 |
 Previous Article | Next Article 
The Journal of Neuroscience, February 1, 1998, 18(3):905-913
RGS4 Inhibits Signaling by Group I Metabotropic Glutamate
Receptors
Julie A.
Saugstad,
Michael J.
Marino,
Julie A.
Folk,
John R.
Hepler, and
P. Jeffrey
Conn
Department of Pharmacology, Emory University, Atlanta, Georgia
30322
 |
ABSTRACT |
Metabotropic glutamate receptors (mGluRs) couple to heterotrimeric
G-proteins and regulate cell excitability and synaptic transmission in
the CNS. Considerable effort has been focused on understanding the
cellular and biochemical mechanisms that underlie regulation of
signaling by G-proteins and their linked receptors, including the
mGluRs. Recent findings demonstrate that regulators of G-protein
signaling (RGS) proteins act as effector antagonists and
GTPase-activating proteins for G subunits to inhibit
cellular responses by G-protein-coupled receptors. RGS4 blocks
Gq activation of phospholipase C and is expressed broadly in rat brain. The group I mGluRs (mGluRs 1 and 5) couple to
Gq pathways to regulate several effectors in the CNS. We
examined the capacity of RGS4 to regulate group I mGluR responses. In
Xenopus oocytes, purified RGS4 virtually abolishes the
mGluR1a- and mGluR5a-mediated but not the inositol
trisphospate-mediated activation of a calcium-dependent chloride
current. Additionally, RGS4 markedly attenuates the mGluR5-mediated inhibition of potassium currents in hippocampal CA1 neurons. This inhibition is dose-dependent and occurs at concentrations that are
virtually identical to those required for inhibition of phospholipase C
activity in NG108-15 membranes and reconstituted systems using purified proteins. These findings demonstrate that RGS4 can modulate mGluR responses in neurons, and they highlight a previously unknown mechanism for regulation of G-protein-coupled receptor signaling in the
CNS.
Key words:
RGS4; RGS proteins; metabotropic glutamate receptor; hippocampus; G-proteins; synaptic regulation; Xenopus
| |
INTRODUCTION |
Glutamate is the principal
excitatory amino acid neurotransmitter in the CNS, and its actions are
mediated by specific interactions with two distinct families of
receptors: the ionotropic and metabotropic glutamate receptors (mGluRs)
(Hollmann and Heinemann, 1994 ). The mGluRs couple to heterotrimeric
GTP-binding proteins (G-proteins) and regulate cell excitability and
synaptic transmission at glutamatergic synapses throughout the brain
(Saugstad et al., 1995a; Conn and Pin, 1997). The mGluR family consists
of at least eight distinct receptors, many with alternatively spliced
isoforms, that are classified into three major subgroups (Conn and Pin,
1997). Members of the group I mGluRs (mGluRs 1 and 5) couple to the
Gq signaling pathway, whereas members of the group II
(mGluRs 2 and 3) and III (mGluRs 4, 6, 7, and 8) mGluRs couple to
Gi/Go signaling pathways in heterologous
expression systems.
Net transmission through glutamatergic circuits can be impacted
dramatically by the mGluRs present at a given synapse, and signaling by
mGluRs is dynamically regulated under various physiological and
pathological conditions (Akiyama et al., 1987; Seren et al., 1989;
Yamada et al., 1989; Aronica et al., 1991; Chen et al., 1992; Mayat et
al., 1994). Delineation of the mechanisms involved in regulating mGluR
function will be critical for development of a complete understanding
of regulation of transmission at glutamatergic synapses.
Considerable effort has been focused on understanding the cellular and
biochemical mechanisms that underlie regulation of signaling by
G-proteins and their linked receptors, including the mGluRs. Recent
findings demonstrate that G-proteins interact directly with a newly
identified family of regulatory proteins, regulators of G-protein
signaling (RGS) proteins, that block G-protein functions (Dohlman and
Thorner, 1997; Koelle, 1997). Although RGS proteins were first
identified by genetic analysis of lower eukaryotic organisms, more than
20 unique mammalian RGS isoforms have been identified by molecular
cloning, with predicted molecular mass ranging from 140 to 17 kDa
(Druey et al., 1996; Koelle and Horvitz, 1996). Biochemical evidence
indicates that RGS proteins serve as GTPase-activating proteins (GAPs)
to markedly accelerate G-catalyzed GTP hydrolysis
(Berman et al., 1996; Hunt et al., 1996; Watson et al., 1996) or as
effector antagonists (Hepler et al., 1997) to negatively regulate
G-protein signaling by members of the Gq or Gi
families, but not Gs or G12.
We reported recently that RGS4, a protein found exclusively in brain
(Druey et al., 1996), blocks Gq activation of phospholipase C in vitro (Hepler et al., 1997) and receptor and
Gq-directed inositol lipid signaling in intact cells (Huang
et al., 1997; Yan et al., 1997). To determine whether RGS4 could play
an important role in regulation of mGluR signaling, we examined the
effects of RGS4 on characteristic cellular responses stimulated by
group I mGluRs in Xenopus oocytes or neurons. We report that
RGS4 blocks the mGluR1a- and mGluR5a-mediated activation of the
calcium-dependent chloride current
(ICl(Ca)) in Xenopus oocytes
and the mGluR5-mediated inhibition of calcium-activated potassium
currents (IAHP) in hippocampal CA1
neurons. This is the first demonstration that RGS proteins can modulate
mGluR responses, and it reveals a previously unknown mechanism for the
regulation of G-protein-coupled receptor (GPCR) responses in the
CNS.
| |
MATERIALS AND METHODS |
Reagents. 3,5-Dihydroxyphenylglycine (DHPG) was
purchased from Tocris Cookson (Bristol, UK). Glutamate, inositol
1,4,5-trisphosphate [Ins(1,4,5)P3], and
isopropylthio--galactoside (IPTG) were obtained from Sigma (St.
Louis, MO). RNA transcription kits were obtained from Stratagene (La
Jolla, CA), 32P-dCTP was purchased from New England
Nuclear, and Xenopus laevis were purchased from Nasco, Ft.
Atkinson, WI. Purified recombinant PLC1 was a generous gift of Drs.
R. Ball and P. Sternweis (University of Texas Southwestern Medical
Center). Recombinant hexahistidine-tagged Gq was
prepared and purified as described (Hepler et al., 1996). The
polyclonal antiserum that specifically recognizes RGS4 (R374) was
kindly provided by Dr. A. G. Gilman (University of Texas
Southwestern Medical Center) and was made in rabbits against the
synthetic peptide YKGAKSSADCTSLVPQ that corresponds to the C-terminal
residues 189-203 of RGS4. The cDNA clones were generously shared by
Dr. A. Gilman (University of Texas Southwestern Medical Center; RGS4),
Dr. E. Mulvihill (Darwin; mGluR1a), Dr. J. P. Pin (Centre
CNRS-INSERM de Pharmacologie-Endocrinologie; mGluR5a), Dr. H. Lester
(California Institute of Technology; Kir3.4), and Dr. J. Adelman
(Vollum Institute; Kir3.1).
