From the The members of a recently identified protein
family termed regulators of G-protein signaling (RGS) act as
GTPase-activating proteins for certain G Numerous biological processes such as vision, olfaction, and
many hormonal responses generate signals that are transduced through
heterotrimeric guanine nucleotide (GTP)-binding proteins (G-proteins)
(1). The regulatory mechanisms that control G-protein signaling
intracellularly have not been well characterized, but the variability
of signal strength and the specificity of a given stimulus among
different cell types suggest that cell type-specific interacting
proteins participate (2). A newly discovered family of regulators of
G-protein signaling (RGS
proteins)1 may play a role in
cell type-specific desensitization. RGS proteins were originally
identified by genetic complementation of a yeast homologue (Sst2p) and
by the identification of a closely related homologue in
Caenorhabditis elegans (EGL10) that regulated G-protein signaling (3-6). Subsequently, transfection experiments and
biochemical studies with recombinant proteins have shown that RGS
family members likely down-regulate signaling through G-protein-coupled
receptors (GPCRs) by acting as GTPase-activating proteins (GAPs) for
some heterotrimeric G-protein Although several RGS proteins such as RGS1, RGS4, RGS10, and
RGS-GAIP accelerate the GTPase activity of Gi In accord with this possibility, we show in this study that the
majority of RGS4 localizes to the cytoplasmic compartment and not at
the plasma membrane, in both transfected cells and in the NG108-15 cell
line. This result contrasts with the RGS family members GAIP, RGS9, and
the yeast RGS homologue Sst2p, which have been reported to be
predominantly membrane-associated (5, 15, 16). Moreover, we found that
co-expression of a GTPase-deficient Gi Cell Lines--
PC12 cells were obtained from ATCC and
grown in Dulbecco's modified Eagle's medium plus 10% fetal calf
serum and 5% horse serum at 37 °C. The 293T cells were the kind
gift of David Baltimore. NG108-15 cells were obtained from Werner Klee
(17) and grown in complete Dulbecco's modified Eagle's medium
supplemented with hypoxanthine/aminopterin/thymidine. The Burkitt
lymphoma cell lines CA46 and MC116 were the kind gift of Dr. Ian
Magrath (NCI, NIH), and the NALM-6 cell line was the gift of Dr. Thomas
Tedder (Duke University). All suspension cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics.
Co-immunoprecipitations--
NG108-15 cells were solubilized in
a 1% C12E10-containing buffer. Cell lysates were then incubated with
GDP alone (30 µM) or GDP and
AlF4 Antibodies, RGS4 Immunoblotting, and Cell
Fractionation--
Antibodies to rat RGS4 were generated by immunizing
rabbits with an N-terminal peptide unique to RGS4 among mammalian RGS proteins (LRASAKDMKHRLGFLC) coupled to keyhole limpet hemocyanin. For
immunoblotting, the cells were lysed in 150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA, and 1% Nonidet
P-40 with a mixture of protease inhibitors (Boehringer Mannheim) for
1 h on ice, and the detergent-insoluble fraction was removed by
microcentrifugation. 75-150 µg of protein was fractionated by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. Membranes were blocked with 10% milk in TTBS
(Tris-buffered saline with 0.05% Tween 20) for 2 h and then
incubated with the appropriate antibody for 2 h. For peptide
blocking studies, the antisera were incubated with a control peptide
(FLAG) or the immunizing peptide for 30 min at room temperature before
dilution of the antibody. The final concentration of the peptide was 10 µg/ml. The blots were washed twice with TTBS before the addition of a
biotinylated goat anti-rabbit immunoglobulin (DAKO). Following a 1-h
incubation, the blot was washed twice with TTBS and then incubated with
streptavidin conjugated to horseradish peroxidase (DAKO). The signal
was detected by ECL following the recommendations of the manufacturer
(Amersham Pharmacia Biotech). For cell fractionation, the method of
Chakrabarti (18) was used. The anti-syntaxin antibody was the gift of
Paul Roche (NCI, NIH), and the pan 14-3-3 antibody was purchased from
Santa Cruz Biotechnology.
