basal
levels by 8 hr. Thus, the RGS2 mRNA increase occurs rapidly and
transiently in discrete brain regions.
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Figure 2.
In situ analysis of RGS2 expression
in brain. A, Postseizure time course of RGS2 mRNA
induction in the whole brain. Adult rats received a single MECS and
were killed 0.5, 1, or 2 hr later. RGS2 mRNA rapidly increases within
0.5 hr after stimulation, and peak mRNA levels are detected 0.5-1 hr
after stimulation. The increase of RGS2 mRNA is seen in regions
including the cerebral cortex, hippocampus, caudate putamen, and
amygdala nucleus. The same hybridization patterns were repeated in
independent experiments (n = 2-3). Postseizure
time course failed to detect a change in the levels of RGS4 and RGS7
mRNA expression (data not shown). Ctx, Cortex;
Hip, hippocampus; Am, amygdala;
ST, striatum. B, Postseizure time course
of RGS2 mRNA in the hippocampus. Adult rats received a single MECS and
were killed 0.5, 1, 2, 4, or 8 hr later. RGS2 mRNA rapidly increases in
the pyramidal layer of hippocampus and the dentate gyrus within 0.5 hr
after stimulation, and peak mRNA levels are detected 0.5-2 hr after
stimulation and return to basal levels by 8 hr. The same hybridization
patterns were repeated in independent experiments
(n = 2-3). Pyr, Pyramidal cell
layer; DG, dentate gyrus. C, Induction of
RGS2 mRNA in LTP paradigm. Chronically implanted awake and behaving
animals received a high frequency (HF) synaptic
stimulus to the left dentate gyrus and an identical
number of stimuli at low frequency (LF) to the
right. Rats were killed 1 hr after the HF stimulus. RGS2
mRNA is strongly induced in the hippocampal granule cells by the HF
stimulus. The bottom brain image is a composite that
includes a half brain from naive control (C side) and
MECS-stimulated (S side) rats. Identical results were
observed in four independent experiments. The LTP studies failed to
detect a change in the levels of RGS4 and RGS7 mRNA using adjacent
sections (data not shown). D, Induction of RGS2 mRNA by
administration of haloperidol (haldol) and
cocaine. Adult rats were injected with haloperidol (1 mg/kg, i.p.) or
cocaine (20 mg/kg, i.p.) and were killed 0.5, 1, or 2 hr later. Peak
induction occurred 30 min after drug administrations. RGS2 mRNA is
induced in the striatum by haldol (approximately twofold), but a
minimal change was seen after this dose of cocaine. The same results
were repeated in two independent experiments (n = 2). CP, Caudate putamen.
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RGS2 mRNA is induced in brain neurons by synaptic activity in
association with synaptic enhancement
The rapid induction of RGS2 mRNA after MECS suggests that RGS2 may
be regulated by excitatory synaptic mechanisms. To test this
hypothesis, granule cells of the adult hippocampus were synaptically stimulated by activating their major afferent projections from the
entorhinal cortex using a chronic in vivo
electrophysiological preparation in unanesthetized awake behaving rats
(McNaughton et al., 1986). The intensity and frequency of the synaptic
stimulus can be precisely controlled in this preparation, and it is
used extensively to study mechanisms of LTP. Stimulation administered at 0.1 Hz at an intensity level sufficient to activate granule cell
action potentials did not result in increased RGS2 mRNA expression in
these cells (Fig. 2C, top left). In contrast,
stimuli of the same intensity, when administered at a HF (400 Hz),
resulted in a rapid and robust induction of RGS2 mRNA in the granule
cells (Fig. 2C, top right). RGS2 mRNA was induced
in each of four animals killed 0.5-1 hr after the HF stimulus
(n = 4). Thus, the rapid time course of RGS2 mRNA
induction after the HF stimulus is similar to that produced by
seizure-induced neuronal activation in the hippocampus. RGS2 mRNA
induction was blocked by pretreatment of rats with the NMDA-type
glutamate receptor antagonist MK-801 (1 mg/kg; n = 2), indicating that induction in this paradigm is dependent on
synaptic activation of the NMDA receptor. The magnitude of the RGS2
mRNA induction by the HF stimulus was also comparable to that after the
seizure-induced activation (Fig. 2C); however, unlike MECS,
which induced RGS2 mRNA in the cerebral cortex and hippocampus (Fig.
