From the Department of Molecular and Cellular
Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan, the ¶ Hamamatsu
University School of Medicine, Hamamatsu, Aichi 431-3192, Japan, the
Department of Health Science, Jichi Medical School and CREST,
3311-1 Yakushiji Minamikawachi, Tochigi 329-0498, Japan, the
** Department of Biochemistry, Institute for Developmental Research,
Aichi Human Service Center, Kasugai, Aichi 480-03, Japan, the
Department of Neurophysiology, Tokyo
Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi,
Tokyo 183-8526, Japan, and the §§ Department of
Physiology, Tokyo Medical and Dental University, Graduate School and
Faculty of Medicine, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Received for publication, August 1, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Functional roles of the
NH2-terminal region of RGS (regulators of G protein
signaling) 8 in G protein signaling were studied. The deletion of the
NH2-terminal region of RGS8 ( RGS1 (regulators of G
protein signaling) proteins comprise a large family of more than 20 members that modulate heterotrimeric G protein signaling (1, 2). This
protein family was originally identified as a pheromone desensitization
factor in yeast (3). Many members of RGS protein family were
subsequently identified by virtue of a common stretch of 120 amino
acids termed the RGS domain in organisms ranging from yeast to human
(1, 2, 4, 5). It was shown that several RGS proteins (RGS1, RGS3, RGS4, and GAIP) attenuate G protein signaling in cultures (4, 6, 7).
Biochemical studies demonstrated that RGS members function as a
GTPase-activating protein for We previously searched for RGS proteins specifically expressed in
neural cells using a culture system of neuronally differentiating P19
cells. We isolated cDNA of RGS8 and identified it as a RGS protein
induced in differentiated P19 cells (11). In addition, since RGS7 had
been reported to be expressed predominantly in the brain (5), we also
isolated a full-length cDNA of RGS7 (12). Biochemical studies
indicated that RGS8 binds to G Yeast Pheromone Response Assay--
A bioassay was used to
measure the sensitivity of the pheromone response in yeast that
expresses RGS proteins as described (20). A DNA fragment containing the
Myc tag (MEQKLISEEDLSRGS) was introduced into pTS210 yeast expression
vector under the control of a galactose-inducible promoter. By
polymerase chain reaction amplification, cDNA fragments containing
the coding sequence of RGS8 or Western Blotting of Epitope-tagged RGS Proteins--
Single
colonies of yeasts transformed with Myc-tagged RGS constructs were
inoculated into ura Immunoprecipitation--
By polymerase chain reaction
amplification, cDNA fragments for RGS8 and Cell Fractionation--
A Syrian hamster leiomyosarcoma cell
line, DDT1MF2, was grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. RGS8 and Chimeric cDNAs of RGS8 and Fluorescent Protein--
We
expressed RGS8 and Two-electrode Voltage Clamp--
Two-electrode voltage clamp
analysis was done as described previously (11). The curve fittings of
turning on and off phases were done by a fitting function of pClamp
software (Axon) based on the Simplex method. The turning on phases were
fitted with a single-exponential function. As the turning off phases of
RGS8 and The NH2-terminal Domain of RGS8 Is Required for Its
Full Ability to Function in Yeasts--
In the
NH2-terminal region of RGS4 and RGS16 outside of the RGS
domains, the sequence conservation was found, and it was shown that the
deletion of this region reduced the effect to attenuate pheromone
signaling in yeasts (17, 18). Sequence comparison revealed that the
NH2 terminus of RGS8 is similar to the conserved NH2-terminal sequences found in RGS4, RGS16, and also RGS5
(Fig. 1). However, the conserved cysteine
residues (Cys-2, Cys-12) which were reported to be palmitoylated (18)
are absent in the NH2 terminus of RGS8. To define the role
of the NH2-terminal domain of RGS8, we analyzed the ability
of RGS8 mutant lacking the NH2-terminal 35 amino acids
( NH2-terminal Deletion Shifts the
Distribution of RGS8 from the Particulate to Cytosolic
Fraction--
How can the NH2-terminal domain modulate the
function of RGS8 without changing the properties of interaction with
G NH2-terminal Domain Is Required for the Nuclear
Localization and the G Protein-activated Translocation of RGS8--
To
further investigate the cellular distribution of RGS8 in cultured cells
and also to examine roles of its NH2 terminus, two
different fluorescent proteins were fused to RGS8 and
RGS proteins interact directly with G RGS8-induced Desensitization of the Receptor-activated GIRK Current
Was Abolished by Deletion of Its NH2-terminal
Domain--
GIRK channels are known to be activated directly by
G In this study, we demonstrated the functional significance of the
NH2-terminal domain of RGS8. First, we compared this region of RGS8 with those of RGS4, RGS5, and RGS16 and identified a conserved sequence in the NH2 terminus. By using a yeast halo assay,
we showed that the deletion of the NH2-terminal domain of
RGS8 results in a partial loss of function to inhibit the pheromone
response pathway. We used pTS210, a single-copy expression vector under the control of a galactose-inducible promoter. When a higher
