From the Metabolic Diseases Branch and the Diabetes
Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and the ¶ Department of Molecular Genetics, University of Illinois
at Chicago, College of Medicine, Chicago, Illinois 60607
Received for publication, October 10, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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The role that G Seven transmembrane-spanning receptors respond to extracellular
signals and in turn regulate intracellular processes through their
interaction with signal-transducing heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) in eukaryotic cells
(1). Complementary DNAs from five G protein Although G To this end this work was undertaken to examine the intracellular
distribution of G Cell Culture--
For maintenance, rat pheochromocytoma PC12
cells were grown in 75-cm2 flasks grown at 37 °C and 5%
CO2 containing DMEM supplemented with 10% horse serum, 5%
fetal bovine serum, 4 mM L-glutamine, 1 × penicillin/streptomycin (Biofluids, Rockville, MD) (supplemented DMEM)
without the addition of nerve growth factor (NGF).
Immunocytochemistry and Confocal Laser Microscopy--
Cells
were processed for immunofluorescent staining as described previously
(17). Briefly, PC12 cells were plated onto
poly-D-lysine-precoated covered chamber slides (Lab-Tek II,
Nalge) and grown in supplemented DMEM containing 50 ng/ml NGF at
37 °C for 16 h. The medium was discarded, and the cells were
washed and then fixed in 2% (v/v) formalin in phosphate-buffered
saline. The slides were then incubated with one or more primary
antibodies in phosphate-buffered saline, 10% fetal calf serum (v/v),
and 0.075% (w/v) saponin for 1.5 h, washed, and then incubated
with appropriate labeled secondary antibody (fluorescein
isothiocyanate-conjugated donkey anti-rabbit IgG and rhodamine
red-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Labs, West
Grove, PA)) in the same buffer for 45 min. For imaging of nuclei, cells
were treated with the DNA-binding cyanine dye YOYO (18). After
staining, 1-2 drops of Permount (Vector Labs, Burlingame, CA) were
added to the sample surface. For epifluorescent imaging, cells were
viewed in a Zeiss Axiophot inverted microscope (Carl Zeiss Inc.,
Thornwood, NY), and images were captured with a PentaMAX camera
(Princeton Instruments Inc., Trenton, NJ) utilizing IP Labs
software (Scanalytics Inc., Fairfax, VA). For confocal imaging, cells
were viewed with a Zeiss LSM 510 laser scanning microscope. Final
images were processed using Adobe Photoshope (Adobe Systems Inc.,
Mountain View, CA) software.
Subcellular Fractionation of Mouse Brain and PC12 Cells--
CD1
mouse brain (1 g, wet weight) or a PC12 cell pellet (~1 × 107 cells) were homogenized (30 strokes) with a Dounce
homogenizer with a B-type pestle in 5 ml of Buffer A (50 mM
triethanolamine HCl, pH 7.5; 25 mM KCl; 5 mM
MgCl2; 0.5 mM dithiothreitol; 17 µg/ml AEBSF;
2 µg/ml each aprotinin, leupeptin, and pepstatin; and 1 µg/ml
soybean trypsin inhibitor) containing 0.25 M sucrose on
ice. For PC12 cells the homogenate was further passed five times
through a 25-gauge needle to ensure that the majority of cells were
broken. An aliquot of the homogenate was then stained with trypan blue,
and the integrity of nuclei was verified under a light microscope. The
nuclear, cytosolic, and membrane fractions were prepared as described
by Schilling et al. (19) with the following modifications.