2C, lower left), the HF stimulus induced RGS2 only in the hippocampus. The HF stimulation parameters determined to
induce RGS2 are identical to those determined previously to induce LTP,
and in each of our preparations, the HF stimulus induced robust
synaptic enhancement. However, no change was detected in the level of
RGS4 and RGS7 mRNA by HF stimulus in adjacent tissue sections (data not
shown). These observations indicate that RGS2 is rapidly and
transiently regulated by physiological synaptic activity.
The effects of haloperidol and cocaine on RGS2 mRNA level
in striatum
To evaluate whether RGS genes might be regulated by
dopamine-dependent mechanisms, we examined the levels of three RGS
mRNAs in the rat striatum after the administration of
haloperidol or cocaine. Rats were injected with haloperidol or
cocaine and were killed 0.5, 1, or 2 hr later. Haloperidol is a
dopamine receptor antagonist and is widely used as an antipsychotic
drug (Carlson et al., 1986; Kandel, 1991). RGS2 mRNA expression was
rapidly induced in the caudate putamen within 30 min after the
haloperidol administration, suggesting regulation by dopamine receptor
mechanisms (Fig. 2D). In contrast, cocaine (20-30
mg/kg, i.p.), an inhibitor of catecholamine reuptake by neurons that
induces several other IEGs in the brain (Bhat and Baraban, 1993;
Brakeman et al., 1997), caused a minimal level of change in RGS2 mRNA
(Fig. 2D). In the analysis of time courses, the
largest level of induction was seen 30 min after the administration of
these drugs. No change was detected in the level of RGS4 and RGS7 mRNA
after administration of these drugs (data not shown).
Extensive codistribution of RGS2, RGS4, and RGS7 in cortex
and hippocampus
We compared the localization of mRNAs for RGS2, RGS4, and RGS7 by
in situ hybridization (Fig.
3). Comparisons were performed using
paired half brains from naive control and MECS-stimulated (30 min)
rats. This analysis demonstrated an extensive anatomic distribution of
RGS2 induction by MECS in cortex, amygdala, hippocampus, and caudate
putamen. Expression of RGS4 and RGS7 in these brain regions was
unaffected by MECS. In the naive control brain (Fig. 3, C
side), RGS2, RGS4, and RGS7 showed unique and discrete
localizations throughout the brain, with highest levels of expression
in the cerebral and cerebellar cortex. RGS4 mRNA was enriched in the pyriform cortex, caudate putamen, amygdala nucleus, Purkinje cell layers of cerebellum, and the pyramidal cell layer of the hippocampus. In addition, RGS4 mRNA was enriched in the principal nuclei of the
thalamus, and this contrasts with RGS2, which was not enriched in
thalamus. The distribution of RGS7 mRNA is similar to RGS4 and is the
most widespread of the three surveyed RGS mRNAs. Overall levels of RGS7
hybridization were less than either RGS2 or RGS4. RGS7 is expressed in
cerebral neocortex, pyriform cortex, principal nucleus of thalamus,
hippocampus, cerebellar granule cell layer, and both the pyramidal cell
layer and dentate gyrus of the hippocampus. RGS7 is distinct from RGS4
in the dense staining pattern of the hippocampus and cerebellar granule
cell layer. For each RGS subtype, the hybridization patterns were
identical between rats (n = 2-5) in independent
hybridizations (n  2). The results of a recent report
of localization of RGS4 and RGS7 by in situ hybridization in
the rat brain agree with our findings (Gold et al., 1997). This report
clearly revealed the laminar expression pattern of RGS4 in the
neocortex, with the highest concentration of nine RGS subtypes
(RGS3-RGS11).
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Figure 3.