concentration of galactose or multicopy vector was used, we could not
detect a clear difference in the sensitivity to the mating pheromone of
yeasts carrying RGS8 and We unexpectedly found that RGS8 is predominantly localized in the
nuclei of DDT1MF2 cells. This nuclear localization of RGS8 was shown by
its fusion protein with a fluorescent protein (GFP or RFP) and further
confirmed immunocytochemically with anti-RGS8 antibody. Similar nuclear
localization of RGS8 was observed in 3T3 and HEK293 cells transfected
with RGS8 cDNA. We, moreover, showed that the
NH2-terminal domain of RGS8 is required for its nuclear
localization within cells. In the case of RGS4, despite having a
similar conserved sequence in the NH2 terminus (Fig. 1), a
predominant localization in the cytoplasm has been reported using
HEK293 cells transfected with RGS4 cDNA (24). Considering the
subcellular distributions of RGS8 and RGS4, it is possible that the
first 7 amino acids in the NH2 terminus of RGS8 are
responsible for the unique nuclear localization. Indeed, we found a
potential NLS sequence at amino acids 7-11. Quite recently, a
truncated isoform of RGS3, termed RGS3T, has been reported to be
localized to the nucleus and induce apoptosis in RGS3T-transfected
Chinese hamster ovary cells (25). Two potential nuclear localization signal sequences were found in the NH2 terminus of RGS3T
and truncation of the NH2 terminus resulted in a reduction
of nuclear localization. RGS8 in nucleus might also function in
cellular processes such as apoptosis.
We showed that RGS8 is translocated from the nucleus to the plasma
membrane structures by coexpression of G When coexpressed in Xenopus oocytes with a G protein-coupled
receptor and GIRK1/2, What is the mechanism behind the desensitization in the presence of
RGS8? If we assume that there are only two forms of G protein
(nonactive and active) and two rate constants (NRGS8) resulted in a
partial loss of the inhibitory function in pheromone response of
yeasts, although G
binding was not affected. To examine roles in
subcellular distribution, we coexpressed two fusion proteins of
RGS8-RFP and
NRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active G
o resulted in translocation of
RGS8 protein to the plasma membrane. In contrast,
NRGS8-GFP was
distributed diffusely through the cytoplasm in the presence or absence
of active G
o. When coexpressed with G protein-gated
inwardly rectifying K+ channels,
NRGS8
accelerated both turning on and off similar to RGS8. Acute
desensitization of G protein-gated inwardly rectifying K+
current observed in the presence of RGS8, however, was not induced by
NRGS8. Thus, we, for the first time, showed that the NH2
terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of heterotrimeric G
proteins (8, 9, 10). Therefore, RGS proteins are proposed to
down-regulate G protein signaling in vivo by enhancing the rate of G
GTP hydrolysis.
o and G
i3,
and that RGS7 binds to G
o, G
i3, and
G
z. To examine effects of each RGS protein on G protein
signaling, we coexpressed a G protein-coupled receptor and a G
protein-coupled inwardly rectifying K+ channel (GIRK1/2)
(13-15) in Xenopus oocytes and analyzed the activation and
deactivation kinetics. We observed that RGS8 significantly speeds up
both activation and deactivation of GIRK current (11). Doupnik et
al. (16) reported the similar accelerated kinetics of GIRK current
by RGS1, RGS3, or RGS4. We further observed that RGS8 induces acute
desensitization of receptor-activated GIRK current in the presence of
ligands (11). In the case of RGS7, activation of GIRK current was
clearly accelerated as with RGS8, but the acceleration effect on
deactivation was significantly weaker than that of RGS8. The acute
desensitization of receptor-activated GIRK observed with RGS8 was not
apparent with RGS7. Thus, RGS7 and RGS8 were shown to accelerate G
protein-mediated modulation of GIRK current differentially (12). What
is the structural basis for the weaker off acceleration and reduced
desensitization in the case of RGS7? One possibility is that a
difference in the NH2-terminal domain contributes to the
differential modulation of GIRK current. The conserved
NH2-terminal domains of RGS4 and RGS16 were recently shown
to be important for membrane association and biological function by the
analysis of the ability to inhibit pheromone signaling in the budding
yeast (17-19). A homologous domain was also found in the
NH2 terminus of RGS8. Therefore, we investigated functional
roles of the NH2-terminal domain of RGS8 in G protein
signalings. We examined effects of deletion of this
NH2-terminal domain on the pheromone signaling of yeasts, the G
binding, the subcellular distribution, and the modulation of
the receptor-activated GIRK current.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NRGS8 (mutant RGS8 which lacks
NH2-terminal 35 amino acids) were isolated. After
confirmation by sequencing analysis, they were fused in-frame
immediately downstream of the Myc tag in pTS210. The sst2 deletion
mutant yeast SNY86 (21) was transformed with each cDNA in pTS210
and selected on ura
dropout plates. Independent colonies
of each yeast transformant were grown and a halo bioassay was performed.