Immunofluorescence--
Cells were plated in 6-well tissue
culture dishes containing glass coverslips. Cells were fixed in 50%
methanol/50% acetone for 1 h at 4 °C. Fixed cells were blocked
for 1 h at room temperature in PBS plus 10% goat serum and 2%
bovine serum albumin. The cells were then incubated with the
appropriate antiserum for 2 h at room temperature. Following two
washes with PBS, the cells were incubated with fluorescein
isothiocyanate-conjugated goat anti-rabbit immunoglobulins (Tago) for
1 h at room temperature. The cells were washed twice with PBS,
air-dried, mounted on silanized glass slides, and examined under the
fluorescence microscope.
Immunoelectron Microscopy--
NG108-15 cells were plated on
ThermanoxTM (Nunc) coverslips, fixed in 0.1 M
lysine-HCl-NaP04/0.1% glutaraldehyde, and then incubated with the preimmune control or RGS4 antiserum in PBS/0.005% saponin for
2 h. The cells were secondarily labeled with sheep anti-rabbit IgG
Fab (Jackson Laboratories) in PBS/0.005% saponin for 1 h at room
temperature. After three rinses with PBS, the cells were fixed in 1.5%
glutaraldehyde/0.1 M NaCacodylate/5% sucrose for 1 h
at room temperature. ImmunoPure Metal Enhanced DAB substrate (Pierce)
was then added for 5 min at room temperature according to the
manufacturer's instructions. Cells were then fixed with 4%
paraformaldehyde/2.5% glutaraldehyde/0.1 M cacodylate and
processed for electron microscopy essentially as described by Scidmore
et al. (19).
In Situ Hybridization--
After euthanization, rat brains were
rapidly removed and frozen in isopentane (Fluka) chilled on dry ice.
Brains were cut in 12-µm sections on a cryostat (Frigocut 2800E,
Cambridge Instruments) and thaw-mounted onto gelatin/chrome alum-subbed
slides. Slides were post-fixed in 4% formaldehyde/0.9% NaCl for 10 min, acetylated in fresh 0.25% acetic anhydride in 0.1 triethanolamine/0.9% NaCl, pH 8, for 10 min, dehydrated in an
ascending series of alcohols, delipidated in chloroform, and then
rehydrated in a descending series of alcohols. Slides were air dried
and stored at RGS4 Interacts with Several G Laboratory of Immunoregulation and the
** Laboratory of Molecular Biology,
Department of Cell Biology and Physiology,
Washington University School of Medicine,
St. Louis, Missouri 63310
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunits
in vitro, but their physiological effects in cells are
uncertain in the face of similar biochemical activity and overlapping
patterns of tissue expression. Consistent with its activity in in
vitro GTPase-activating protein assays, RGS4 interacts
efficiently with endogenous proteins of the Gi and
Gq subclasses of G
subunits but not with
G12
or Gs
. Unlike other RGS proteins such
as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely
membrane-associated, a majority of cellular RGS4 is found as a soluble
protein in the cytoplasm. However, the expression of a GTPase-deficient
Gi
subunit (Gi
2-Q204L) resulted in the
translocation of both wild type RGS4 and a
non-Gi
-binding mutant (L159F) to the plasma membrane.
These data suggest that RGS4 may be recruited to the plasma membrane
indirectly by G-protein activation and that multiple RGS proteins
within a given cell might be differentially localized to determine a
physiologic response to a G-protein-linked stimulus.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-subunits (7-11).
subunits
with apparently similar ability, the precise affinities of the various RGS proteins for individual G
subunits is only slowly
emerging, and almost nothing is known about the physiologic regulation
of these proteins (8-11). Although previous studies documented the inability of RGS1, RGS4, and RGS-GAIP to enhance Gs
or
G12
GTPase activity (7-9), recent experiments have
demonstrated that either recombinant RGS4 or RGS-GAIP accelerates GTP
hydrolysis by Gq
in reconstituted GPCR-G-protein
phospholipid vesicles and inhibits second messenger generation through
a Gq
-coupled GPCR when added to membranes prepared from
the neural cell line NG108-15 (11). In addition, either the transient
expression of RGS4 in COS-7 cells or its permanent expression in HEK293
cells inhibits Gq-mediated signaling (12, 13). However, the
response to gonadotropin-releasing hormone (GnRH) in COS-7 cells
transfected with the GnRH receptor, which also couples to
Gq
, was markedly inhibited by RGS3 but not by RGS4 (14).