Comparative localizations of RGS2, RGS4, and
RGS7 mRNA in brain. The brains are composites of half brains from a
naive control rat (C side) and a rat that received a
single MECS stimulation 1 hr before being killed (S
side). Note that the laterality of the cerebellum is opposite
of the cerebrums in the bottom panels. In the control
brains, RGS2, RGS4, and RGS7 individually show discrete neuronal
localization throughout the brain with high concentrations in the
cortex. See Results for a more detailed discussion of the
comparative distributions. Seizure produced a modest induction of RGS4
that is restricted to granule cells of the posterior hippocampus,
whereas RGS7 is unchanged. RGS2 mRNA is induced by seizure in the
cerebral cortex and hippocampus. For each RGS subtype, the
hybridization patterns were virtually indistinguishable between rats
(n = 2-5) and independent hybridizations
(n 2). Neo, Neocortex;
Pi, pyriform cortex; CP, caudate putamen;
Am, amygdala; Gr, granule cell layer;
Th, thalamus; MG, medial geniculate
nucleus; Pur, Purkinje cell layer; DG,
dentate gyrus; Pyr, pyramidal cell layer.
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RGS2 selectively accelerates GTP hydrolysis by Gq in
solution assays
The dynamic changes in RGS2 expression that occur in models of
neural plasticity lead us to examine the effect of RGS2 upregulation on
neuronal cell function. Because RGS family members are known to
accelerate the rate of GTP hydrolysis of various receptor-coupled G-proteins, we began with a biochemical characterization that examined
the G-protein specificity of RGS2 using single turnover GTPase assays
(Berman et al., 1996). Although such assays have been described
previously for members of the Gs, Gi, and
G12 subfamilies, difficulties in preparing GTP-Gq
have precluded such simple measurements with this substrate. Rather,
effects of RGS proteins on the GTPase activity of Gq
have required reconstitution of Gq heterotrimer into
phospholipid vesicles with an appropriate receptor (e.g., M1 mAChR) to
stimulate nucleotide exchange. Berman et al. (1996) recently noted that
RGS4 could accelerate the GTPase activity of the
GTPase-deficient R178C mutant of Gi. Accordingly, the
analogous mutation was made in Gq (R183C), and the
resultant reduction of basal GTPase activity permitted preparation of
R183C Gq-GTP for use in single turnover assays.
Figure 4, A-D, demonstrates
that RGS4 accelerates the rate of GTP hydrolysis by R183C
Gq, Go, and Gi (but not
Gs). These experiments substantiate previous results
with RGS4 and demonstrate the utility of R183C Gq for
single turnover assays. In contrast, RGS2 was an effective GAP with
Gq but failed to have any significant effect on GTP
hydrolysis by Go, Gi, or
Gs.
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Figure 4.
GAP activity of RGS2 for Gq.
A-D, Examination of in vitro GTPase
activity of 0.2 nM R183C Gq
(A), 0.85 nM Go
(B), 1.2 nM Gi1
(C), and 1.2 nM Gs
(D) in the presence of RGS2 and RGS4. GTPase
activity was measured in the absence (open squares) or
presence of purified RGS protein [RGS2, 50 nM (open
circles), 150 nM (open triangles),
or 500 nM (filled circles); RGS4, 150 nM (filled squares) final
concentration]. E, The effect of increasing RGS2
concentrations on the GTPase activity of 0.2 nM R183C
Gq. The data for Gs and Gi
are plotted as the average ± SEM of duplicate measurements; the
data for Gq and Go are single points. The
data are representative of four separate assays performed for each G
protein.
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Further examination of RGS2 activity using a constant concentration of
R183C GTP-Gq (0.2 nM) with increasing
concentrations of RGS2 (0-500 nM) revealed that enhanced
GTPase activity could be detected with RGS2 concentrations as low as
0.5 nM (Fig. 4E). Kinetic analysis using
a two-site exponential curve fit to determine the initial rate of GTP
hydrolysis revealed a 200-fold increase in the catalytic rate constant
for GTP hydrolysis from 0.005/min to 1/min at maximal concentrations of
RGS2. The second site from the curve-fitting analysis is consistent
with the low intrinsic GTPase activity of R183C Gq. The
results of a recent report demonstrated that RGS2 selectively inhibited
Gq-mediated activation of phospholipase C in cell membranes
(Heximer et al., 1997). This observation is consistent with the
capacity of RGS2 to act as a GAP on Gq.