dropout medium supplemented with 2%
galactose or glucose and were grown to an identical density
(A600 = 1). Yeast cells were precipitated and
lysed in SDS sample buffer. Proteins were extracted by sonication,
separated on SDS-polyacrylamide gels, and transferred to nitrocellulose
membranes. Expression levels of Myc-tagged RGS proteins were then
examined by Western blotting using anti-Myc tag monoclonal antibody
(9E10, BabCO). Signals were detected with an ECL system (Amersham
Pharmasia Biotech).
NRGS8 were isolated.
For
NRGS8, a forward primer that included the ATG start codon was
used. After confirmation of their nucleotide sequences, they were
cloned into pCXN2 expression vector provided by Professor Miyazaki
(22). Biotinylated proteins were generated by in vitro
transcription/translation of the resultant plasmid DNAs using the
TNT-coupled reticulocyte system (Promega) and biotinylated lysine-tRNA
complex (TranscendTM tRNA, Promega). Each biotinylated
protein was mixed with purified bovine G
o in binding
buffer containing 20 mM HEPES, pH 8.0, 0.1 M
NaCl, 1 mM dithiothreitol, 6 mM
MgCl2, 10 µM GDP, 30 µM
AlF4
(30 µM
AlCl3, 10 mM NaF), and 0.1% polyoxyethylene
10-lauryl ether (C12E10). After incubation for
4 h at 4 °C, the reaction mixture was precleared with 50%
(v/v) protein G-Sepharose for 1 h at 4 °C, incubated with
anti-bovine G
o antibody for 2 h at 4 °C, and
then cleared with 50% (v/v) protein G-Sepharose for an additional
2 h. Protein G beads were washed four times in the binding buffer,
suspended in SDS sample buffer, and boiled for 5 min. Proteins were
separated by SDS-polyacrylamide gel and transferred to nitrocellulose
paper. Co-immunoprecipitation of biotinylated protein was examined with
streptavidine-horseradish peroxidase (Promega). Immunoprecipitation of
G
o was confirmed by Western blotting using
anti-G
o antibody (Santa Cruz Biotechnology). Signals were detected with an ECL system (Amersham Pharmasia Biotech).
NRGS8 expression
constructs were described in the previous section on
immunoprecipitation. The plasmid DNAs of these expression constructs
were transfected into DDT1MF2 cells by the CaPO4 methods
(23). After selection in the presence of G418 (0.8 mg/ml, Life
Technologies, Inc.), stable lines were established. The cultured cells
were sonicated in 50 mM Tris acetate buffer, pH 7.5, containing 10 mM MgCl2, 1 mM
dithiothreitol, 1 mM EDTA, and 0.1 mM
phenylmethylsulfonyl fluoride and the homogenate was centrifuged
(44,000 × g, 20 min, 4 °C). The resultant
supernatant and precipitate were mixed with SDS sample buffer and used
as cytosolic and particulate fractions.
NRGS8 as a chimeric protein with fluorescent
protein at the carboxyl terminus. By polymerase chain reaction
amplification, cDNA fragments for RGS8 and
NRGS8 were isolated.
For
NRGS8, a forward primer that included the ATG start codon was
used. After confirmation by sequencing analysis, RGS8 cDNA was
fused in-frame to red fluorescent protein (RFP) in pDsRed1-N1 (CLONTECH) or in one particular experiment to green
fluorescent protein (GFP) in pEGFP-N1 (CLONTECH).
NRGS8 was ligated in-frame to the beginning of GFP in pEGFP-N1.
Resultant plasmid DNAs were transiently transfected into DDT1MF2 cells
using FuGENE 6 (Roche Molecular Biochemicals). At 48 h after the
transfection, transfected cells were fixed by treatment with 4%
paraformaldehyde for 30 min. Confocal microscopy was performed with a
Zeiss microscope connected with an LSM410 Laser Scanning Confocal
Microscope (Zeiss). The confocal images were collected using a ×63
objective. For a three-dimensional reconstructed image, transfected
cells were examined with a LSM510 Laser Scanning Confocal Microscope
(Zeiss) and stacks of 35 images spaced by 0.35 µm were recorded.
