RGS7 and RGS8 Differentially Accelerate G Protein-mediated
Modulation of K+ Currents*
Osamu
Saitoh
§,
Yoshihiro
Kubo¶,
Megumi
Odagiri
,
Masumi
Ichikawa
,
Kanato
Yamagata**, and
Toshiaki
Sekine
From the
Department of Molecular and Cellular
Neurobiology, the ¶ Department of Neurophysiology, the
Department of Anatomy and Embryology, and the ** Department of
Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience,
2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan
 |
ABSTRACT |
The recently discovered family of RGS (regulators
of G protein signaling) proteins acts as GTPase activating proteins
which bind to
subunits of heterotrimeric G proteins. We previously showed that a brain-specific RGS, RGS8 speeds up the activation and
deactivation kinetics of the G protein-coupled inward rectifier K+ channel (GIRK) upon receptor stimulation (Saitoh,
O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997)
Nature 390, 525-529). Here we report the isolation of a
full-length rat cDNA of another brain-specific RGS, RGS7. In
situ hybridization study revealed that RGS7 mRNA is
predominantly expressed in Golgi cells within granule cell layer of
cerebellar cortex. We observed that RGS7 recombinant protein binds
preferentially to G
o, G
i3, and
G
z. When co-expressed with GIRK1/2 in
Xenopus oocytes, RGS7 and RGS8 differentially accelerate G
protein-mediated modulation of GIRK. RGS7 clearly accelerated
activation of GIRK current similarly with RGS8 but the acceleration
effect of deactivation was significantly weaker than that of RGS8.
These acceleration properties of RGS proteins may play important roles
in the rapid regulation of neuronal excitability and the cellular
responses to short-lived stimulations.
 |
INTRODUCTION |
Numerous extracellular signals such as hormones,
neurotransmitters, and odors stimulate seven transmembrane-spanning
receptors that activate heterotrimeric G proteins. These G proteins
function as signal transducing molecules by regulating cellular
effectors including enzymes and ion channels (1, 2). The regulatory mechanisms that control G protein signaling have not been fully studied. Recently, a new family of regulators of G protein signaling (RGS)1 was identified in
organisms ranging from yeast to mammals (3, 4). Genetic screenings for
negative regulators for pheromone response pathway in yeast identified
a protein, Sst2 (5). By genetic and biochemical analyses, Sst2 was
revealed to interact directly with G protein
subunit (6). In the
last few years, full or partial sequences of 22 RGS proteins have
been identified in mammals. All of them share a conserved RGS domain of
~120 amino acids (7-15). It has been shown that several RGS proteins
(RGS1, RGS3, RGS4, GAIP) attenuate G protein signaling in cultures (9, 16, 17). Biochemical studies have demonstrated that some RGS members
(RGS1, RGS4, RGS10, GAIP, RGSr/RGS16, RET-RGS1) function as
GTPase-activating proteins (GAPs) for the Gi family of
subunit, including G
o, G
i, and transducin (10, 14,
18-20). Hence, these characterized RGS proteins are proposed to
down-regulate G protein signaling in vivo by enhancing the
rate of G
GTP hydrolysis. However, whether other RGS proteins
regulate G protein signalings in a similar manner remains to be established.
Because there are many G protein signaling pathways which regulate
important functions such as neural transmission in the brain, it is
possible that certain RGS proteins might determine a mode of G protein
signaling that control neural functions. We searched RGS proteins
specifically expressed in neural cells using neuronally differentiating
P19 cells in culture. RGS8 was induced in neuronally differentiated P19
cells. Biochemical studies indicated that RGS8 functions as a GAP for
G
o and G
i3.To examine effects of RGS8 on
G protein signaling, we co-expressed a G protein-coupled receptor and a
G protein-coupled inwardly rectifying K+ channel (GIRK1/2)
(21-23) in Xenopus oocytes and analyzed turning on and off
upon agonist application. We found that RGS8 significantly accelerates
both turning on and off (24). Do other neural-tissue-specific RGS
proteins function in a similar manner ? RGS4 and RGS7 have been
reported to be expressed predominantly in the brain (8, 9). Doupnik
et al. showed similar electrophysiological results on RGS1,
3, and 4 as RGS8 (25). As only partial sequence with incomplete 5' end
of RGS7 cDNA has been known (8), we isolated a cDNA clone
encoding full-length RGS7 for functional analysis. We determined
distribution of RGS7 mRNA and binding character of the core domain of
RGS7 and analyzed effects of RGS7 on turning on and turning off
kinetics of GIRK current.