Preparation of RGS protein. Bacterial cultures (JM109)
harboring the hexahistidine-tagged RGS4 cDNA in a bacterial expression vector (His6-RGS4/pQE60) were grown to OD6000.5, and
protein production was induced by the addition of 0.1 mM
IPTG. Cells were grown for 12 hr at 30°C with constant shaking (150 rpm) and harvested by centrifugation. Cell lysates were prepared, and
the supernatant containing RGS4 was recovered in Buffer A [50
mM HEPES, pH 8.0, 20 mM -mercaptoethanol,
100 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)]. This material was applied to a column containing 5 ml of Ni-NTA affinity resin (Qiagen, Hilden, Germany), and the column
was washed with 100 ml of Buffer A containing 500 mM NaCl and 15 mM imidazole. RGS4 was eluted with 20 ml of Buffer A
containing 150 mM imidazole. Eluted protein was
concentrated to 1 ml and further enriched by size exclusion
chromatography. Sample was applied to a column containing 50 ml
Sephacryl S-200 media (Pharmacia, Piscataway, NJ) equilibrated with 50 mM HEPES, pH 7.4, 100 mM KCl, and 1 mM DTT, and fractions enriched with RGS4 were pooled, concentrated, and stored at  80°C.
Reconstitution of RGS4 with NG108-15 membranes. NG108-15
membranes were prepared exactly as described previously (Hepler et al.,
1997). Confluent NG108-15 cells were grown at 37°C in DMEM supplemented with 10% fetal bovine serum, 0.1 mM
hypoxanthine, 0.4 µM aminopterine, and 16 µM thymidine, and harvested in buffer containing 50 mM Na-HEPES, pH 8.0, 1 mM EDTA, 150 mM NaCl, and 0.1 mM PMSF. Homogenates were
prepared by nitrogen cavitation and centrifugation at 500 × g to remove nuclei and unbroken cells. Supernatants were
centrifuged at 100,000 × g, and membranes were suspended in Buffer B (50 mM Na-HEPES, pH 7.2, 1 mM EDTA, 3 mM EGTA, 5 mM
MgCl2, 150 mM NaCl, 2 mM
dithiothreitol, and 0.1 mM PMSF) and then stored at
80°C. Reactions were performed as described previously (Hepler et
al., 1997) with minor modifications. NG108-15 membranes (6.5 µg/assay) in 10 µl of Buffer A were mixed with an equal volume of
RGS4 in Buffer C (50 mM Na-HEPES, pH 7.2, 3 mM
EGTA, 100 mM NaCl2, 2 mM
dithiothreitol, and 80 mM KCl). Membranes and RGS4 were
incubated at 4°C for 30 min and then mixed with 30 µl of sonicated
phospholipid vesicles containing [3H]phosphatidyl
inositol 4,5-bisphosphate and phosphatidylethanolamine (Hepler et al.,
1993) and 100 µM GTP S in Buffer C. Reactions were
initiated by the addition of 10 µl of 9 mM
CaCl2 in Buffer C, and assays were performed for 30 min at
30°C. Reactions were stopped, and the samples were processed as
described (Hepler et al., 1993).
Reconstitution of RGS4 with Gq and phospholipase
C1. Reactions were performed essentially as described (Hepler
et al., 1997) with minor modifications. Assays were performed in a
final volume of 60 µl. Purified recombinant Gq was
activated with 1 mM GTPS for 1 hr at 30°C in Buffer A. Activated Gq (10 µl; 6 nM) was mixed with
RGS4 in 10 µl of Buffer C. The Gq/RGS sample was
incubated for 30 min at 4°C and then mixed with 10 µl of 9 mM CaCl2 in Buffer B. The reactions were
started by the addition of 30 µl of sonicated phospholipid vesicles
containing [3H]phosphatidyl inositol
4,5-bisphosphate and phosphatidylethanolamine (Hepler et al.,
1993) and purified recombinant PLC1 (1 ng) in Buffer B. Assays were
performed for 20 min at 30°C. Reactions were stopped, and the samples
were processed as described (Hepler et al., 1993).
RNA isolation and analysis. Total RNA was isolated from
adult rat brain tissue using RNA-Stat 60 (Tel-Test, Friendswood, TX). The RNA was separated on agarose/formaldehyde gels and transferred by
capillary action to membrane overnight. The resulting RNA blots were
stained with methylene blue to visualize the ribosomal RNA bands and
then hybridized with RGS4 cDNA probe labeled with 32P-dCTP
at 42°C for 12-16 hr. The blots were washed to a final stringency of
65°C in 0.2× SSC (1× = 150 mM NaCl, 15 mM
sodium citrate), 1.0% SDS to remove unbound probe, and the RNA was
visualized by autoradiography using Kodak BiomaxMS film.
Oocyte electrophysiology. Oocytes were harvested from
anesthetized Xenopus laevis and enzymatically defolliculated
as described previously (Saugstad et al., 1995b). Stage V-VI oocytes
were injected with 50-100 ng of a cRNA consisting of mGluR1a or
mGluR5a in combination with Kirs 3.1 and 3.4. The oocytes were
maintained at 16°C in 1× Barth's culture solution (90 mM NaCl, 1 mM KCl, 10 mM HEPES, 4 mM NaOH) supplemented with 100 µg/ml gentamicin and 50 µg/ml penicillin/streptomycin. Electrophysiological recordings were performed 72-96 hr after injection of the cRNA mixtures. For the RGS4
experiments, 50 nl of a 48 µM stock of purified RGS4
protein or heat-inactivated RGS4 protein (30 min/100°C) was injected
into each oocyte, and recordings were performed 30 min to 1 hr after injection. Patch pipettes with tip diameters of 1-2 µm were used as
electrodes and filled with 3 M KCl. Oocytes were
voltage-clamped at 60 mV in a two-electrode voltage-clamp mode
(Oocyte Clamp OC-725C, Warner Instrument Corporation). Currents were
acquired at 5 kHz with Axotape 2.0.2. The oocytes were placed in a 1 ml chamber and bathed continuously in 1× Barth's culture containing 1.5 mM CaCl2 and 1.5 mM
MgCl2 at ~2 ml/min. Solutions were changed using a
solenoid valve controller (Valvelink 8, Automate Scientific) with an
exchange time of 30-45 sec. Glutamate diluted to a final concentration
of 100 µM into 1× Barth's bathing solution was used to
elicit ICl(Ca). For
IGirk, oocytes were initially bathed in 1× Barth's and then changed to a high potassium (hK) solution (2 mM NaCl, 96 mM KCl, 10 mM HEPES,
1.5 mM CaCl2, 1.5 mM
MgCl2). Glutamate diluted to a final concentration
of 100 µM into hK solution was applied when the basal
high potassium current reached equilibrium. The hK current was
subtracted from the total current to obtain the agonist-induced
current. Current amplitudes were measured off line, and statistical
analysis was performed using SigmaPlot 4.0 (SPSS). A value of <0.05
was considered significant.