NRGS8 data were fitted better with a two-exponential
function, the fittings of them were done with a two-exponential
function. When the turning off time course of the data of RGS(
) was
fitted with a double-exponential function, the contribution of the
slower component was small, and the time constant could not be
determined reliably. Thus, the turning off phases of the data of
RGS(
) were fitted with a single-exponential function.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NRGS8) to inhibit the pheromone response pathway in
Saccharomyces cerevisiae, as shown in Fig.
2A. Wild type RGS8 inhibited
the growth arrest response to mating pheromone. Deletion of the
NH2 terminus 35 amino acids resulted in a partial loss of
function; i.e.
NRGS8 could not efficiently attenuate the
effect of pheromone signaling. This effect of removal of the
NH2 terminus on RGS8 function was confirmed by comparing
the size of the halos of growth inhibition of cell lawns grown on agar
plates (Fig. 2B). Thus, it is clear that the
NH2-terminal region of RGS8 is required for its full
ability to function in yeasts. Expression levels of wild and mutant
RGS8 proteins in yeast cells grown in galactose were determined by
Western blotting using anti-Myc antibody (Fig. 2C). Similar
expression levels were detected, indicating that the functional
difference was not due to impairment of expression. The specificity of
the antibody was tested by using yeasts grown in glucose (data not
shown).
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Fig. 1.
Conservation of NH2-terminal
sequences of RGS4, RGS16, RGS5, and RGS8. The
NH2-terminal sequences of rat RGS4 (4), mouse RGS16 (28),
mouse RGS5 (28), and rat RGS8 (11) are aligned. Conserved amino acid
residues are boxed in black.
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Fig. 2.
Effect of NRGS8 on
the response of yeasts to mating pheromone.
A, cells of the sst2 strain (SNY86)
carrying pTS210 vector, RGS8-pTS210, and
NRGS8-pTS210 were plated on
soft agar containing 0.5% galactose. Sterile filter disks were placed
on the nascent lawn, and synthesized
-pheromone was applied to the
discs. Plates were incubated at 30 °C for 36 h. The amount of
-pheromone added to each disc was: 0 ng (top left), 0.2 ng
(top right), 2 ng (bottom right), 20 ng
(bottom left). B, the effect of removal of
NH2 terminus on RGS8 function was statistically confirmed.
The size of the halo of growth inhibition of cell lawns grown on agar
plates was calculated by measuring its diameter. Results were expressed
as a percentage of the control halo with vector alone. The mean and
S.D. (n = 4) were as follows. RGS(
): 100 ± 2.6%, RGS8(+): 8.2 ± 4.5%,
NRGS8(+): 18.1 ± 2.8%.
Values of
NRGS8 were significantly different from those of wild type
RGS8 (p value = 0.0096) by Student's unpaired
t test. C, two colonies isolated from each yeast
(SNY86) transformed with pTS210 vector, RGS8-pTS210, and
NRGS8-pTS210 were cultured in galactose-containing medium.
Expression of RGS8 was detected by Western blotting with anti-Myc
antibody.
NRGS8 Retains G protein Binding Activity--
We examined
whether NH2-terminal deletion affects the G protein binding
activity of RGS8. Biotin-labeled proteins of the wild type and
NRGS8
were generated by in vitro transcription and translation. Each labeled RGS8 protein was incubated with purified bovine
G
o in the presence of GDP and
AlF4
. The complex containing
G
o was immunoprecipitated using anti-G
o antibody. Similar to wild type RGS8,
NRGS8 was recovered with G
o (Fig. 3). From these
results, it was clearly demonstrated that the NH2-terminal
deletion of RGS8 does not affect G protein binding activity.
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Fig. 3.
NRGS8 retains G protein binding
activity. Biotinylated proteins of RGS8 and
NRGS8 were
generated by in vitro transcription/translation and mixed
with purified bovine G
o. After incubation for 4 h
at 4 °C, the complex containing G
o was
immunoprecipitated with anti-bovine G
o antibody as
described under "Experimental Procedures." Precipitated proteins
were separated on SDS-polyacrylamide gel and transferred to
nitrocellulose paper. Co-immunoprecipitation of biotinylated protein
was examined with streptavidine-horseradish peroxidase
(top). The left three lanes contained in
vitro translated products without immunoprecipitation.