RGS2 accelerates GTP hydrolysis by both Gq and
Gi in reconstituted lipid vesicles
Because all other members of the RGS protein family tested to date
act as GAPs on members of the Gi subfamily of G-protein subunits, we explored the activity of RGS2 further in a more complex
system containing Gi heterotrimer and M2 mAChRs
reconstituted into phospholipid vesicles (Parker et al., 1991). The
rate of GTP hydrolysis by Gi was measured in the
presence of the receptor agonist carbachol and the receptor antagonist
atropine (Fig. 5). As anticipated,
carbachol produced a sixfold increase in the rate of GTP hydrolysis in
the absence of an RGS protein. Consistent with previous results (Berman
et al., 1996), addition of RGS4 caused a further 23-fold increase in
the rate of GTP hydrolysis in the presence of carbachol. Surprisingly,
RGS2 also stimulated GTP hydrolysis dramatically in this system,
demonstrating that this RGS protein also acts as a GAP for both
Gi and Gq if assayed under appropriate
conditions.
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Figure 5.
GAP activity of RGS2 with M2
mAChR(Gi) vesicles. Gi1 were
monitored in reconstituted vesicles containing the M2 mAChR and
Gi heterotrimer in the presence or absence of RGS2 or RGS4.
The activation state of Gi was controlled with the receptor
agonist carbachol (1 mM) or the receptor antagonist
atropine (20 µM). The final concentrations of the
proteins were 0.5 nM M2 mAChR, 2 nM
Gi, and 500 nM RGS2 or RGS4. The data
are representative of two similar experiments.
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RGS2 and RGS4 inhibit MAP kinase activation by
mAChR activation
Based on biochemical studies, we examined the hypothesis that RGS2
upregulation might modulate intracellular signaling cascades consequent
to receptor activation. One of the signaling events induced by several
different G-proteins is the phosphorylation and activation of MAP
kinase (Faure et al., 1994). The phosphorylation of MAP kinase leads to
its translocation to the nucleus in which it phosphorylates several
transcription factors that, in turn, control IEG expression (Xia et
al., 1996). For our studies, we selected the M1 and M2 mAChRs, because
they are well characterized and yield robust activation of
Gq and Gi, respectively, in heterologous expression systems (Wan et al., 1996). Furthermore, mAChRs are major
excitatory receptors in the hippocampus and cerebral cortex and may
therefore be the natural target of RGS2 in stimulated neurons. Both
Gq- and Gi-coupled receptors stimulate MAP
kinase activation via distinct signaling pathways (Faure et al., 1994; Hawes et al., 1995). M2 mAChR(Gi)-mediated
MAP kinase activation is believed to be transmitted through G
subunits, which is the same pathway used by yeast to mediate pheromone
signaling. subunits activate MAP kinase through the sequential
interaction of the signaling proteins phosphoinositide 3-kinase ,
tyrosine kinase, Shc, Grb2, Sos, Ras, and Raf (Della Rocca et al.,
1997; Lopez-Ilasaca et al., 1997). In contrast, Gq
mediates M1 mAChR(Gq)-stimulated MAP kinase
activation by increasing phosphatidylinositol turnover (Hawes et al.,
1995).
Initial experiments confirmed robust agonist-dependent activation of
MAP kinase in COS cells that expressed M1 or M2 mAChRs (Fig.
6). COS cells were transiently
transfected with combinations of mAChR and epitope-tagged MAP kinase
(HA-ERK1). Transfected cells were exposed to the agonist carbachol for
5 min before immunoprecipitation of HA-ERK1, and HA-MAP kinase
activities were determined by the phosphorylation of MBP. Treatment of
cells expressing M1 or M2 mAChR with carbachol caused a 20- or 40-fold
increase in ERK1 kinase activity in these cells. Coexpression of RGS2
(or RGS4) inhibited both M1- and M2-dependent activation of MAP kinase. RGS2 inhibited M1-dependent activation of MAP kinase by ~50% and inhibited M2-dependent activation of MAP kinase by ~35%. The similar activity of RGS2 for both M1- and M2-dependent MAP kinase activation is
consistent with our observation that RGS2 can act as a GAP for both
Gq and Gi.
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Figure 6.
RGS2 and RGS4 inhibit mAChR-mediated MAP
kinase activation. COS cells were transfected with an HA-tagged ERK1
plasmid, an M1 mAChR plasmid, or an M2 mAChR plasmid, in conjunction
with empty vector or RGS expression plasmids. Transfected cells were
exposed to carbachol (100 µM) for 5 min before lysis and
immunoprecipitation of HA-ERK1. The activity of HA-MAP kinase
(KINASE) was then assayed using MBP as the substrate.