Whole-cell electrophysiology. For patch-clamp studies, thick
(400 µm) transverse hippocampal slices were prepared and maintained as described previously (Gereau and Conn, 1994a,b). Slices were maintained fully submerged on the stage of a brain slice chamber and
perfused continuously with artificial cerebrospinal fluid (ACSF; 1 ml/min). ACSF contained 124 mM NaCl, 2.5 mM
KCl, 1.3 mM MgSO4, 2.0 mM
CaCl2, 1.0 mM
NaH2PO4, 26 mM
NaHCO3, and 10 mM glucose, equilibrated
with 95%O2/5%CO2. DHPG was applied
through the perfusion system to give a final concentration of 10 µM. Whole-cell recording was performed as described
(Gereau and Conn, 1994b,c). Briefly, patch electrodes (3-7 M ) were
pulled from thin-walled borosilicate glass and filled with 150 mM potassium gluconate, 5 mM HEPES, 2 mM ATP, 0.1 mM GTP, 0.1 mM DTT, pH
adjusted to 7.4 with 0.5 M KOH. In preliminary experiments
with RGS4 protein we found that the inclusion of EGTA/calcium buffer in
the patch solution caused rapid clogging of the electrode, possibly by
promoting aggregation of the protein. The patch solution used here
allows for recording of IAHP for >45 min, with
no discernible rundown; however, the IAHP
exhibits faster kinetics than is usually reported because of the
absence of calcium buffer in our patch solution (Engisch et al., 1996).
Recordings from CA1 pyramidal cells were established using the
"blind" patch/slice technique described in detail (Blanton et al.,
1989) using a Warner PC-501A patch-clamp amplifier.
IAHP was measured by recording tail currents
after a 100 msec step to 0 mV from a holding potential of 50 mV. We found that this protocol and filling solution produced a robust and
stable IAHP that was modulated by DHPG. For
protein dialysis, the tips of patch electrodes were filled with normal
patch solution and then filled with active RGS4 (0.25-2.5
µM in patch pipette) or heat-inactivated RGS4 (2.5 µM) in normal patch solution. Series resistance was
monitored throughout the experiment and often increased as protein
dialyzed into the cell. Cells were not included if series resistance
increased above 20 M or increased during drug application.
| |
RESULTS |
Purified RGS4 blocks Gq-directed activation of
phospholipase C1 in cell membranes and reconstituted systems
Members of the Gq family of G-proteins link a wide
variety of neurotransmitter receptors, including the group I mGluRs 1 and 5, to activation of the isoforms of phospholipase C (PLC)
(Hepler and Gilman, 1992). We have shown previously that RGS4 is a GAP for Gq and that it blocks receptor and Gq
functions in reconstituted systems (Hepler et al., 1997) or when
overexpressed in intact cells (Huang et al., 1997; Yan et al., 1997).
To examine possible roles for RGS4 as a regulator of mGluR signaling,
we first prepared purified recombinant RGS4 and examined its capacity
to block Gq functions (Fig.
1). RGS4 synthesized in Escherichia
coli was purified to near homogeneity and is recognized by a
specific anti-RGS4 sera (Fig. 1A). NG108-15
neuroblastoma X glioma cells express Gq and
G11 (Gutowski et al., 1991), and activation of endogenous Gq/11 in NG108-15 cell membranes with GTPS,
a poorly hydrolyzable analog of GTP, stimulates activation of
endogenous PLC isoforms (Gutowski et al., 1991; Hepler et al.,
1997). Although RGS4 is an activator of Gq- and
Gi-catalyzed GTP hydrolysis, it also can inhibit
Gq functions by binding directly to the GTPS-activated form of Gq to occlude interactions
with PLC (Hepler et al., 1997). Reconstitution of purified RGS4 with
NG108-15 membranes completely blocked the capacity of
GTPS-Gq/11 to stimulate PLC activity (Fig.
1B). This observation implies that RGS4 directly blocks Gq interactions with PLC1, and this idea was
confirmed in complementary experiments demonstrating that RGS4 blocks
GTPS-Gq activation of PLC1 when reconstituted as
purified proteins with phospholipid vesicles (Fig. 1C).
These findings are consistent with our previous observations (Hepler et
al., 1997) and demonstrate that the recombinant RGS4 can block
Gq functions in vitro. This material was used
for all subsequent experiments.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 1.
Purified RGS4 blocks
G-protein-directed activation of phospholipase C in NG108-15 membranes
and Gq-directed activation of purified phospholipase
C1. A, Purified RGS4 (0.5 µg) was resolved by
SDS-PAGE and visualized by staining with Coomassie blue (left, stain) or transferred to nitrocellulose and visualized by
immunostaining with specific anti-RGS4 sera R374 (right,
blot). B, NG108-15 membranes (6.5 µg) were
mixed with [3H]phosphatidyl inositol
4,5-bisphosphate-containing phospholipid vesicles and incubated for 30 min at 30°C with 100 µM GTPS (open circles) or GTPS and various concentrations of RGS4
(closed circles). Synthesis of
[3H] inositol 1,4,5-trisphosphate
([3H]InsP3) was measured, and
[3H]InsP3 accumulation in the absence
of NG108-15 membranes (blank = 118 pmol) was
subtracted from each value. The data presented are duplicate values and
representative of three independent experiments, each with similar
results. C, Purified recombinant Gq was activated with 1 mM GTPS for 1 hr at 30°C. Activated
Gq (1 nM final) was mixed with purified
recombinant PLC1 (1 ng) and in the absence (open
circles) or presence of various concentrations of RGS4
(closed circles). Synthesis of
[3H]InsP3 was measured, and
[3H]InsP3 accumulation in the absence
of PLC1 (blank = 170 pmol) was subtracted from
each value. The data presented are duplicate values and representative
of three independent experiments, each with similar results.
|
|
Distribution of RGS4 messenger RNA in adult rat brain
To determine the relative distribution and levels of expression of
RGS4 mRNA within the CNS, we isolated total RNA from adult rat whole
brain and eight well defined brain subregions and subjected these
samples to RNA blot analysis using a 32P-labeled RGS4 cDNA
as a probe (Fig. 2). RGS4 mRNA is
detected in all brain regions examined, with the highest levels of RNA expression in cerebral cortex, brainstem, and thalamus. The relative levels of 28S ribosomal RNA are also shown to confirm equal loading of
the RNA.
View larger version (79K):
[in this window]
[in a new window]
|
Figure 2.