 |
EXPERIMENTAL PROCEDURES |
Cloning of RGS7 cDNA--
Oligonucleotide primers
(5'-TTGGCAGTGGAGGACCTGAAGAAAAG-3', 5'-CAGCTTGTAAATGTGCTCCTGAGCAT-3')
were synthesized based on the reported sequence of rat RGS7 (8). Total
RNA was isolated from rat brain, and the first strand cDNA was
synthesized as a template of PCR. The amplified 200-base pair DNA was
cloned into pGEM-T vector (Promega), and its sequence was determined.
This PCR-amplified fragment of RGS7 was used to screen rat hippocampus
cDNA library. The longest cDNA clone was sequenced on both strands.
Northern Blot--
Northern blot analysis was performed as
described previously (26). Digoxigenin-labeled cRNAs, which had been
synthesized using RGS7 and RGS8 cDNAs, were utilized as a probe. To
detect mRNA of neurofilament protein 140K and
glyceraldehyde-3-phosphate dehydrogenase, digoxigenin-labeled DNA
probe was also prepared by random priming method.
In Situ Hybridization--
In situ hybridization was performed
as described (27) with the following modifications. Sagittal frozen
sections of rat brain were treated with 5 µg/ml proteinase K for 5 min at 37 °C. To remove excess cRNA probes after hybridization,
RNase A treatment (20 µg/ml, 30 min, 37 °C) was carried out.
Expression and Purification of His-tagged RGS7 Core
Domain--
Recombinant protein of RGS core domain of RGS7 was
expressed with hexahistidine tag at the N terminus in Escherichia
coli. Bacterial expression plasmid of His-tagged RGS7 was
constructed as follows. The coding region corresponding to amino acids
303-470 of RGS7, which includes RGS core domain, was PCR-amplified
using RGS7 cDNA as a template. Nucleotide sequence of the amplified DNA was confirmed by sequencing both strands. The confirmed DNA fragment was cloned into pQE30 (Qiagen). The resultant plasmid was
transformed into E. coli, M15. His-tagged RGS7 core domain, which was induced by IPTG treatment (5 mM, 1 h), was
purified using Ni2+-NTA-agarose as described previously
(24).
Binding Assay of RGS7--
Binding assay between RGS protein and
G protein was carried out as previously described (24). His-tagged RGS
protein (10 µg) and rat brain membrane fractions (0.5 mg) were
incubated for 30 min at 5 °C in 20 mM HEPES, pH 8.0, 0.38 M NaCl, 3 mM dithiothreitol, 6 mM MgCl2, 10 µM GDP, 30 µM AlF4
(30 µM
AlCl3, 10 mM NaF), 40 mM imidazole.
After solubilization with 1% cholate for 1 h at 4 °C and
centrifugation at 30,000 rpm for 20 min, Ni2+-NTA-agarose
beads were added to detergent-soluble extract, incubated for 30 min at
4 °C, and washed five times with 20 mM HEPES, pH 8.0, 3 mM dithiothreitol, 0.1% polyoxyethylene 10-lauryl ether (C12E10), 1 µM GDP, 30 µM
AlF4
, and 40 mM imidazole.
Complexes containing His-tagged RGS protein were eluted from
Ni2+-NTA-agarose with SDS sample buffer and examined by
SDS-polyacrylamide gel electrophoresis. The identification of proteins
bound to RGS protein was performed by immunoblotting with antibodies to
G
i1, and G
i2, G
i3,
(Calbiochem-Novabiochem), G
o, G
z, and
G
q/11 (Santa Cruz Biotechnology). Signals were detected
with ECL system (Amersham Pharmacia Biotech).