The radioactivity of MBP was determined by scintillation spectroscopy
of excised bands and is presented as a percentage relative to the empty
vector controls to M2 mAChR (2.2 × 105 cpm) or
M1 mAChR (5.0 × 105 cpm) controls. Note that
RGS2 inhibited ~70% of ERK activation by mAChR1 and ~50% of
activation by mAChR2. RGS4 produced similar levels of inhibition in
these assays. Data are mean ± range of duplicate determinations
from a representative experiment. The same results were repeated in two
to four independent experiments. Equivalent ERK1
(HA-ERK1) levels were detected in HA-immunoprecipitates
by immunoblot (WB) analysis.
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DISCUSSION |
A central goal of neurobiology is to understand how neurons effect
long-term changes in response to synaptic activity. In the present
study, we demonstrate that RGS2 is a neuronal IEG and provide evidence
for its contribution to cellular signaling. RGS2 gene expression is
dynamically responsive to synaptic activity, as illustrated using the
in vivo LTP paradigm. Induction in this paradigm is
dependent on activation of NMDA receptors and is associated with
long-term synaptic plasticity. RGS2 mRNA is also rapidly induced in the
striatum by acute administration of haloperidol, suggesting regulation
by dopamine-dependent mechanisms. The dynamic regulation of RGS2
appears to be unique among RGS family members that are expressed in
brain. Although other members of the RGS family, including RGS4 and
RGS7, are expressed in brain neurons and show extensive codistribution
with RGS2 in cortex, amygdala, hippocampus, and striatum, they are not
induced by MECS or haloperidol or in association with LTP. RGS2 is
additionally notable because it functions as a GAP for both
Gi and Gq in reconstituted lipid vesicles.
These observations suggest that RGS2 induction may modify the signaling
properties of stimulated neurons by reducing the magnitude or duration
of Gi- and Gq-dependent ligand-receptor systems. Our studies do not define which signaling systems are most
affected by RGS2 upregulation; however, the major Gq-linked receptors of cortical and hippocampal granule cell neurons include M1
mAChRs, group I metabotropic glutamate receptors, 1 adrenergic receptors, bradykinin receptors, D2b dopamine receptors, and 5-HT2 serotonin receptors. Because these receptors are typically linked to
phosphatidylinositol turnover, it is anticipated that RGS2 upregulation
will modulate receptor-mediated release of intracellular calcium from
inositoltrisphosphate-sensitive pools. This notion is supported by the
recent report demonstrating that RGS4 inhibits calcium signaling by
group I metabotropic glutamate receptors (Saugstad et al., 1998).
Our studies further demonstrate that RGS2 upregulation may impact
signaling events leading to activation of MAP kinase. In cell assays,
RGS2 shows inhibitory effects on both M1- and M2-dependent receptor
activation of MAP kinase and is similar in efficacy to RGS4. This
effect of RGS2 is consistent with its GAP activity for both
Gq and Gi. Because MAP kinase activation is
linked to gene expression in neurons (Xia et al., 1996) and has been
demonstrated to play an essential role in a model of activity-dependent
plasticity (Bailey et al., 1997; Kornhauser and Greenberg, 1997; Martin
et al., 1997), upregulation of RGS2 may impact events at the nucleus, as well as the plasma membrane.
Members of the RGS family are rapidly upregulated in response to
specific stimuli in other systems. SST2 is expressed at low constitutive levels in naive yeast and is transcriptionally induced by
pheromone. Induction of SST2 is responsible for desensitization to
pheromone signaling (Dietzel and Kurjan, 1987). Similarly, RGS1
expression is induced by a platelet-activating factor (PAF) in human
B-cell lymphoma cell line, and elevated concentrations of RGS1 block
PAF-induced MAP kinase activation (Druey et al., 1996). Furthermore,
the level of EGL-10 activity quantitatively regulates the G-protein
signaling in C. elegans (Koelle and Horvitz, 1996),
suggesting that expression may be tightly controlled to regulate
G-protein signaling. These examples, together with our present
observations, support the notion that dynamic transcriptional control
of specific RGS proteins may represent a general principle important in
controlling the duration or intensity of G-protein-coupled receptor
signaling.