The homogenate was centrifuged at 500 × g for 5 min at
4 °C to remove tissue debris (first pellet). The first supernatant
was then centrifuged at 2,000 × g for 15 min at
4 °C. The second pellet was saved, and the second supernatant was
further centrifuged at 100,000 × g for 1 h at
4 °C. The third supernatant was taken as cytosol extract and the
third pellet as membrane fraction that was extracted by resuspension in
Buffer A containing 0.5% (w/v) Genapol C-100 and incubating on a
rocker at 4 °C for 2 h. The second pellet was resuspended in 1 ml of Buffer A and mixed with 2 ml of Buffer B (Buffer A containing 2.3 M sucrose). The mixture was carefully loaded on top of 1 ml of Buffer B, and the nuclei was pelleted by centrifugation at 12,400 × g for 1 h at 4 °C. The pellet was
washed once with Buffer A containing 0.25 M sucrose and
labeled as nuclear extract. For independent experimental replication,
the membrane, nuclear, and cytosol fractions of both mouse brain and
PC12 cells were also obtained commercially (Geneka Biotechnology,
Quebec, Canada). Proteins from different fractions were quantified by
both the Bradford method (20) and Coomassie Blue staining of
SDS-polyacrylamide gels.
Limited Proteolysis of PC12 Nuclear Extract--
PC12 nuclear
extract prepared as above was dialyzed against 20 mM
Tris-HCl and 5 mM dithiothreitol overnight at 4 °C, then loaded onto a Mono Q anion exchange column (Amersham Pharmacia Biotech)
equilibrated in the same buffer. The column was eluted in a gradient
from 0 to 0.6 M NaCl, and the fractions containing a broad
peak of G Gel Electrophoresis, Immunoblotting, and
Immunoprecipitation--
Protein samples were separated on 4-20%
gradient slab gels (Novex) by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membranes, and after primary and
secondary antibody incubation, chemiluminescent signal was detected
using the Lumi-Light PlusTM Western blot kit (Roche) according to the
manufacturer's instructions. Primary antibodies used were:
affinity-purified rabbit ATDG polyclonal antibody against N terminus of
G Construction of Stable PC12 Cell Transfectants--
To establish
a PC12 cell line stably transfected with tetracycline-inducible
HA-tagged G Preparation of cDNAs Encoding GFP Fusion Proteins and RGS7
for Transient Transfection--
For construction of a
GFP-G Transient Expression of GFP Fusion Proteins in PC12 Cells--
1
day prior to transient transfection of GFP fusion plasmids, PC12 cells
at 90% confluence were harvested and plated in 2 ml of supplemented
DMEM containing 50 ng/ml NGF into the chambers of
poly-D-glycine-coated chamber slides as detailed above. The number of cells was adjusted to 70% of their density at harvest. After
overnight incubation of the cells, 2 µg of plasmid DNA and 8 µl of
LipofectAmine 2000 reagent (Life Technologies), diluted in 200 µl of
Opti-MEMI medium, were mixed, incubated at room temperature for 20 min,
and added directly to each chamber. After mixing gently, the culture
was incubated for 24-48 h. The expression of each fusion construct was
then verified by immunoblotting, and the fluorescence of the GFP fusion
proteins was determined by fluorescence microscopy as detailed above.
Subcellular Localization of G Dual Immunofluorescence Analysis of G Subcellular Localization of G
Tight complexes between native G Subcellular Localization of GFP Fusions with Wild-type and Mutant
G
All three GFP fusion proteins were expressed in transiently transfected
PC12 cells and had the expected immunoreactivity according to their
chimeric composition as evidenced by immunoblots (Fig. 4C).
Furthermore, the effect of the L22P,L26P mutations in G
Like G The nuclear localization documented here illustrates another novel
property of G The molecular basis for the nuclear targeting of G The demonstration here that mutational perturbation of the N-terminal
region of G In PC12 cells the nuclear staining with RGS7 antibody was consistently
fainter and less uniform than the G Is the distribution of G The function of G5
regulator of G protein signaling (RGS) complexes play in signal
transduction in brain remains unknown. The subcellular localization of
G
5 and RGS7 was examined in rat PC12 pheochromocytoma
cells and mouse brain. Both nuclear and cytosolic localization of
G
5 and RGS7 was evident in PC12 cells by
immunocytochemical staining. Subcellular fractionation of PC12 cells
demonstrated G
5 immunoreactivity in the membrane,
cytosolic, and nuclear fractions. Analysis by limited proteolysis
confirmed the identity of G
5 in the nuclear fraction.