Distribution of RGS4 RNA in rat brain. RNA blot
analysis of adult rat brain hybridized with a 32P-labeled
RGS4 cDNA probe and visualized by autoradiography. RGS4 mRNA was
detected as one size class of ~3.5 kb in all brain regions, with the
highest levels seen in cortex, brainstem, and thalamus.
|
|
The distribution of group I mGluR mRNA and protein in various adult rat
brain regions has been described (Masu et al., 1991; Abe et al., 1992;
Martin et al., 1992; Baude et al., 1993; Romano et al., 1995). Both
mGluR1 and mGluR5 are expressed broadly within rat brain, and mGluR1 is
particularly abundant in cerebellum, thalamus, hippocampus, and
olfactory bulb, whereas mGluR5 is enriched in cortex, hippocampus, and
olfactory bulb. These distribution patterns indicate that group I
mGluRs and RGS4 coexist in some of the same brain regions, supporting
our hypothesis that RGS4 could serve as a modulator of mGluR signaling
in neurons.
RGS4 blocks mGluR1a-mediated activation of ion currents in
Xenopus oocytes
We next examined the capacity of purified RGS4 to regulate
mGluR1a-mediated ion currents in Xenopus oocytes. The cRNA
encoding mGluR1a was coexpressed in oocytes with the cRNAs encoding
Kirs 3.1 and 3.4, the two protein subunits that form the functional G-protein-coupled inward-rectifying potassium channel Girk (Dascal et
al., 1993; Kubo et al., 1993; Krapivinsky et al., 1995). Two-electrode voltage-clamp recording was performed 3-4 d after injection. In these
oocytes, the application of glutamate (100 µM) elicits
two large inward currents; the ICl(Ca) that is
mediated by activation of PLC and the subsequent
Ins(1,4,5)P3-induced release of calcium, and
IGirk, the potassium current carried by
activation of the exogenously expressed Kir channels (Fig.
3A, left). The
ICl(Ca) is only slightly reduced by pertussis
toxin (PTX) treatment (Houamed et al., 1991; Masu et al., 1991),
suggesting that the current is mediated through the Gq
signaling pathway. In contrast, the IGirk
current is inhibited by PTX treatment (Saugstad et al., 1996) and is
most likely mediated by G  (Reuveny et al., 1994;
Wickman et al., 1994) released from
Gi/Go. Microinjection of purified RGS4
protein (50 nl of 48 µM) markedly reduced the peak
ICl(Ca) amplitude (82.4% of control) (Fig.
3A, middle). Microinjection of RGS4 also reduced,
to a lesser extent, the observed peak current amplitude for the
IGirk (59.3% of control), consistent with
previous reports that RGS4 acts as a GAP for Gi proteins
(Berman et al., 1996; Watson et al., 1996). These effects were caused
by active RGS4 protein, because microinjection of heat-inactivated RGS4 failed to elicit a statistically significant reduction of either the
mGluR1a-mediated ICl(Ca) or
IGirk (Fig. 3A, right).
These data show that RGS4 blocks the mGluR1a-mediated
Gq-linked activation of ICl(Ca)
(Fig. 3B), and to a lesser extent the mGluR1a-mediated Gi/o-linked activation of IGirk
(Fig. 3C).
View larger version (21K):
[in this window]
[in a new window]
|
Figure 3.
Differential effects of RGS4 on
mGluR1a-induced currents in Xenopus oocytes.
A, Oocytes coinjected with mGluR1a and Kirs 3.1 and 3.4 were recorded under two-electrode voltage clamp at 60 mV. Application
of 100 µM glutamate in 1× Barth's solution to a control
oocyte (left) resulted in the activation of the
ICl(Ca). Subsequently, the bathing solution
was changed to a high potassium solution (hK),
and the application of 100 µM glutamate in hK resulted in
a large agonist-induced IGirk. In a separate
oocyte injected with 50 nl of purified RGS4 (48 µM) at
least 30 min before recording (middle), glutamate
application did not result in the activation of
ICl(Ca). The hK solution evoked an inward
current attributable to basally activated potassium channels, whereas
the subsequent addition of glutamate in hK resulted in activation of
IGirk. In an oocyte injected with 50 nl of
heat-inactivated RGS4 (right), the
ICl(Ca) was induced by application of
glutamate, as was the IGirk. Each
trace is one representative oocyte. B,
Bar graph depicting the effect of RGS4 on the mGluR1a-induced
activation of ICl(Ca) in control,
RGS4-injected, or heat-inactivated RGS4-injected oocytes. RGS4 resulted
in an 82.4% reduction in the
ICl(Ca), whereas the heat-inactivated
RGS4 produced currents that were not significantly different from the
control, as determined by unpaired Student's t test.
The numbers for each value are in parentheses, and *
indicates a p value of <0.05, as determined by
unpaired Student's t test. C, Bar
graph depicting the effect of RGS4 on the mGluR1a-induced activation of IGirk in control,
RGS4-injected, or heat-inactivated RGS4-injected oocytes. Injection of
RGS4 resulted in a 59.3% reduction in the
IGirk, and the boiled RGS4 also
resulted a small reduction in current compared with control. The
numbers for each value are in parentheses, and *
indicates a p value of <0.05, as determined by unpaired
Student's t test.
|
|
RGS4 blocks mGluR5a-mediated activation of
ICl(Ca) in Xenopus oocytes
To extend the observation that RGS4 blocks Gq-linked,
mGluR-mediated responses in oocytes (Fig. 3), we examined the effect of
RGS4 on cellular responses mediated by mGluR5a, another group I mGluR
linked to Gq signaling (Abe et al., 1992). In oocytes expressing mGluR5a, the application of 100 µM glutamate
elicited a robust ICl(Ca) (Fig.
4A, left).
Microinjection of purified active RGS4 protein (50 nl of 48 µM) nearly abolished the ICl(Ca)
(93.7% of control) (Fig. 4A, middle),
whereas heat-inactivated RGS4 was without effect (Fig.
4A, right). The summary data (Fig.
4B) indicate that RGS4 blocked the mGluR5a-mediated
activation of ICl(Ca). These results demonstrate
that RGS4 blocks signaling by both of the Gq-coupled group
I mGluRs in Xenopus oocytes, consistent with the idea that
RGS4 acts directly on Gq.
View larger version (11K):
[in this window]
[in a new window]
|
Figure 4.
RGS4 blocks mGluR5a-induced calcium-activated
chloride currents in Xenopus oocytes. A,
Oocytes expressing mGluR5a were recorded under two-electrode voltage
clamp at 60 mV. Application of 100 µM glutamate in 1×
Barth's solution to a control oocyte (left) resulted in
the activation of the
ICl(Ca). In a separate oocyte injected with 50 nl of purified RGS4 (48 µM) 30 min to 1 hr before recording (middle), glutamate application did
not result in the activation of
ICl(Ca); however, sister oocytes
injected with 50 nl of heat-inactivated RGS4 (right)
were able to evoke currents similar to those in control oocytes. Each
trace is one representative oocyte. B,
Bar graph depicting the effect of RGS4 on the mGluR5a-induced activation of ICl(Ca) in control,
RGS4-injected, or heat-inactivated RGS4-injected oocytes. RGS4 resulted
in a 93.7% reduction in the ICl(Ca),
whereas the current amplitudes evoked in oocytes injected with
heat-inactivated RGS4 were not significantly different from the
control, as determined by unpaired Student's t test.