Immunoprecipitation of G
o was confirmed by Western
blotting using anti-G
o antibody (bottom). In
the lane indicated with a star, isolated G
o
was applied.
? It has been demonstrated that the NH2-terminal
domains of RGS4 and RGS16 serve as a membrane targeting sequence in
yeasts (18, 19). To determine whether the NH2-terminal
domain of RGS8 influences its intracellular distribution or not, wild
type RGS8 or
NRGS8 cDNA was transfected to DDT1MF2 cells, which
do not express endogenous RGS8, and their cell homogenates were
fractionated. Cytoplasmic and particulate fractions were subjected to
Western blotting analysis using the RGS8-specific antibody (Fig.
4). The specificity of the antibody was
determined by Western blotting of whole cell extracts from control
cells and cells transfected with RGS8 cDNA. Wild type RGS8 was
present in the particulate fraction. In contrast,
NRGS8 was
abundantly detected in the cytoplasm. In both transfected cells,
G
q/11 was exclusively present in the particulate
fraction. These results clearly demonstrated that the
NH2-terminal domain of RGS8 is required for its subcellular
distribution.
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Fig. 4.
NH2-terminal deletion shifts the
distribution of RGS8 from the particulate to cytosolic fraction.
A, DDT1MF2 cells expressing RGS8 or NRGS8 were
cultured and fractionated. The resultant cytosolic (C) and
particulate (P) fractions were separated on
SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue.
B, after electrophoresis, both fractions were also
transferred to nitrocellulose paper. Western blotting using anti-RGS8
antibody (top) and anti- G
q/11 antibody
(bottom) was performed.
NRGS8, respectively, and they were coexpressed in DDT1MF2 cells. RGS8 was
expressed as a chimeric protein with RFP at the carboxyl
terminus and
NRGS8 was coexpressed as a chimeric protein with GFP.
Surprisingly, RGS8-RFP was highly concentrated in nuclei of most
transfected cells (Fig. 5,
upper). On the other hand,
NRGS8-GFP was diffusely distributed through the cytoplasm within identical cells (Fig. 5,
middle). These observations were consistent with the
analysis using the cell fractionation method (Fig. 4), since the
particulate fraction contains nuclei and cell membranes. These results
clearly demonstrated that RGS8 is localized in nuclei in DDT1MF2 cells, and that the NH2-terminal domain of RGS8 is required for
this unique nuclear localization. Sequence comparison revealed that the
NH2 terminus of RGS8 is similar to the conserved
NH2-terminal sequences of RGS4 and RGS16, but that the
first 7 amino acids of RGS8 are unique (Fig. 1). A search for the
subcellular localization sites of protein identified a putative nuclear
localization signal sequence, PRRNK at amino acids 7-11. This nuclear
localization signal sequence was thought to function in the
distribution of RGS8 in nonactivated DDT1MF2 cells. The nuclear
localization of RGS8 was further confirmed by an immunofluorescence
method. DDT1MF2, NIH3T3, and HEK293 cells were transfected with
cDNA of wild type RGS8 without fluorescent protein and were
immunostained with anti-RGS8 antibody. In all cases, similar nuclear
localization of RGS8 was observed (data not shown).
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Fig. 5.
Nuclear localization of RGS8.
RGS8 and NRGS8 were transiently coexpressed in DDT1MF2 cells. RGS8
was expressed as a chimeric protein with RFP at the carboxyl terminus
(RGS8) and
NRGS8 was coexpressed as a chimeric protein with GFP
(
NRGS8). At 48 h after transfection, confocal images were
collected using a × 63 objective lens. Bar, 10 µm.
in its active state (9, 10)
and the heterotrimeric G protein complex is mainly associated with cell
membranes. Since RGS8 was found to be localized to nuclei in DDT1MF2
cells, it is considered to be unable to efficiently interact with G
in the cells activated by stimulation of G proteins. To investigate the
subcellular localization of RGS8 after G protein activation, a
constitutively active G
was further coexpressed in DDT1MF2 cells. We
previously showed that RGS8 binds preferentially to G
o
and G
i3 (11). When G
o Q205L was further
coexpressed for 48 h, a marked translocation of RGS8 to the plasma
membrane was observed (Fig. 6,
upper). In many cells, RGS8-RFP was concentrated to unique
membrane structures that may correspond to membrane ruffles or
microprojections and to the cell periphery. The nuclear localization of
RGS8-RFP, which was intense in unstimulated cells, tended to decrease
or disappear in G
o Q205L-expressing cells. The
distribution of
NRGS8-GFP, however, was not significantly influenced
by expression of active G
o and
NRGS8-GFP showed a relatively uniform pattern of distribution (Fig. 6, middle).