The apparent failure of RGS4 to impair GnRH Gq-mediated
signaling could be explained if insufficient levels of RGS4 localized
at the plasma membrane, especially because a relatively low amount of
the RGS4 expression plasmid (0.8 µg) was used.
mutant
(Gi
2Q207L) shifted both the cytoplasmic wild type RGS4
and a non-Gi
-binding mutant of RGS4 (L159F) to the
plasma membrane. These results indicate that some RGS proteins may be
recruited from the cytoplasm to the plasma membrane to modulate
G-protein-mediated signaling pathways and that this shift might be an
indirect result of the activation of G
subunits rather
than a simple physical association.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(30 µM) for 30 min
at room temperature. Recombinant His6 RGS4 (20 µg),
prepared as described previously (9), was added to the lysates for 30 min at room temperature. 50 µl of nickel-nitriloacetic acid superflow
beads (Qiagen) was then added for an additional 20 min. After three
washes with buffer, bound proteins were eluted in SDS sample buffer and
boiled for 5 min. After fractionation on a 10% SDS gel, proteins were
transferred to a nitrocellulose filter and immunoblotted for various
G
subunits using antisera against Gi
1-2
(the kind gift of Allen Spiegel and Paul Goldsmith, NIDDK, NIH),
Go
, Gq
, G12
(Santa Cruz), or Gs
(Calbiochem).
20 °C. Oligonucleotide probes were generated
complementary to bases 61-100 and 661-700 of the published rat RGS4
cDNA (4) and diluted in hybridization buffer (0.6 M
NaCl, 80 mM Tris, pH 7.5, 4 mM EDTA, 0.1%
sodium pyrophosphate, 10% dextran sulfate, 0.2% SDS, 0.02% heparin
sulfate, 50% formamide, 100 mM dithiothreitol) to obtain
2 × 106 cpm/100 ml. The probe was incubated with the
rat forebrain sections for 12 h at 37 °C. The slides were then
washed four times in 1× SSC at room temperature and then three times
in 2× SSC/50% formamide at 40 °C. Slides were washed twice for 30 min in 1× SSC at room temperature, air dried, and applied to
autoradiography film for 2 weeks at room temperature.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Subunits in Neural
Cells--
Recombinant RGS1, RGS4, and RGS-GAIP bind both recombinant,
purified G-protein
-subunits of the Gi subclass and
G-proteins in bovine brain membrane fractions in vitro (9).
The addition of GDP and tetrafluoroaluminate enhances binding
severalfold, suggesting that RGS proteins bind to the transition state
conformation of G
to accelerate GTP hydrolysis. No RGS
protein (including RGS4) has been reported to have GAP activity toward
Gs
or G12
subunits. To examine the
interaction of RGS4 with endogenous G-proteins, we used cell lysates
from NG108-15 cells, which express high levels of RGS4 (see below).
His-tagged RGS4 immobilized on nickel-nitriloacetic acid beads was used
to pull down endogenous G-proteins in cellular lysates in the presence
of GDP or GDP and AlF4
(Fig.
1). Strong interactions between RGS4 and
Gi
1-2 (using a cross-reactive antibody),
Go
, and Gq
were found in the presence of
GDP and AlF4
. In contrast, despite the
abundance of both Gs
and G12
in these
cells, we did not detect binding of RGS4 to either of these
G
subtypes.
View larger version (25K):
[in a new window]
Fig. 1.
Interaction between RGS4 and G
subunits. NG108-15 cells were lysed in 1% C12E10 buffer,
activated with GDP-AlF4
or not, and
then incubated with recombinant (His6)RGS4 and
Ni2+-NTA beads. After three washes with lysis buffer, bound
proteins were eluted in SDS sample buffer, boiled, fractionated on a
10% SDS gel, and immunoblotted for various G
subunits
as indicated to the right.