The numbers for each value are in parentheses, and *
indicates a p value of <0.05, as determined by unpaired
Student's t test.
|
|
RGS4 does not act directly on the calcium-dependent
chloride channel in Xenopus oocytes
Figure 1 demonstrates that RGS4 directly blocks Gq
functions. These findings, together with those of the mGluR1a- and mGluR5a-mediated ICl(Ca) in oocytes, suggest
strongly that RGS4 blocks the Gq-directed formation of
Ins(1,4,5)P3 and calcium release rather than directly
blocking the endogenous oocyte chloride channel. To strengthen this
argument, we examined the capacity of RGS4 to block the
Ins(1,4,5)P3-mediated activation of the calcium-dependent chloride channel in Xenopus oocytes.
Ins(1,4,5)P3 was microinjected directly into control
oocytes or oocytes that had been microinjected with active RGS4 or
heat-inactivated RGS4. The injection of 10-15 nl of 1 mM
Ins(1,4,5)P3 into control oocytes evoked a small inward current attributable to the local release of calcium from
Ins(1,4,5)P3-sensitive intracellular stores and activation
of ICa(Cl) (Fig.
5A, left). In
oocytes injected with either active RGS4 (50 nl, 48 µM)
or heat-inactivated RGS4, subsequent microinjection of
Ins(1,4,5)P3 also elicited an inward current equal to or
greater than that observed in the absence of RGS4 (Fig. 5A,
middle and left). The summary data (Fig.
5B) show that RGS4 does not directly block chloride channel
functions in oocytes, and they support the idea that RGS4 acts upstream
of Ins(1,4,5)P3 formation, most likely by blocking
Gq function directly.
View larger version (12K):
[in this window]
[in a new window]
|
Figure 5.
Direct activation of the calcium-activated
chloride channel by inositol 1,4,5-trisphosphate is not blocked by RGS4
in Xenopus oocytes. A, Uninjected oocytes
(control, left) were two-electrode voltage-clamped at
60 mV. Ins(1,4,5)P3 (1 mM) was injected
directly into the oocytes, and the resulting calcium-activated chloride currents were measured. Oocytes that had been injected with 50 nl of
purified RGS4 (48 µM) for 30 min to 1 hr before recording (middle) also resulted in the activation of
ICl(Ca) in response to the direct injection
of Ins(1,4,5)P3. In addition, oocytes injected with 50 nl
of heat-inactivated RGS4 (right) resulted in the
activation of ICl(Ca) by
Ins(1,4,5)P3. Each trace represents one
individual oocyte. B, Bar graph depicting the direct
activation of ICl(Ca) in control,
RGS4-injected, or heat-inactivated RGS4-injected oocytes. The numbers
for each value are in parentheses, and each set of
conditions was analyzed by unpaired Student's t test
and shown not to be statistically different.
|
|
RGS4 blocks mGluR5-mediated inhibition of
IAHP in hippocampal neurons
Results in oocytes demonstrate that RGS4 can block signaling by
group I mGluRs in an experimental model system. As shown in Figure 2,
RGS4 is expressed in various regions of rat brain where it could
regulate mGluR signaling. This includes expression of RGS4 mRNA in
hippocampus, an area that is particularly enriched in mGluR5 (Abe et
al., 1992). In the hippocampus, a calcium-activated potassium current
(IAHP) that underlies a slow
afterhyperpolarization is known to be inhibited by activation of mGluRs
(Charpak et al., 1990; Desai and Conn, 1991). This modulation is
mediated by the phosphoinositide-coupled mGluRs and requires
intracellular calcium release but not protein kinase C (PKC) or protein
kinase A (PKA) (Abdul-Ghani et al., 1996). Pharmacological studies
suggest that inhibition of IAHP in hippocampal
CA1 pyramidal cells is mediated by a group I mGluR (Gereau and Conn,
1995; Manzoni and Bockaert, 1995). In addition, immunocytochemistry
experiments reveal that mGluR5 is expressed in CA1 pyramidal cells
(Romano et al., 1995; Lujan et al., 1996), whereas mGluR1a is not
expressed in these cells (Martin et al., 1992). To determine whether
RGS4 can regulate signaling by group I mGluRs in a native system, we
tested the hypothesis that RGS4 could block the mGluR5-mediated
modulation of IAHP by the group I-selective
mGluR agonist DHPG (Ito et al., 1992). IAHP was
recorded immediately after a 100 msec depolarization to 0 mV from a
holding potential of 50 mV. This protocol produces a robust and
stable IAHP, presumably by promoting
calcium influx and subsequent activation of calcium-activated potassium
channels. Application of 10 µM DHPG reduced the
IAHP (Fig.
6A,B) by ~45% of
control, on average. Introduction of active RGS4 by whole-cell perfusion (0.25, 1.0, or 2.5 µM in patch pipette)
attenuated the mGluR5-mediated reduction in IAHP
in a dose-dependent manner (Fig. 6C, filled
circles). DHPG (10 µM) applied to cells perfused
with 0.25, 1.0, or 2.5 µM RGS4 produced on average 46, 27, and 12% inhibition of IAHP,
respectively. This indicates that 2.5 and 1 µM RGS4
inhibited the mGluR5-mediated inhibition of IAHP
by 74% and 41%, respectively, whereas 0.25 µM RGS4 was
without significant effect. Heat-inactivated RGS4 was also without
effect (Fig. 6C, filled triangle), indicating
that the blockade of mGluR5-mediated IAHP
modulation was not caused by nonspecific effects of cell loading. The
concentration-dependent inhibition of the DHPG-induced inhibition of
IAHP by RGS4 occurs at protein amounts that are virtually identical to those required for RGS4 inhibition of
phospholipase C activation in in vitro reconstitution
studies (Fig. 1B,C). These results extend the
observation that RGS4 blocks signaling by group I mGluRs in oocytes to
show that RGS4 also inhibits mGluR5-mediated actions in a
concentration-dependent manner in hippocampal CA1 neurons, suggesting a
role for RGS proteins in the regulation of GPCR responses in the
CNS.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 6.
RGS4 blocks group I-mediated
IAHP inhibition in CA1 pyramidal cells.
A, AHP currents were recorded from CA1 pyramidal cells by whole-cell voltage clamp. Currents were elicited by a 100 msec step
from 50 mV to 0 mV. The truncated currents elicited by the step have
been eliminated for clarity. After 30 min of cell dialysis, application
of 10 µM DHPG produced ~50% reduction in
IAHP (left). Addition of 2.5 µM RGS4 to the patch pipette markedly reduced the effect
of DHPG (middle), whereas addition of 2.5 µM heat-inactivated RGS4 had no effect. Each
panel is an average of three traces. B,
Time course from representative cells of the effect of 2.5 µM RGS4 on DHPG-induced reduction of AHP currents.