Two Electrode Voltage Clamp--
Two electrode voltage clamp
analysis was carried out as described previously (24).
 |
RESULTS AND DISCUSSION |
Isolation of Rat RGS7 cDNA--
The sequence of RGS domain of
rat RGS7 was previously reported (8), and the corresponding DNA
fragment was obtained by PCR amplification using rat brain cDNA as
a template. This PCR-amplified fragment was utilized to screen rat
hippocampus cDNA library. The nucleotide sequence of the isolated
longest clone was determined. Rat RGS7 cDNA of 2243 base pairs
encoded a protein of 477 amino acids. While performing this work,
isolation of bovine and mouse RGS7 was reported (28). Comparison of
amino acid sequence revealed that our rat RGS7 was 94.5% identical to
mouse RGS7 (Fig. 1). The conserved region
among RGS proteins, the RGS domain, was present near C-terminal tail
(amino acids 330-447). As reported for bovine RGS7 (28), a domain of
~190 amino acids near the N terminus showed a considerable sequence
identity to bovine RGS9 and Caenorhabditis elegans EGL10
(8).

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Fig. 1.
Rat RGS7 Sequence. Predicted amino acid
sequence of RGS7 derived from the rat RGS7 cDNA sequence was
aligned with that of mouse RGS7. Amino acids identical in both
sequences are boxed. The amino acid sequences
underlined correspond to RGS domain.
|
|
RGS7 Expression Is Developmentally and Regionally
Regulated--
Expression levels of rat RGS7 mRNA in various
tissues of the brain were examined by Northern hybridization, and
brain-specific expression of RGS7 was confirmed (Fig.
2A). Whole brains were isolated from developing rats, and expression patterns of RGS7 and RGS8
mRNAs were compared. RGS7 expression was detected in 17-day
embryos, increased gradually in later embryos, and peaked in 13-day
neonates. In the case of RGS8, expression was detectable even in 13-day
embryos and increased to adults (Fig. 2B). Brain stem,
cerebral cortex, and cerebellum were dissected, and levels of mRNA
expression of three RGS proteins, which are predominant in brains
(RGS4, RGS7, and RGS8) were examined (Fig. 2C). Both mRNAs of RGS7 and RGS8 were most abundantly expressed in
cerebellum, although RGS4 mRNA was abundant in cerebral cortex.

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Fig. 2.
Northern blot analysis of expression of RGS7
and RGS8 mRNAs. A, total RNA was isolated from
various rat tissues: brain (1), heart (2), lung
(3), stomach (4), spleen (5), liver
(6), kidney (7), testis (8), and back
muscle (M. Latissimus dorsi, 9). 20 µg of isolated RNA was
electrophoresed, transferred, and then hybridized with full-length rat
RGS7 cRNA, full-length rat RGS8 cRNA, or glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA. Arrowheads
indicate the positions of 28S rRNA. B, total RNA (10 µg)
was isolated from developing brains; heads of 13-day embryos
(1), brains of 14-day, 15-day, 17-day, 19-day, 21-day
embryos (2-6), 6-day, 13-day neonates (7 and
8), and adults (9). Expression levels of RGS7,
RGS8, and neurofilament protein 140K (NF140K) mRNAs were
examined by Northern hybridization. Arrowheads indicate the
positions of 28S rRNA. C, brain stem (1),
cerebral cortex (2), and cerebellum (3) were
dissected from adult brains, and total RNA (10 µg) was isolated.