Detection of Endogenous RGS4 Protein and mRNA in Neural Tissue-- Because previous Northern blots had demonstrated RGS4 mRNA predominantly in the brain, we sought to identify RGS4 in neural cells and brain tissue (4). To detect endogenous RGS4, we generated a polyclonal antiserum raised against a unique RGS4 peptide. The specificity of this antibody first was tested by immunoblotting cell lysates of 293T cells versus 293T cells transiently transfected with an HA-RGS4 plasmid (Fig. 2). Expression of HA-RGS4 was verified by blotting with HA antibody (left panel) and with the anti-RGS4 antiserum incubated with an unrelated peptide (middle panel), which both detected a band of approximately 30 kDa. In contrast, when the antiserum was incubated with the immunizing peptide prior to blotting, the RGS4 band was absent (right panel). Next, we screened lysates from various cell lines for RGS4 and detected an approximately 30-kDa band in lysates prepared from several cell lines, primarily those of neural origin (Fig. 3A). One cell line expressed unusually high amounts of RGS4, the mouse glioblastoma × rat neuroblastoma cell line NG108-15. We had noted previously that these cells contained high amounts of RGS4 mRNA.2 We did not detect RGS4 in fibroblast or hematopoietic cell lines with the exception of the pre-B cell line Nalm-6. To determine where RGS4 mRNA transcripts localized in the brain, we performed in situ hybridization. Rat forebrain sections were processed for histochemistry and incubated with oligonucleotide probes (40 base pairs) complementary to bases 61-100 and 661-700 of the RGS4 cDNA. As shown in Fig. 3B, prominent staining is seen in gray matter areas such as the cortex, striatum, and nucleus of the diagonal band. Labeling is low or absent in other forebrain neurons such as the globus pallidus, and RGS4 appears to be absent in white matter tracts such as the corpus callosum. These results are similar to those of Gold et al. (20), who found high expression of RGS4 mRNA in the neocortex, piriform cortex, caudoputamen, and ventrobasal thalamus.
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Subcellular Localization of Endogenous RGS4 in NG108-15
Cells--
The previously noted failure of RGS4 to impair signaling
through the GnRH receptor despite significant GAP activity toward Gq in vitro prompted us to examine whether
RGS4 localized to the cell membrane similar to RGS-GAIP, RGS9, and
Sst2p using a combination of cell fractionation, indirect
immunofluorescence, and immunoelectron microscopy. To determine the
relative proportion of RGS4 in the cytoplasm versus cell
membranes in NG108-15 cells, we immunoblotted
of the
supernatant fraction (cytoplasm, Fig.
4A, third lane) and
all of the pellet fraction (membranes, Fig. 4A, fourth
lane) following hypotonic lysis in the absence of detergent and
high speed centrifugation (100,000 × g). A similar
intensity RGS4 band in the two preparations suggests that the cytoplasm
contains approximately 10-fold more RGS4 than do the cell membranes.
Reprobing the immunoblot with an antibody against syntaxin, which is
almost exclusively membrane-associated (21), verified the integrity of
the fractions (Fig. 4A, lower panel).
Immunofluorescent staining of endogenous RGS4 using the N-terminal RGS4
antiserum and NG108-15 cells revealed a diffuse pattern of expression
in the cytoplasm and perinuclear region, whereas only background
staining was seen with a preimmune serum (Fig. 4B). Next, we
performed immunoelectron microscopy to localize RGS4 ultrastructurally.
NG108-15 cells were reacted with the N-terminal antipeptide antiserum;
thin sections were prepared and evaluated by electron microscopy. RGS4
appears to be distributed diffusely in the cytosol as well as at the
plasma membrane (Fig. 5). In contrast,
only background staining was observed with a preimmune antiserum. These
results, along with the immunofluorescent staining and immunoblotting
of cellular fractions, indicate that RGS4 is predominantly localized in
the cytoplasm as a soluble protein.
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Co-expression of a GTPase-deficient Gi Mutant
Results in Translocation of RGS4 to the Plasma Membrane--
Initial
attempts to demonstrate a shift of cytoplasmic RGS4 to the membrane by
activating cells through GPCRs or G-protein activators were
unsuccessful, perhaps because only a small minority of the cellular
RGS4 is recruited. We reasoned, therefore, that the overexpression of
an activated, GTPase-deficient G
subunit (Gi
2Q207L), might result in a translocation that would
be more easily detectable. Indirect immunofluorescent staining of HA-tagged RGS4 in HEK293 cells detected by confocal microscopy demonstrated that like endogenous RGS4, most of the transfected protein
is cytoplasmic (Fig. 6, far left
column). Co-expression of the GTPase-deficient Gi
2
mutant, however, resulted in a dramatic shift of RGS4 out of the
cytoplasm to the plasma membrane (Fig. 6, second column from
left). To determine whether the membrane recruitment of RGS4 was
the direct result of physical association with an activated G-protein,
we repeated the experiment in HEK293 cells transfected with an RGS4
mutant that does not bind Gi
, RGS4(L159F) (22), with or
without the G-protein mutant. Surprisingly, we saw the same membrane
shift of the nonbinding RGS4 mutant (Fig. 6, two right-hand
columns). To confirm this observation, we transfected these cells
with an HA-RGS4 plasmid alone or with Gi
2(Q207L) and
fractionated the cells as previously.