In control cells transfected with RFP cDNA, a diffuse and sometimes punctuate pattern was observed and the distribution was clearly different from that of RGS8-RFP. Coexpression of G
o
Q205L had almost no effect on the distribution of RFP. Thus, RGS8 was
shown to be translocated to plasma membrane by G protein-activated
signalings. It is clear that the NH2-terminal domain of
RGS8 is required for this translocation.
NRGS8 could bind to
G
o, but it could not translocate to the membrane on
coexpression of G
o Q205L. Therefore, it was thought that
the membrane recruitment of RGS8 was not a direct result of the
physical association with G
. We further investigated the
morphological structure of the region to which RGS8 was translocated on
coexpression of G
o Q205L by confocal microscopy.
Three-dimensional reconstitution of confocal slice images revealed that
the structures where RGS8 accumulated as indicated in Fig. 6,
left, were fine projections or microspikes present on the
surface of DDT1MF2 cells (Fig. 7).
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Fig. 6.
Translocation of RGS8 by expression of active
G o. A constitutively active
G
o was coexpressed with RGS8-RFP (RGS8) and
NRGS8-GFP
(
NRGS8) in DDT1MF2 cells. At 48 h after transfection, confocal
images were collected using a × 63 objective lens.
Bar, 10 µm.
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Fig. 7.
Three-dimensional reconstructed image of RGS8
translocated by G protein activation. RGS8 was expressed as a
chimeric protein with GFP in DDT1MF2 cells. A constitutively active
G o cDNA (RGS8 + G
o Q205L) or control
vector (RGS8+vector) was co-transfected. At 48 h after
transfection, confocal images were collected and a three-dimensional
reconstructed image was obtained. The obtained image was rotated.
subunits released by the Gi family. They are
activated by G protein-coupled receptors such as m2 muscarinic and D2
dopamine receptors. We previously coexpressed GIRK1/GIRK2
heteromultimer and m2 muscarinic receptor with or without RGS8 in
Xenopus oocytes and analyzed the effects of RGS8 on G
protein-mediated modulation of K+ currents. We reported
that RGS8 speeds up the activation and deactivation kinetics of GIRK
upon receptor stimulation. We also reported the RGS8-induced acute
desensitization of the receptor-activated GIRK current (11, 12). Here,
we examined the functional role of the NH2-terminal domain,
which determines subcellular localization, in the RGS8-mediated
modulation of on-off kinetics of GIRK current. We coexpressed RGS8 or
NRGS8 with GIRK1/GIRK2 heteromultimer and m2 muscarinic receptor in
Xenopus oocytes.
NRGS8 coexpression accelerated both the
turning on and off to a similar extent as the wild type RGS8. The acute
desensitization of receptor-activated GIRK current, however, was much
less clear in oocytes expressing
NRGS8 (Fig.
8A). These observations were
confirmed quantitatively by comparing the time constants of the
exponential functions used for the fitting of the activation
(
on) and deactivation (
off) phases
(detail in the legends of Fig. 8B) and the extent of
desensitization (Fig. 8B). Thus, we identified a novel
function of the NH2-terminal domain of RGS8 to cause acute
desensitization of GIRK current activated by receptor stimulation.
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Fig. 8.
Effects of RGS8 and
NRGS8 on turning on and turning off kinetics, and
on the desensitization of GIRK current upon stimulation of m2
muscarinic receptor. A, GIRK1/2 and m2
muscarinic receptor without (upper trace) or with RGS8
(middle trace) or with
NRGS8 (lower trace)
were coexpressed in Xenopus oocytes. Current traces at a
holding potential of
80 mV are shown. 10 µM ACh was
applied at the time indicated by the bars. B, Comparison of
the time constants
on (top) and
off (middle), and the desensitization level
(bottom) of the GIRK 1/2 current upon stimulation of m2
muscarinic receptor in the absence (left) or presence of
RGS8 (middle) or
NRGS8 (right). The turning on
phases of three groups were fitted with a single-exponential function,
and the time constants of them were compared (
on). The
turning off phases of RGS(
) data were fitted with a single
exponential function. In contrast, a two-exponential function was used
to fit those of the data with RGS8 and
NRGS8 satisfactorily. The
time constants of the fast (and major) component of them were compared
with that of the single time constant of RGS(
) data
(
off).
on and
off values
of RGS8 and
NRGS8 were significantly different from those without
RGS protein (average and S.D. are shown, n = 9 or 10, p value < 0.05) by Student's unpaired t
test. The level of desensitization was measured as a percentage of the
reduction of the induced current after 1 min of ligand application.