Expression levels of RGS7, RGS8, and RGS4 mRNAs were examined by
Northern hybridization. Arrowheads indicate the positions of
28S rRNA.
|
|
We next examined the cellular distribution of transcripts of RGS7 and
RGS8 by in situ hybridization using nonradioactive probes. RGS7 mRNA was expressed in cerebral cortex, especially in layers 2 and 3. RGS7 was also detected in hippocampus and cerebellum (data not
shown, Fig. 3A). Similar
distribution patterns of RGS7 were previously reported with
35S-labeled probes (29, 30). In addition, we observed that
RGS7 mRNA was most strongly expressed in medium-sized cells in the granule cell layer of cerebellar cortex. These cells were larger in
size than the granule cells and were identified as Golgi cells (Fig.
3A). The probe for RGS8 mRNA densely labeled Purkinje
cells of cerebellum (Fig. 3B). Thus, it is clearly
demonstrated that RGS7 and RGS8 mRNAs are expressed in distinct
inhibitory neurons in cerebellum.

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Fig. 3.
Localization of RGS7 mRNA to Golgi cells
in cerebellar cortex. In situ hybridization of sagittal
sections of rat cerebellar cortex using RGS7 antisense (A),
RGS8 antisense (B), or RGS7 sense (C) riboprobes
labeled with digoxigenin.
|
|
RGS7 Binds G
o, G
i3, and
G
z--
By biochemical in vitro binding
method, we examined the specificity of RGS7. Popov et al.
demonstrated functional similarity between the full-length RGS4 protein
and its RGS domain in regard to affinities toward activated G
,
catalytic activities, and acceleration values. Moreover, they also
showed that specificity of the RGS domains of RGS4, GAIP, and RGS10 for
G
subunits are similar to the reported specificity of full-length
RGS proteins (31). Thus, RGS domain is generally considered to be
sufficient to determine the basic properties of RGS proteins. We
prepared recombinant protein corresponding to amino acids 303-470,
covering the RGS domain of RGS7, using E. coli as a
hexahistidine-tagged protein. This RGS7 core domain (RGS domain)
protein (RGS7d) was purified and incubated with brain membranes treated
with GDP and AlF4
. Complexes containing RGS7d
were precipitated with Ni2+-NTA-agarose and were analyzed
by SDS-polyacrylamide gel electrophoresis. After Coomassie Blue
staining, we observed that a 40-kDa protein recovered with RGS7d (Fig.
4A). To determine which G
is recognized with RGS7, we performed immunoblotting using antisera
specific for different subtypes of G
subunit. When proteins
recovered with RGS7d were examined, we found that G
i3
and G
z were also recovered with RGS7d in addition to
G
o (Fig. 4B). Thus, it was clarified that
RGS7 recognizes G
o, G
i3, and
G
z. We previously demonstrated that RGS8 binds only
G
o and G
i3 (24). G
z is a
member of Gi family of
subunit, and interaction with
G
z has been reported for GAIP, RGS4, and RGS10 (20, 31,
32). Here we observed that RGS7 also binds G
z,
indicating that RGS7 and RGS8 have different selectivity of G
bindings in brain membranes where various types of G
exist.

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Fig. 4.
Interaction of RGS7 with G
subunit. His-tagged protein of RGS core domain of RGS7
(RGS7d) was incubated with rat brain membranes treated with GDP and
AlF4 . After extraction with detergent,
complexes containing His-tagged RGS7d were purified by
Ni2+-NTA-agarose. A, proteins bound to 10 µg
of RGS7d were resolved by SDS-PAGE and detected by staining with
Coomassie Brilliant Blue. 1, recovered His-tagged RGS7d from
buffer alone; 2, proteins recovered in the absence of RGS7d;
3, proteins recovered in the presence of His-tagged RGS7d.
40-kDa protein recovered with His-tagged RGS8 is indicated by
arrow. B, identification of G subunit bound to
RGS7d. Rat brain membrane proteins (upper) and proteins
bound to 40 µg of His-tagged RGS7d (lower) were analyzed
by immunoblotting with antibodies to G i1 and
G i2 (1), G i3 (2),
G o (3), G z (4), and
G q/11 (5).
|
|
It was reported that RGS7 inhibited G
q-coupled calcium
mobilization (30). We, however, could not detect G
q by
immunoblotting of proteins precipitated with the RGS7d. It is possible
that RGS7 interacts with G
q weakly but that their
binding affinity is not strong enough for in vitro
co-precipitation. Because an apparent and intense band was visualized
by immunoblotting of brain membranes with G
q antibody
(Fig. 4B, top panel), the possibility that the G
q present in the used membranes was degradated was excluded.