Subcellular fractionation of mouse brain demonstrated G
5
and RGS7 but not G
2/3 immunoreactivity in the nuclear
fraction. RGS7 and G
5 were tightly complexed in the
brain nuclear extract as evidenced by their coimmunoprecipitation with anti-RGS7 antibodies. Chimeric protein constructs containing green fluorescent protein fused to wild-type G
5 but not green
fluorescent fusion proteins with G
1 or a mutant
G
5 impaired in its ability to bind to RGS7 demonstrated
nuclear localization in transfected PC12 cells. These findings suggest
that G
5 undergoes nuclear translocation in neurons via
an RGS-dependent mechanism. The novel intracellular
distribution of G
5·RGS protein complexes suggests a
potential role in neurons communicating between classical
heterotrimeric G protein subunits and/or their effectors at the plasma
membrane and the cell nucleus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit genes
(G
1-5) have been identified by molecular cloning. Whereas the G
1-4 isoforms are highly homologous
(80-90%) and widely expressed (2), the G
5 isoform
exhibits much less homology with other isoforms (~50%) and is
preferentially expressed in brain (3). A splice variant of
G
5, G
5-long (G
5L), is present in retina, which contains a 42-amino acid N-terminal
extension (4).
5 can be shown to interact with classical
components of heterotrimeric G proteins signaling pathways such as
G
2 (3, 5, 6), G
q (6), and phospholipase C-
(3, 5, 7, 8) when
tested in vitro, no such interactions have been demonstrated
in native tissues. Instead, G
5 has been purified from
retinal cytosol bound to regulator of G protein signaling (RGS)1 protein-7 (RGS7) (9,
10) and from brain bound to RGS6 (11) and RGS7 (10, 11). Additionally,
a tight native complex between G
5L and RGS9 was isolated
from retinal rod outer segment membrane extracts (12). Studies of
recombinant proteins in vitro show that a tight interaction
with RGS proteins 6, 7, and 11 is demonstrable for G
5
and G
5L but not for the other G
isoforms, mediated by a G
-like (GGL) domain present in a subfamily of RGS proteins (13-16). These recent novel observations underscore the view of G
5 as a unique and highly specialized G protein subunit
but leave open the question of its function within the brain.
5. The results demonstrate that
G
5, along with RGS7, is expressed prominently in the
neuronal nucleus, as well as the cell membrane and cytosol. This
distribution pattern suggests that G
5·RGS
complexes may shuttle information between classical G protein-signaling
elements at the plasma membrane and the cell nucleus.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 C-terminal antibody SGS/1 (5)
immunoreactivity eluting at ~0.15 M NaCl were identified
and pooled. Samples of partially purified nuclear extract and parallel
samples of mouse brain membrane extract were treated with
endoproteinase Lys-C or Glu-C (V8 protease) (Calbiochem) at an
enzyme:protein ratio of 1:50 (w/w) for 30 min at 30 °C and analyzed
by SGS antibody immunoblotting as described (5).
5 (11), rabbit polyclonal antibody SGS against the C
terminus of G
5 (5), goat anti-RGS7 C-19 and R-20 IgG
(Santa Cruz), rabbit anti-TFIID (TBP) N-12 IgG (Santa Cruz), anti-GFP
monoclonal antibody (CLONTECH), and
affinity-purified rabbit KT polyclonal antibody against the N terminus
of G
1 (21). Immunoprecipitation from brain nuclear extracts or PC12 cell lysates employed goat anti-RGS7 C-19 or normal
goat IgG following the previously described methodology (11).