Values of RGS8 were significantly different from those with
NRGS8 or
from those without RGS (average and S.D. are shown, n = 9 or 10, p value < 0.05) by Student's unpaired
t test.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NRGS8. Under these conditions of overexpression, a proper subcellular distribution might not be necessary for sufficient function of RGS8 because of high amount within
the cells.
o Q205L in
DDT1MF2 cells. The NH2-terminal domain of RGS8 was shown to
play critical roles in this membrane recruitment, since mutant RGS8
lacking the NH2-terminal sequence of 35 amino acids could
not translocate to the membrane within identical cells. This
NRGS8
contains an intact RGS domain, which is sufficient for the interaction
with G
. Therefore, the mechanism of the membrane translocation was considered to be independent of RGS8-G protein interaction. RGS4 was
also reported to be recruited from the cytoplasm to the plasma membrane
on the expression of activated, GTPase-deficient G
i2 (24). It was shown that a non-G
binding mutant of RGS4 could translocate like wild type RGS4, indicating that this translocation was
not a direct result of physical association with an activated G
. It
is possible that G protein activation triggers binding of unidentified
cellular factors to the subcellular localization signals of the
NH2 terminus of RGS proteins for translocation to the membrane.
NRGS8 accelerated the turning on and off of
GIRK current upon receptor stimulation similar to wild type RGS8. Wild
type RGS8 induced significant levels of acute desensitization of the
response during receptor activation, but the desensitization in oocytes
expressing
NRGS8 was similar to that in control oocytes. These
results demonstrated that the NH2-terminal domain of RGS8 is required to cause acute desensitization of GIRK. Indeed, we previously reported that RGS7, which contains the characteristic long
NH2 terminus different from that of RGS8, showed slower
desensitization. Kovoor et al. (26) also made similar
recordings of receptor-activated GIRK current in Xenopus
oocytes expressing RGS proteins. Their data indicated that RGS4 induces
acute desensitization, but that RGS7 and RGS9 do not cause significant
desensitization. RGS4 has a similar NH2-terminal sequence
to RGS8, and both RGS7 and RGS9 possess the long NH2
terminus containing DEP (Dishevelled/EGL10/pleckstrin homology) and GGL
(G protein
subunit-like) domains instead of the short conserved
NH2 terminus of RGS8.
, on-rate;
,
off-rate), the current amplitude is expected to increase single exponentially with a time constant of 1/(
+
). In this case, no desensitization is expected to occur. To explain the presence of
desensitization in the two-state model, it is necessary to assume a
gradual decrease in the on-rate or a gradual increase in the off-rate
in the course of the receptor stimulation. As the turn-on speed,
determined dominantly by
, was not significantly decreased by the
second ligand application immediately after the initial trial (data not
shown), it appears that the on-rate was not decreased during the ligand
stimulation. If the off-rate is increased during the response, the
turning off after ligand washout should be faster with RGS8, which
showed a more intense desensitization. As the turning off speed after
ligand washout was similar between RGS8 and
NRGS8, the off-rate was
not increased during the response. Thus, the desensitization could not
be explained by a gradual change of the on- or off-rate. What then is
the mechanism of desensitization in the presence of RGS8? With the
assumption that there are three states, a nonactive nonready state, a
nonactive ready state, and an active state, the desensitization could
be explained by the depletion of the ready pool as discussed by Chuang
et al. (27). The presence of a large ready pool enables the
acceleration of the turning on, but at the same time the depletion of
the ready pool during the response could result in the acute
desensitization even if the on-rate and off-rate are not changed. If
this is the case, the NH2-terminal region of RGS8 is
thought to be necessary for the formation of the ready pool and for its
depletion during the response. It is speculated that the
NH2 terminus of RGS8 controls the G protein pool by
controlling its subcellular distribution, although further study is
required to understand how this enlarged G protein pool is regulated by
RGS proteins.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Professor M. Lazdunski for
GIRK2 cDNA, Dr. A. Connolly for m2 muscarinic receptor cDNA,
and Dr. J. D. Jordan for Go Q205L cDNA. We
thank Dr. H. Nakata for helpful discussion, Dr. K. Nishi for use of a
confocal microscope, M. Odagiri for technical assistance, and M. Kato
for advice and help with confocal images.
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FOOTNOTES |
---|
* This work is supported in part by research grants from the Ministry of Education, Science, Sports and Culture of Japan (to O. S. and Y. K.) and from the Kato Memorial Bioscience Foundation (to O. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Molecular and Cellular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan. Tel.: 81-423-25-3881 (ext. 4058); Fax: 81-423-21-8678; E-mail: osaito@tmin.ac.jp.