of the cytosol
fractions and 1/2 of the membrane fraction were separated by
SDS-polyacrylamide gel electrophoresis and immunoblotted with an
anti-HA antibody. As shown in Fig. 7,
expression of the Gi
mutant resulted in a significant
decrease in the amount of both wild type RGS4 (top panel)
and RGS4(L159F) (third panel from top) seen in the cytoplasm
compared with the membrane fraction, consistent with the
immunofluorescence. By densitometric analysis, the cytosol/membrane ratio of the wild type RGS4 protein was decreased from 96 to 3.8 in
those cells expressing activated G
. Likewise, the ratio of RGS4(L159F) went from 0.44 to 0.16. Of note, there appeared to be
more pellet-associated RGS4(L159F) than was the case with wild type
RGS4. However, the indirect immunofluorescence clearly shows that the
majority of RGS4(L159F) is cytoplasmic. We hypothesize that some of the
pelleted material may represent insoluble cytoplasmic aggregates that
precipitated with high speed centrifugation rather than true
membrane-associated protein. Nonetheless, the shift of RGS4(L159F) is
still apparent from the cytosol/membrane ratio in the presence of
activated Gi
. Reprobing the blot with an antibody
against 14-3-3 proteins confirmed that the overall ratio of protein in
the two fractions remained constant (second panel from top).
The expression of Gi
2(Q207L) is shown in the
bottom panel.
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DISCUSSION |
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In this study, we show that by adding recombinant RGS4 to
GDP-AlF4-treated cellular lysates, we
could efficiently retrieve endogenous Gi
and
Gq
proteins, but neither Gs
nor
G12
, which is consistent with its activity toward these
G
subfamilies in GAP assays. However, our main
observation is that in unexpected contrast to RGS-GAIP and RGS9, other
RGS proteins that have similar GAP activity as RGS4 and that localize
predominantly at the plasma membrane, most of the cellular RGS4 is
apparently soluble in the cytoplasm. However, it can be recruited to
membranes by expression of an activated G
subunit. This
finding has several implications for the activity of RGS4 that make it
unique among RGS proteins characterized so far.
First, because a relatively small proportion of cellular RGS4 is
membrane-associated at any given time, the trafficking of RGS4 to the
plasma membrane could be an important regulatory mechanism capable of
modulating both the intensity and duration of a signal generated
through Gi- and Gq-coupled receptors. We used
several different methods including cellular fractionation and
immunoblotting, immunofluorescent staining with confocal microscopy,
and immunoelectron microscopy to visualize the relatively low levels of
RGS4 at the plasma membrane despite the high total cellular levels of
RGS4. Consistent with our findings, the addition of exogenous RGS4 to NG108-15 membranes raised the basal level of adenylyl cyclase activity
and reversed enkephalin-mediated inhibition of cAMP synthesis triggered
by prostaglandin E1 (11). Because RGS family members act catalytically,
i.e. a relatively small amount of RGS4 should be able to
enhance GTP hydrolysis by an excess of Gi subunits, the
endogenous levels of RGS4 present in the NG108-15 membrane preparation
must be quite low. We speculate that RGS family members may be either
membrane-associated such as RGS-GAIP, Sst2p, and RGS9, where they
likely set a threshold for signaling, or largely soluble in the
cytoplasm such as RGS4, from where they may be recruited to the
membrane, possibly by the G-protein-linked signal itself. Indeed, if
the entire NG108-15 cellular content of RGS4 was localized at the
plasma membrane, signaling through Gi- and Gq-linked receptors would be predicted to be severely
compromised. Consistent with the differential intracellular
localization of various RGS family members is the recent identification
of a novel RGS protein, RET-RGS1, which is likely localized to the
plasma membrane by a transmembrane segment to enhance the GTPase
activity of retinal transducin (23).