RGS7 and RGS8 Differentially Accelerate G Protein-mediated
Modulation of K+ Currents--
GIRK are known to be
activated directly by G
subunits released from pertussis
toxin-sensitive G proteins of Gi family including Gi and Go (33, 34).
They are activated by various G protein-coupled receptors such as m2
muscarinic and D2 dopamine receptors. The expression patterns of GIRK
family (GIRK1
GIRK4) mRNAs in the brain are known in detail. It
was shown that the brain type GIRKs (GIRK1 and GIRK2) are widely
distributed and expressed in hippocampus and cerebral cortex (35).
Thus, expression of GIRK1 and GIRK2 partly overlaps with RGS7
expression. We coexpressed GIRK1/GIRK2 heteromultimer and m2 muscarinic
receptor with or without RGS protein in Xenopus oocytes and
analyzed the speed of turning on and off upon agonist application under
two-electrode voltage clamp (Fig.
5A). As previously reported,
coexpression of RGS8 obviously accelerated the speed of both turning on
and off. RGS7 coexpression accelerated the turning on process similarly
with RGS8. The effect of RGS7 on off-acceleration, however, was much
weaker than that of RGS8. This observation was statistically confirmed
by comparing the time constants for the fitted single exponential
function of the activation and deactivation phases,
on
and
off (Fig. 5, B and C).

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Fig. 5.
Effects of RGS7 and RGS8 on turning on and
turning off kinetics and on the dose-response relationships of GIRK
current upon stimulation of m2 muscarinic receptor. A,
GIRK1/2 and m2 muscarinic receptor without (upper trace) or
with RGS7 (middle trace) or with RGS8 (lower
trace) were co-expressed in Xenopus oocytes. Current
traces at a holding potential of 80 mV are shown. 10 µM
ACh was applied at the times indicated by the bars.
B and C, comparison of the time constants
on (B) and off (C)
of the GIRK1/2 current upon stimulation of m2 muscarinic receptor, in
the absence or presence of RGS7 or RGS8. From the traces in panel
A, the increasing and decreasing phases were fitted by a single
exponential function and on and off were
obtained. The mean and standard deviation of on
(n = 12) were as follows: RGS( ), 3971 ± 865 ms;
RGS7(+), 1834 ± 418 ms; and RGS8(+), 1544 ± 395 ms. Those
of off (n = 12) were as follows:
RGS( ), 15126 ± 2431 ms; RGS7(+), 10322 ± 992 ms; and
RGS8(+), 4827 ± 1299 ms. Both on and
off of RGS7 and RGS8 were significantly different from
control (RGS( )) values (p value < 0.05) by
Student's unpaired t-test. off of RGS7 and
RGS8 were also significantly different (p value < 0.05) from each other. D, comparison of dose-response
relationships upon stimulation of the m2 receptor in the absence or
presence of RGS7 or 8. Responses to all doses were tested in a single
oocyte and normalized. The average and S.D. of seven oocytes were
plotted. Kd values and Hill coefficients for fitting
were 0.19 µM, 0.85 for RGS( ); 0.12 µM,
0.70 for RGS7(+); and 0.23 µM, 0.82 for RGS8(+).
|
|
We previously reported that RGS8 has a function to increase the on rate
besides the GAP activity and that the on-acceleration cannot be
explained by the GAP activity itself. Chuang et al. (36)
also showed that RGS4 has both a negative regulator function as a GAP
and a positive regulator function to increase the available G protein
pool. They discussed various possible mechanisms of the latter,
including GAP activity itself. Our observation that on acceleration and
off acceleration are not parallel modulated by RGS7 and RGS8 supports
that on and off acceleration is regulated by distinct mechanisms. The
biochemical basis of on acceleration remains to be elucidated.