Control = filled circles; RGS4 = open
circles. C, Dose-response curve depicting the
mean data for the effect of RGS4 on DHPG-induced inhibition of
IAHP. Filled circles indicate
the average percent inhibition of IAHP
induced by 10 µM DHPG in control cells or cells dialyzed with purified RGS4 (250 nM, 1 µM, or 2.5 µM), whereas the filled triangle
represents cells dialyzed with 2.5 µM RGS4 that has been heat-inactivated. Data are represented as mean ± SEM;
n = 4 for each set (* = p < 0.05 relative to control; unpaired Student's t
test).
|
|
| |
DISCUSSION |
mGluRs and other G-protein-coupled receptors have been shown to
play a critical role in the regulation of neuronal excitability, synaptic transmission, and various other functions of neurons and glia.
Because of the important roles of these receptors in regulation of cell
function, their activity is tightly regulated by mechanisms such as
desensitization, receptor expression levels, and the specific
localization of receptors in the cell. We now report that mGluR
signaling through Gq-mediated pathways can be regulated by
a new family of proteins that regulate G-protein signaling, the RGS
proteins. One family member, RGS4, is widely expressed in the rat brain
and is shown to inhibit phospholipase C activation in vitro
in a dose-dependent manner. When microinjected into oocytes expressing
either of the Gq-coupled mGluRs, mGluR1a or mGluR5a, RGS4
completely blocks activation of ICl(Ca) by
mGluRs. This inhibition is attributable to a block of the
Gq signaling, because RGS4 did not block activation of
ICl(Ca) by direct injection of
Ins(1,4,5)P3 into oocytes. On the basis of the calculated
volume of an oocyte, we estimate the final concentra-tion of RGS4
inside the oocyte to be ~1-5 µM. At this concentration
of RGS4, the extent of RGS4 inhibition of the
ICl(Ca) in oocytes (80-90%) correlates very well with the extent of RGS4 blockade of
Gq-activation of PLC observed in NG108-15 membranes
(Fig. 1B). It should be noted that RGS4 was
synthesized in E. coli, and although the protein is clearly
active, we cannot rule out the possibility that potential post-translational modifications on RGS4 could increase its affinity for the plasma membrane and/or target G subunits,
thereby decreasing its effective concentration in a native system. To date, however, RGS4 has not been isolated from native brain tissue for
comparative study.
In addition to blocking mGluR effects on the
Gq-regulated ICl(Ca), we
found that RGS4 also attenuates effects of group I mGluRs on the
G-regulated IGirk. RGS4 is a
GAP for both Gq (Hepler et al., 1997; Huang et al.,
1997; Yan et al., 1997) and members of the Gi family of
G subunits (Berman et al., 1996), and RGS4 blocks
receptor and Gi-directed inhibition of adenylyl cyclase
activity in cell membranes and intact cells (Hepler et al., 1997; Huang
et al., 1997; Yan et al., 1997). We propose that the inhibitory effect
of RGS4 on IGirk is attributable to deactivation
of Xenopus Gi/o, which in turn serves as a
sink for free G and promotes formation of inactive G-protein heterotrimer. Evidence to support this idea can be found in
yeast, where the prototypical RGS protein Sst2 clearly acts on
G to block the signaling functions of
G (Dohlman et al., 1996).
In addition to the oocyte expression system, RGS4 was also shown to
modulate mGluR responses in hippocampal neurons. Activation of mGluRs
has a number of physiological effects in the hippocampus (Pin and
Duvoisin, 1995; Saugstad et al., 1995a; Conn and Pin, 1997) that are
likely to play a critical role in regulating transmission through the
hippocampal circuit and to be important for both normal hippocampal
function and in certain pathological states involving the hippocampus,
such as temporal lobe epilepsy. In addition to their inhibition of
IAHP, evidence suggests that group I
mGluRs mediate the other direct excitatory effects of mGluR activation on hippocampal pyramidal cells (Charpak et al., 1990, Davies et al.,
1995; Gereau and Conn, 1995; Manzoni and Bockaert, 1995; Fitzjohn et
al., 1996). Although we focused on modulation of
IAHP in the current study, it is likely that
RGS4 would block other group I mGluR-mediated effects in CA1 pyramidal
cells, including inhibition of a leak potassium current, inhibition of
the M-current, potentiation of NMDA receptor currents, and activation
of a nonselective cation current.
Although the present findings clearly demonstrate that RGS4 is capable
of inhibiting signaling by group I mGluRs, the exact role of endogenous
RGS4 in regulation of mGluR-mediated responses and the conditions under
which endogenous RGS proteins regulate mGluR signaling are not yet
clear. If the cellular levels of RGS4 are elevated in resting neurons
or glia, then RGS4 likely serves an important role in tonic regulation
of signaling events by mGluRs and other GPCRs. In this case,
mGluR-mediated responses could be fine-tuned by regulating levels of
RGS4 or other RGS proteins under different physiological and
pathological conditions. For instance, signaling by group I mGluRs is
known to be tightly regulated during postnatal development so that
mGluR-mediated increases in phosphoinositide hydrolysis are much
greater in neonatal than in adult animals. Furthermore, a number of
manipulations can lead to long-term increases in mGluR-mediated
phosphoinositide hydrolysis in adult that are not correlated with
changes in the levels of group I mGluRs (Iversen et al., 1994; Rosdahl
et al., 1994; Akbar et al., 1996). The present findings suggest that
mGluR signaling could be regulated by altering levels of RGS4 or
another RGS protein that regulates Gq-mediated responses.
Additional studies will be needed to determine whether developmental
regulation of resting cellular levels of RGS4 participates in
developmental regulation of mGluR-mediated responses.
Another possible role of RGS proteins is in receptor-mediated
desensitization of mGluR responses. If RGS4 levels are low in resting
neurons, activation of mGluRs and other Gq-linked
neurotransmitter receptors may increase the cellular levels of RGS4 or
other RGS proteins and thereby inhibit subsequent
Gq-mediated signaling events. For instance, Desai et al.
(1996) reported recently that incubation of cells expressing mGluR1a
with an mGluR agonist results in a long-term desensitization of
mGluR1a-mediated increases in phosphoinositide hydrolysis. Ongoing
protein synthesis was required for this desensitization, and the loss
of response did not appear to be caused by downregulation of
cell-surface mGluR1a receptors. In another recent report Balazs et al.
(1997) showed that treatment of cortical astrocytes with mGluR agonists
results in a biphasic pattern of desensitization of mGluR5, which
includes a rapid desensitization phase that arises within 1-3 hr, and
a delayed phase, which peaks at 24 hr. It is tempting to speculate that
these delayed forms of desensitization of group I-mediated responses
are mediated by mGluR-induced increases in levels of RGS proteins. It
is possible that Gq-linked receptors act to regulate the
cellular levels of RGS4 and other RGS proteins to serve a role in
heterologous desensitization of neurotransmitter signaling. What RGS4
and other RGS proteins contribute to desensitization events relative to
other well studied mechanisms (e.g., phosphorylation, internalization)
remains unexplored, and clarification of these issues in future studies
will prove to be of great interest.