EGL-10 is enriched in neuronal dendrites and is relatively excluded
from the cell body or axon (Koelle and Horvitz, 1996). This subcellular
targeting appears to be mediated by its N-terminal 120 amino acids
(Koelle and Horvitz, 1996). This demonstration of subcellular targeting
suggests that other RGS members may similarly be targeted to discrete
subcellular regions. This may be important in understanding why
multiple family members with shared G-protein specificity are
coexpressed in neurons. Sequence comparisons indicate significant
homology between the N terminus of EGL-10 and RGS7 (64%) and RGS9
(33%), whereas the N terminus of RGS4 shows similarity to RGS5 (67%),
RGS16 (55%), and RGS14 (51%). By contrast, the N terminus of RGS2 is
not homologous to other RGS family members and may be hypothesized to
confer unique targeting. We are not presently able to examine this
issue because of the lack of specific immunoreagents. However,
independent of a uniquely localized effect, upregulation of RGS2 might
be anticipated to increase Gs signaling because of the
reciprocal relationship between Gi and Gs. The late phase of hippocampal LTP is known to involve a persistent increase
in protein kinase A activity (Frey et al., 1993) and to be dependent on
protein synthesis (Frey and Morris, 1997). Upregulation of RGS2 might
contribute to this phenomenon.
Biochemical characterization of the GAP activity of RGS2 revealed a
paradox. All RGS proteins assayed previously have displayed GAP
activity toward members of the Gi subfamily of G
proteins in solution. In contrast, demonstration of the GAP activity of RGS2 toward Gi1 required reconstitution of the G-protein
with receptors in phospholipid vesicles. We do not as yet understand the nature of this requirement. Reconstitution into vesicles may simply
increase the effective concentrations of Gi and RGS2. Other possibilities include a more specific requirement for
phospholipid for the RGS-G-protein interaction or, perhaps, the
formation of a complex of RGS2 and Gi that includes
receptor (Doupnik et al., 1997). A practical consequence of this result
is to decrease substantially the significance of previous negative
observations on RGS-G-protein interactions that were examined only
in solution.
Dynamic regulation of RGS2 occurs in the context of other mechanisms
that modulate G-protein-dependent signaling. Agonist-dependent phosphorylation of receptors by protein kinases and the subsequent binding of arrestin-like molecules contribute to signal desensitization (Freedman and Lefkowitz, 1996). This type of receptor phosphorylation appears to be involved in the rapid receptor trafficking processes that
occur within minutes of agonist exposure. Another inhibitory mechanism
involves downregulation of G-protein-coupled receptors, which may play
a role in long-term desensitization of signaling (Hausdorff et al.,
1990). In our studies of activity-regulated genes, we identified a
novel IEG, termed Homer (Brakeman et al., 1997), which binds to the C
terminus of group I metabotropic glutamate receptors. Group I
metabotropic glutamate receptors are coupled to Gq and
function to release intracellular calcium pools. Thus, both RGS2 and
Homer represent IEG responses to effect modulation of
Gq-dependent signaling. The present study reinforces the
important link between neuronal activity and long-term changes in
cellular signaling and defines a novel mechanism in this process.
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FOOTNOTES |
Received April 7, 1998; revised July 6, 1998; accepted July 8, 1998.
This work is supported by National Institutes of Health Grants MH01152
(P.F.W.), MH53608 (P.F.W.), GM34497, and GM30355, a grant from the
National Alliance for Research on Schizophrenia and Depression
(P.F.W.), Carol A. Barnes Grant AG09219, the Raymond and Ellen Willie
Distinguished Chair and Molecular Neuropharmacology Award (A.G.G.), and
Robert A. Welch Foundation Grant I-0982 (E.M.R.). We thank M. Papapavlou for excellent technical assistance, and R. Kim for help in
studies of RGS subtypes in the brain. We thank Dr. Yaping Tu for
providing M2 mAChR-Gi vesicles and assistance with the protocol.
Correspondence should be addressed to Dr. Paul F. Worley, Department of
Neuroscience, Johns Hopkins University School of Medicine, 725 North
Wolfe Street, Baltimore, MD 21205.
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Top
Abstract
Introduction