¶¶ Supported by the Mitsubishi Foundation and Core Research for Evolutional Science and Technology of the Japan Science and Technology Corp.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M006917200
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ABBREVIATIONS |
---|
The abbreviations used are: RGS, regulators of G protein signaling; G proteins, heterotrimeric guanine nucleotide-binding proteins; GFP, green fluorescent protein; RFP, red fluorescent protein; GIRK, G protein-gated inwardly rectifying K+ channel.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Dohlman, H. G.,
and Thorner, J.
(1997)
J. Biol. Chem.
272,
3871-3874 |
2. |
Berman, D. M.,
and Gilman, A. G.
(1998)
J. Biol. Chem.
273,
1269-1272 |
3. | Chan, R. K., and Otte, C. A. (1982) Mol. Cell. Biol. 2, 11-20[Medline] [Order article via Infotrieve] |
4. | Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746[CrossRef][Medline] [Order article via Infotrieve] |
5. | Koelle, M. R., and Horvitz, H. R. (1996) Cell 84, 115-125[Medline] [Order article via Infotrieve] |
6. |
Yan, Y.,
Chi, P. P.,
and Bourne, B.
(1997)
J. Biol. Chem.
272,
11924-11927 |
7. |
Huang, C.,
Hepler, J. R.,
Gilman, A. G.,
and Mumby, S. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6159-6163 |
8. | Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452[Medline] [Order article via Infotrieve] |
9. | Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 383, 172-175[CrossRef][Medline] [Order article via Infotrieve] |
10. | Hunt, T. W., Fields, T. A., Casey, P. J., and Peralta, E. G. (1996) Nature 383, 175-177[CrossRef][Medline] [Order article via Infotrieve] |
11. | Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Saitoh, O.,
Kubo, Y.,
Odagiri, M.,
Ichikawa, M.,
Yamagata, K.,
and Sekine, T.
(1999)
J. Biol. Chem.
274,
9899-9904 |
13. | Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 364, 802-806[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Lesage, F.,
Guillemare, E.,
Fink, M.,
Duprat, F.,
Heurteaux, C.,
Fosset, M.,
Romey, G.,
Barhanin, J.,
and Lazdunski, M.
(1995)
J. Biol. Chem.
270,
28660-28667 |
15. | Velimirovic, B. M., Gordon, E. A., Lim, N. F., Navarro, B., and Clapham, D. E. (1996) FEBS Lett. 379, 31-37[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Doupnik, C. A.,
Davidson, N.,
Lester, H. A.,
and Kofuji, P.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10461-10466 |
17. | Chen, C., and Lin, S.-C. (1998) FEBS Lett. 422, 359-362[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Srinivasa, S. P.,
Bernstein, L. S.,
Blumer, K. J.,
and Linder, M. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5584-5589 |
19. |
Chen, C.,
Seow, K. T.,
Guo, K.,
Yaw, L. P.,
and Lin, S.-C.
(1999)
J. Biol. Chem.
274,
19799-19806 |
20. | Saitoh, O., Odagiri, M., Masuho, I., Nomoto, S., and Kinoshita, N. (2000) Biochem. Biophys. Res. Commun. 270, 34-39[CrossRef][Medline] [Order article via Infotrieve] |
21. | Nomoto, S., Adachi, K., Yang, L.-X., Hirata, Y., Muraguchi, S., and Kiuchi, K. (1997) Biochem. Biophys. Res. Commun. 241, 281-287[CrossRef][Medline] [Order article via Infotrieve] |
22. | Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193-200[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Mannual , Cold Spring Harbor Lab., Cold Spring Harbor, NY |
24. |
Druey, K. M.,
Sullivan, B. M.,
Brown, D.,
Fischer, E. R.,
Watson, N.,
Blumer, K. J.,
Gerfen, C. R.,
Scheschonka, A.,
and Kehrl, J. H.
(1998)
J. Biol. Chem.
273,
18405-18410 |
25. |
Dulin, N. O.,
Pratt, P.,
Tiruppathi, C.,
Niu, J.,
Voyno-Yasenetskaya, T.,
and Dunn, M. J.
(2000)
J. Biol. Chem.
275,
21317-21323 |
26. |
Kovoor, A.,
Chen, C.-K.,
He, W.,
Wensel, T. G.,
Simon, M. I.,
and Lester, H. A.
(1999)
J. Biol. Chem.
275,
3397-3402 |
27. |
Chuang, H.-H., Yu, M.,
Jan, Y. N.,
and Jan, L. Y.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11727-11732 |
28. |
Chen, C.,
Zheng, B.,
Han, J.,
and Lin, S.-C.
(1997)
J. Biol. Chem.
272,
8679-8685 |