In the present study we chose to focus on RGS4 because it is one of
only two RGS proteins to date that have been reported to inhibit
Gq-regulated pathways. However, it is important to note
that RGS proteins comprise a large and diverse family of regulatory
proteins. All mammalian family members share a 120 amino acid core
domain that, at least in the case of RGS4, forms a bundle of nine  helices. Specific residues within this helical domain contact G
subunits and act to accelerate G-catalyzed GTP
hydrolysis and block G-effector interactions (Popov et al., 1997; Tesmer et al., 1997). Beyond this shared feature, RGS proteins differ in most other regards. They range in size from 140 to
17 kDa, share little overall amino acid sequence identity outside the
core RGS domain, and are distributed very differently across tissues
and cells (Dohlman and Thorner, 1997; Koelle, 1997). These striking
differences strongly suggest diversity of function and may enable RGS
proteins to interact with target proteins and to incorporate
post-translational modifications that confer important (and as yet
unappreciated) cellular functions independent of their established
roles as regulators of G-protein signaling at the plasma membrane.
All RGS proteins characterized to date, including RGS4, serve as
inhibitors of members of the Gi but not the Gs
or G12 family of proteins. RGS4 was the first family member
identified that also regulates Gq functions (Hepler et al.,
1997). Although poorly understood, roles for RGS4 in desensitization
necessarily are complex because it regulates both Gq- and
Gi-directed signaling pathways. We have recently found that
RGS2 is also a selective and potent inhibitor of Gq
functions (Heximer et al., 1997). Thus, of the RGS proteins
characterized to date, RGS4 and RGS2 are the most likely to be involved
in regulation of signaling by receptors coupled to Gq
pathways, such as the group I mGluRs. Interestingly, RGS4 is expressed
exclusively in brain (Druey et al., 1996), whereas RGS2 is found in
many tissues including brain (Chen et al., 1997). It is likely that
this tissue-specific expression, as well as many other factors,
contribute to RGS regulation of mGluR and GPCR signaling within an
individual neuron or glial cell. Little is known about cellular
localization of RGS proteins and how this process may be regulated.
Whether RGS4 and other RGS proteins are found in the cytosol or plasma
membrane, and whether they are localized to the soma, dendrites, or
axons of neurons is unknown. How individual neurons and glia regulate
the cellular levels and localization of RGS proteins in time and space
will necessarily determine specific roles for these regulatory proteins
in CNS signaling and which GPCR-mediated responses they impact.
| |
FOOTNOTES |
Received Nov. 14, 1997; accepted Nov. 19, 1997.
This work was supported by in part by National Institutes of Health
Grants NS28405, NS31373, and NS34876 (P.J.C.), and the American Heart
Association, Georgia Affiliate, the Pharmaceutical Researchers and
Manufacturers of America Foundation, and the American Cancer
Society/Winship Cancer Center (J.R.H.). We thank Dr. Dieter Jaeger for
helpful discussions and Hazar Awad for help with the RGS4 protein
purification.
Correspondence should be addressed to Dr. P. J. Conn, Department
of Pharmacology, Rollins Research Building, Atlanta, GA
30322.
| |
REFERENCES |
-
Abe T,
Sugihara H,
Nawa H,
Shigemoto R,
Mizuno N,
Nakanishi S
(1992)
Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction.
J Biol Chem
267:13361-13368[Abstract/Free Full Text].
-
Abdul-Ghani MA,
Valiante TA,
Carlen PL,
Pennefather PS
(1996)
Tyrosine kinase inhibitors enhance a Ca2+-activated K+ current (IAHP) and reduce IAHP suppression by a metabotropic glutamate receptor agonist in rat dentate granule neurones.
J Physiol (Lond)
496:139-144[ISI][Medline].
-
Akbar MT,
Rattray M,
Powell JF,
Meldrum BS
(1996)
Altered expression of group I metabotropic glutamate receptors in the hippocampus of amygdala-kindled rats.
Brain Res
43:105-116.
-
Akiyama K,
Yamada N,
Sato M
(1987)
Increase in ibotenate-stimulated phosphatidylinositol hydrolysis in slices of the amygdala/pyriform cortex and hippocampus of rat by amygdala kindling.
Exp Neurol
98:499-508[ISI][Medline].
-
Aronica E,
Frey U,
Wagner M,
Schroeder H,
Krug M,
Ruthrich H,
Catania MV,
Nicoletti F,
Reymann KG
(1991)
Enhanced sensitivity of metabotropic glutamate receptors after induction of long-term potentiation in rat hippocampus.
J Neurochem
57:376-383[ISI][Medline].
-
Balazs R,
Miller S,
Romano C,
de Vries A,
Chun Y,
Cotman CW
(1997)
Metabotropic glutamate receptor mGluR5 in astrocytes: pharmacological properties and agonist regulation.
J Neurochem
69:151-163[ISI][Medline].
-
Baude A,
Nusser Z,
Roberts JDB,
Mulvihill E,
McIlhinney RAJ,
Somogyi P
(1993)
The metabotropic glutamate receptor (mGluR1a) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.
Neuron
11:771-787[ISI][Medline].
-
Berman DM,
Wilkie TM,
Gilman AG
(1996)
GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein subunits.
Cell
86:445-452[ISI][Medline].
-
Blanton MG,
Turco JJL,
Kriegstein AR
(1989)
Whole cell recording from neurons in slices of mammalian cerebral cortex.
J Neurosci Methods
30:203-210[ISI][Medline].
-
Charpak S,
Gahwiler BH,
Do KQ,
Knopfel T
(1990)
Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters.
Nature
347:765-767[Medline].
-
Chen C,
Zheng B,
Han J,
Lin S-C
(1997)
Characterization of a novel mammalian RGS protein that binds to G proteins and inhibits pheromone signaling in yeast.
J Biol Chem
272:8679-8685[Abstract/Free Full Text].
-
Chen SJ,
Winder DG,
Desai MA,
Klann E,
Sweatt JD,
Conn PJ
(1992)
Amygdala kindling alters protein kinase C activity and increases levels of a 17 kDa protein kinase C substrate in the dentate gyrus.
J Neurochem
59:1761-1769[ISI][Medline].
-
Conn PJ,
Pin J-P
(1997)
Pharmacology and functions of metabotropic glutamate receptors.
Annu Rev Pharmacol Toxicol
37:205-237[ISI][Medline].
-
Dascal N,
Schreibmayer W,
Lim NF,
Wang W,
Chavkin C,
DiMagno L,
Labarca C,
Kieffer BL,
Gaveriaux-Ruff C,
Trollinger D,
Lester HA,
Davidson N
(1993)
Atrial G-protein-activated K+ channel: expression cloning and molecular properties.
Proc Natl Acad Sci USA
90:10235-10239[Abstract/Free Full Text].
-
Davies CH,
Clarke VRJ,
Jane DE,
Collingridge GL
(1995)
Pharmacology of postsynaptic metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurons.