Chuang et al. (36) concluded that the acute desensitization
of the response is because of the nucleotide exchange and hydrolysis cycle of G proteins. We also observed that the desensitization speed is
accelerated by RGS proteins similarly with the off acceleration, i.e. the desensitization with RGS8 was faster than that with RGS7.
What is the structural basis for the weaker off acceleration effect of
RGS7? It is most likely that lower GAP activity might contribute to
weak acceleration of off process. However, recombinant protein of RGS7
core domain has been shown to act as a strong GAP for G
i
subfamily proteins in vitro (30), and its high activity does
not seem to be significantly different from that of RGS8, which we
previously reported (24). Another possibility is that domain(s) other
than RGS domain, which for example determine the subcellular
localization of RGS protein, could affect the function. A
characteristic structural feature of RGS7 among RGS proteins is its
long N terminus. Koelle and Horvitz (8) demonstrated that the protein
of EGL10, which is a C. elegans homologue of RGS7 is present
in processes of neurons and in dense body/sarcoplasmic reticulum-like
structures within body wall muscle cells. They demonstrated that
N-terminal region of EGL10 functions to localize the protein at least
within muscle cells using transgenic nematodes. It is possible that
this N-terminal domain of RGS7 may function as a regulatory element of
off acceleration of G protein signaling by changing subcellular
localization of RGS7.
The effect of RGS protein on the dose-response (peak current)
relationships upon receptor stimulation was analyzed (Fig.
5D). As described previously, RGS8 has almost no effects on
the dose-response relationships (24). RGS7 shifted the relationship
curve to lower dose only slightly. Thus, RGS 8 and RGS7 differentially
accelerate the time course of G protein-mediated modulation of GIRK
without significantly influencing the dose-response relationship of the induced currents. These different abilities of RGS proteins to speed up
G protein-signalings may be physiologically important. On acceleration
is thought to be useful for cellular responses to short-lived signals
such as neurotransmitter release in the brains. As Doupnik et
al. (25) described, without RGS proteins, brief pulse application
of agonist leads to only a small amplitude of the GIRK current. On the
other hand, with RGS proteins, brief agonist stimulation can still
cause full activation of GIRK by on acceleration. Thus, with either
RGS8 or RGS7, GIRK can be fully activated even by short application of
agonist. With RGS8, however, because of highly accelerated
deactivation, the integrated K+ current is less than that
with RGS7, which only weakly speeds up the deactivation. The strong
effect of RGS7 on on acceleration and the weak effect on off
acceleration are thought to enhance the integrated response caused by
short agonist application very efficiently.
 |
ACKNOWLEDGEMENTS |
We are grateful to Prof. M. Lazdunski for
GIRK2 cDNA and to Dr. A. Connolly for m2 muscarinic receptor
cDNA. We thank Dr. H. Nakata for helpful discussion and K. Nakata
for preparing sections for in situ hybridization.
 |
FOOTNOTES |
*
This work was supported by research grants from the Ministry
of Education, Science, Sports and Culture of Japan (to O. S. and to
Y. K.) and from the Naito Foundation (to O. S.) and by support from
the Core of Research for Evolutional Science and Technology of the
Japan Science and Technology Corporation (to Y. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence of rat RGS7 reported in this paper has
been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB024398.
§
To whom correspondence should be addressed. Tel.: +81-423-25-3881,
ext. 4058; Fax: +81-423-21-8678; E-mail: osaito{at}tmin.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
RGS, regulators of G
protein signaling;
G proteins, heterotrimeric guanine
nucleotide-binding proteins;
GAP, GTPase-activating protein;
PCR, polymerase chain reaction;
IPTG, isopropyl-
-D-thiogalactoside;
RGS7d, RGS7 core domain
protein;
NTA, nitrilotriacetic acid.
 |
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