5 cDNA, mouse brain G
5 cDNA (3, 5) was used as
template for the polymerase chain reaction employing Pfu
polymerase (Stratagene) and primers encoding a 5' in-frame influenza
virus HA epitope YPYDVPDYA, and cloned between HindIII and
XbaI sites within the multilinker site of pTRE
(CLONTECH) to create pTRE-HAG
5. A PC12 cell line
stably transfected with pTet-On (CLONTECH) was then
subsequently transfected with the pTRE-HAG
5 plasmid or pTRE vector
only as follows. About 5 × 106 of log phase PC12
cells were harvested by centrifugation and resuspended in 0.4 ml of
DMEM without fetal bovine serum. The cell suspension was
transferred to an electroporation cuvette and mixed with 15 µg of
pTRE-HAG
5 plasmid or pTRE vector and 1 µg of pTK-HG plasmid
(encoding the hygromycin resistance gene). After the mixture was
incubated in the hood for 10 min, the cells were electroporated at 220 V and 950 microfarads. Cells were immediately transferred to 10 ml of
fresh DMEM containing 100 µg/ml G418 in a 100-mm dish (biocoated) and
incubated at 37 °C. After a 24-h incubation, hygromycin was added to
a final concentration of 200 µg/ml, and the culture was placed back
in the incubator until individual colonies were visible. Individual
colonies were expanded, and the level of HA-G
5 gene
expression was determined by Western blot analysis. Peak protein
expression upon doxycycline induction (2.5 µg/ml) occurred in 6-12 h.
5 fusion expression plasmid, wild-type
G
5 cDNA was cloned in-frame downstream of a
red-shifted GFP variant in the pEGFP-C2 vector
(CLONTECH) using the Rapid Ligation Kit (Roche).
The starting methionine of G
5 in this construct was eliminated by
mutation into alanine using the QuickChange site-direct mutagenesis kit
(Stratagene, La Jolla, CA). The resultant construct was named
pEGFP-G
5. For construction of the
pEGFP-G
5-L22P,L26P mutant, codons corresponding to
leucine residues 22 and 26 of the wild-type G
5 sequence within the
pEGFP-G
5 construct were altered to prolines using the
QuickChange kit. For construction of the GFP-G
1 fusion
protein, the G
1 cDNA was polymerase chain reaction
amplified using a 5'-primer that converted the starting methionine to
alanine, and the resulting polymerase chain reaction product was
ligated in-frame into the pEGFP-C2 vector. The resultant construct was
named pEGFP-G
1. For transient expression of RGS7, codons
2-469 (full-length except for the starting methionine) of bovine RGS7
(kindly provided by Dr. Vladlen Slepak) were amplified by polymerase
chain reaction employing the Pwo polymerase (Boehringer Mannheim). Primers encoding 5'- and 3'-BamHI linkers were
employed, and after digestion the resulting construct was ligated
in-frame into the BamHI site of pcDNA4/HisMax-C vector
(Invitrogen), which adds N-terminal His6 and Xpress epitope tags. The
DNA sequence of all inserts was verified by dideoxy sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 in PC12
Cells--
Biochemical analysis of fractions prepared from mouse (4,
11) and rat (10, 22) brain homogenates demonstrated previously G
5 association with both membrane and cytosolic
fractions. To resolve and analyze the subcellular distribution of
G
5 better, a homogeneous population of neuronal cells in
continuous culture such as PC12 cells, which upon differentiation
assume a neuron-like phenotype, offers many advantages over brain. The
expression of G
5 mRNA and protein in rat
pheochromocytoma PC12 cells as well as several other cell lines of
neuroendocrine origin was recently documented by ribonuclease
protection and immunoblotting, respectively (23). The levels of
G
5 expression in PC12 cells was not altered significantly by NGF treatment and differentiation into sympathetic neuron-like cells (23). We therefore studied the intracellular distribution of G
5 in NGF-differentiated PC12 cells by
immunocytochemical methods (Fig. 1,
A and B).
Unexpectedly, an epifluorescent signal over the nucleus, in addition to
diffuse cytoplasmic staining, was evident with ATDG antibody
directed against the N terminus of G
5 (11) (Fig.