Br J Pharmacol
116:1859-1869[ISI][Medline].
-
Desai MA,
Conn PJ
(1991)
Excitatory effects of ACPD receptor activation in the hippocampus are mediated by direct effects on pyramidal cells and blockade of synaptic inhibition.
J Neurophysiol
66:40-52[Abstract/Free Full Text].
-
Desai MA,
Burnett JP,
Mayne NG,
Schoepp DD
(1996)
Pharmacological characterization of desensitization in a human mGlu1a-expressing non-neuronal cell line cotransfected with a glutamate transporter.
Br J Pharmacol
118:1558-1564[ISI][Medline].
-
Dohlman HG,
Thorner J
(1997)
RGS proteins and signaling by heterotrimeric G proteins.
J Biol Chem
272:3871-3874[Free Full Text].
-
Dohlman HG,
Song J,
Ma D,
Courshesne WJ,
Thorner J
(1996)
Sst2: a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression localization genetic interaction and physical association with Gp1 (G protein subunit)
Mol Cell Biol
16:5194-5209[Abstract].
-
Druey KM,
Blumer KJ,
Kang VH,
Kehrl JJ
(1996)
Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family.
Nature
379:742-746[Medline].
-
Engisch KM,
Wagner JJ,
Alger BE
(1996)
Whole-cell voltage-clamp investigation of the role of PKC in muscarinic inhibition of IAHP in rat CA1 hippocampal neurons.
Hippocampus
6:183-191[ISI][Medline].
-
Fitzjohn SM,
Irving AJ,
Palmer MJ,
Harvey J,
Lodge D,
Collingridge GL
(1996)
Activation of group I mGluRs potentiates NMDA responses in rat hippocampal slices.
Neurosci Lett
203:211-213[ISI][Medline].
-
Gereau RW,
Conn PJ
(1994a)
A cyclic AMP-dependent form of associative synaptic plasticity induced by coactivation of -adrenergic receptors and metabotropic glutamate receptors in rat hippocampus.
J Neurosci
14:3310-3318[Abstract].
-
Gereau RW,
Conn PJ
(1994b)
Presynaptic enhancement of excitatory synaptic transmission by -adrenergic receptor activation.
J Neurophysiol
72:1438-1442[Abstract/Free Full Text].
-
Gereau RW,
Conn PJ
(1994c)
Potentiation of cAMP responses by metabotropic glutamate receptors depresses excitatory synaptic transmission by a kinase-independent mechanism.
Neuron
12:1121-1129[ISI][Medline].
-
Gereau RW,
Conn PJ
(1995)
Roles of specific metabotropic glutamate receptor subtypes in hippocampal CA1 pyramidal cell excitability.
J Neurophysiol
74:122-129[Abstract/Free Full Text].
-
Gutowski S,
Smrcka AV,
Nowak L,
Wu D,
Simon MI,
Sternweis PC
(1991)
Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones.
J Biol Chem
266:20519-20524[Abstract/Free Full Text].
-
Hepler JR,
Gilman AG
(1992)
G proteins.
Trends Biochem Sci
17:383-387[ISI][Medline].
-
Hepler JR,
Kozasa T,
Smrcka AV,
Simon MI,
Rhee SG,
Sternweis PC,
Gilman AG
(1993)
Purification from Sf9 cells and characterization of recombinant Gqand G11.
J Biol Chem
268:14367-14375[Abstract/Free Full Text].
-
Hepler JR,
Biddlecome GH,
Kleuss C,
Camp LA,
Hofmann SL,
Ross EM,
Gilman AG
(1996)
Functional importance of the amino terminus of Gq.
J Biol Chem
271:496-504[Abstract/Free Full Text].
-
Hepler JR,
Berman DM,
Gilman AG,
Kozasa T
(1997)
RGS4 and GAIP are GTPase-activating proteins for Gq and block activation of phospholipase C by -thio-GTP-Gq.
Proc Natl Acad Sci USA
94:428-432[Abstract/Free Full Text].
-
Heximer SP,
Watson N,
Linder ME,
Blumer KJ,
Hepler JR
(1997)
RGS2/GOS8 is a selective inhibitor of Gq function.
Proc Natl Acad Sci USA
94:14389-14393[Abstract/Free Full Text].
-
Hollmann M,
Heinemann S
(1994)
Cloned glutamate receptors.
Annu Rev Neurosci
17:31-108[ISI][Medline].
-
Houamed KM,
Kuijper JL,
Gilbert TL,
Haldeman BA,
O'Hara PJ,
Mulvihill ER,
Almers W,
Hagen FS
(1991)
Cloning expression gene structure of a G protein-coupled glutamate receptor from rat brain.
Science
252:1318-1321[Abstract/Free Full Text].
-
Huang CF,
Hepler JR,
Gilman AG,
Mumby SM
(1997)
Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells.
Proc Natl Acad Sci USA
94:6159-6163[Abstract/Free Full Text].
-
Hunt TW,
Fields TA,
Casey PJ,
Peralta EG
(1996)
RGS10 is a selective activator of Gi GTPase activity.
Nature
383:175-177[Medline].
-
Ito I,
Kohda A,
Tanabe S,
Hirose E,
Hayashi E,
Mitsunaga S,
Sugiyama H
(1992)
3,5-dihydroxyphenylglycine: a potent agonist of metabotropic glutamate receptors.
NeuroReport
3:1013-1016[ISI][Medline].
-
Iversen L,
Mulvihill E,
Haldeman B,
Diemer NH,
Kaiser F,
Sheardown M,
Kristensen P
(1994)
Changes in metabotropic glutamate receptor mRNA levels following global ischemia: increase of a putative presynaptic subtype (mGluR4) in highly vulnerable rat brain areas.
J Neurochem
63:625-633[ISI][Medline].
-
Koelle MR
(1997)
A new family of G-protein regulators: the RGS proteins.
Curr Opin Cell Biol
9:143-147[ISI][Medline].
-
Koelle MR,
Horvitz HR
(1996)
EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins.
Cell
84:115-125[ISI][Medline].
-
Krapivinsky G,
Gordon EA,
Wickman K,
Velimirovic B,
Krapivinsky L,
Clapham DE
(1995)
The G-protein-gated atrial K+-channel IkAch is a heteromultimer of two inwardly rectifying K+-channel proteins.
Nature
374:135-141[Medline].
-
Kubo Y,
Reuveny E,
Slesinger PA,
Jan YN,
Jan LY
(1993)
Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel.
Nature
364:802-806[Medline].
-
Lujan R,
Nusser Z,
Roberts JDB,
Shigemoto R,
Somogyi P
(1996)
Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus.
Eur J Neurosci
8:1488-1500[ISI][Medline].
-
Manzoni O,
Bockaert J
(1995)
Metabotropic glutamate receptors inhibiting excitatory synapses in the CA1 area of rat hippocampus.
Eur J Neurosci
7:2518-2523[ISI]
|
Top
Abstract
Introduction