Second, because an RGS4 mutant protein that does not interact with
activated Gi is nonetheless able to translocate to the
membrane when a GTPase-deficient Gi
is overexpressed, these results suggest that the recruitment of RGS4 is not a simple result of the physical RGS-G
interaction. Indeed, our
experiments demonstrate that transiently overexpressed
Gi
2(Q207L) is expressed to a large extent in the
cytoplasmic fraction, and as previously stated, some studies have shown
that RGS4 does not interact with nor act as a GAP toward
Gi
2(Q207L) (7, 8). Thus, it is unclear whether the
G-protein recruits RGS4 directly. The physiologic effect of RGS4
overexpression is to impair signaling through Gi- and
Gq-linked transmembrane receptors. Thus, it is likely that
some other mechanism exists to target low levels of RGS4 to the plasma
membrane. RGS4 does not have a transmembrane segment, but it does
contain several N-terminal cysteines that undergo palmitoylation
(24).3 Although this
modification may assist in retaining RGS4 at the membrane, it does not
explain how RGS4 initially localizes at the plasma membrane.
G
subunits have been implicated in targeting other
proteins to the cell membrane; however, studies have indicated that
RGS4 does not bind G
subunits (9). Furthermore, RGS4 lacks SH2,
SH3, or pleckstrin homology domains, which might mediate interactions
with other proteins or phospholipids, so it remains unclear how RGS4 is
co-localized with activated G
subunits at the plasma
membrane.
Third, the cytoplasmic location of RGS4 could also serve as a reservoir
for the recruitment of RGS proteins to other intracellular membranes
where heterotrimeric G-proteins likely have regulatory roles such as in
the exocytic secretory pathway. In LLC-PK1 epithelial cells
Gi3 was found predominantly on Golgi membranes and diffusely throughout the cytoplasm (25). Overexpression of
Gi
3 retarded secretion of heparan sulfate proteoglycan
and resulted in the accumulation of precursors in the
medial-trans-Golgi. Deactivation of Gi
3 by an RGS in
this case would be expected to have the opposite effect, that is, to
enhance secretion. Because RGS proteins increase GTPase activity of
Gi
and Gq
, they may provide valuable
tools for the investigation of the role of these G
subunits in secretory pathways.
Although one mechanism of RGS regulation may be differential localization within the cell, another is differential tissue expression. Some RGS family members such as RGS-GAIP and RGS3 are expressed in many tissues, whereas others such as RGS1 and RGS4 have a limited tissue distribution (3, 4). Northern blot analysis had identified neural tissue as a major site of RGS4 expression. The discovery of RGS4 (and presumably other RGS proteins expressed in brain) offers a novel mechanism by which neurons may sort the signals received from converging G-protein-linked pathways. We found high levels of RGS4 transcripts only in select areas of the brain, such as the nucleus of the diagonal band, a region enriched for cholinergic neurons (26). Similarly, RGS1, which is expressed in B lymphocytes, is confined largely to a subset of the B-cell compartment, the germinal center region of lymphoid tissue.4 That an RGS protein has such a small window of expression within a given tissue suggests that it likely helps regulate very specific responses; the examination of those cell types may provide a clue to its physiological role. Furthermore, if neurons express RGS4 predominantly in the cytoplasm as do NG108-15 cells, an understanding of the intracellular regulation of RGS4 may provide some interesting insights into signal integration in those neurons. As more is learned about the cellular localization, tissue distribution, mechanisms of activation, and G-protein specificity of the various RGS family members, a better understanding of the physiologic roles of this interesting group of proteins should emerge.
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ACKNOWLEDGEMENTS |
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We thank J. S. Gutkind for providing numerous reagents, Paul Roche for technical advice, Mary Rust and Cindy Pagonis for excellent editorial assistance, and A. S. Fauci for support.
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FOOTNOTES |
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* 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.
§ Present address: Molecular Signal Transduction Section, Lab. of Allergic Diseases, NIAID, NIH, Bethesda, MD 20852. Tel.: 301-435-8875.
Supported by National Institutes of Health Grant DK38452.
¶¶ To whom correspondence should be addressed: NIH, Bldg. 10, Rm. 11B-13; 10 Center Dr., MSC 1876. Tel.: 301-496-2031; Fax: 301-402-0070.
1 The abbreviations used are: RGS, regulators of G-protein signaling; GAP, GTPase-activating protein; GPCR, G-protein-coupled receptor; GnRH, gonadotropin-releasing hormone; PBS, phosphate-buffered saline; HA, hemagglutinin.
2 A. Scheschonka, unpublished observation.
3 K. Druey, unpublished results.
4 V. H. Kang and A. Scheschonka, unpublished data.
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REFERENCES |
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