1A). Cells processed in parallel with preimmune antibodies
produced a weaker background signal (Fig. 1B). To confirm that the G
5 immunofluorescent signal over the nucleus
seen by light microscopy was indeed associated with the nucleus,
subcellular fractionation of PC12 cells and immunoblotting analysis
with two different antibodies to G
5 were performed:
N-terminally directed ATDG and the C-terminally directed SGS antibody
(5) (Fig. 1, E and F). In the resulting
immunoblots both antibodies demonstrated a ~39-kDa immunoreactive
band in the nuclear fraction of PC12 cells of identical mobility to the
G
5 present in the membrane and cytosolic fractions (Fig.
1E, upper panel, and 1F). In contrast, G
1 immunoreactivity was confined to the membrane fraction (Fig. 1E, middle panel). To verify the identity of the
~39-kDa immunoreactive band in the PC12 cell nuclear fraction as
G
5, immunoblots with antibody SGS directed against the C terminus of
G
5(5) were performed on control and experimental samples subjected
to limited proteolytic digestion (5) (Fig. 1G). Partial
digestion with endoproteinase Lys-C resulted in major C-terminal
fragments of ~22 kDa and 12 kDa, whereas treatment with V8 protease
resulted in a major ~35-kDa C-terminal immunoreactive fragment (Fig.
1G). The C-terminal fragments were identical in mobility to
those generated in parallel from mouse brain membranes (Fig.
1G) and to those generated from recombinant G
5 as
described previously (5).
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Fig. 1.
Immunohistochemical and biochemical analysis
of G 5 and RGS7 in naïve
PC12 cells. Panels A-D, epifluorescence microscopic
images of untransfected NGF-differentiated PC12 cells processed for
immunohistochemistry with either affinity-purified ATDG
anti-G
5 antibody (11) (A), the corresponding
preimmune rabbit IgG (B), goat anti-RGS7 (C-19) antibody
(C), or control goat IgG (D) as described under
"Experimental Procedures." Panels E and F,
immunoblots of PC12 cell subcellular fractions (M, crude
membrane fraction; C, cytoplasm; N, nuclear
fraction) with the indicated antibodies and the relative mobility of
the major immunoreactive bands indicated on the right in
kDa. Anti-G
5 blots employ either the N-terminally
directed antibody ATDG (11) or the C-terminal antibody SGS (5), as
shown. The subcellular fractionations shown in panels E and
F represent two independent preparations. Panel
G, immunoblot with polyclonal G
5 C-terminal
antibody SGS (5) of mouse brain membrane extract (left
lanes) or partially purified PC12 nuclear extract (right
lanes) either untreated (C) or subjected to limited
proteolysis with either endoproteinase Lys-C (Lys-C) or
Glu-C (V8), as described under "Experimental
Procedures." The relative mobility of the major immunoreactive bands
is indicated on the right in kDa.
5 and RGS7 in
Stably Transfected PC12 Cells by Confocal Microscopy--
To increase
the signal to noise ratio for further immunofluorescence studies, PC12
cells were stably transfected with an N-terminally HA epitope-tagged
G
5 cDNA under the control of an inducible promoter. When induced
by doxycycline in NGF-differentiated PC12 cells, the pattern of G
5
immunoreactivity was the same as that seen by epifluorescence in the
naïve PC12 cells, with strong cytoplasmic and nuclear
expression whether analyzed with ATDG antibody (Fig. 2,
A and C) or anti-HA
antibody (not shown). The nuclear localization was confirmed by
confocal microscopy and dual immunofluorescence analysis with the
DNA-binding cyanine dye YOYO (18) (Fig. 2, D-F). Three
groups have independently isolated tight native complexes of
G
5 bound to RGS7 from rodent brain (10, 11, 24). We therefore examined the cells for evidence of nuclear expression of
RGS7. Confocal microscopic analysis of cells probed with antibody to
the C terminus of RGS7 revealed strong cytoplasmic and a weaker, patchy
nuclear staining pattern(Fig. 2B). A similar distribution of
RGS7 was evident in naïve PC12 cells analyzed with the same antibody (Fig. 1C; compare with 1D). Dual
immunofluorescence analysis with both G
5 and RGS7 antibodies
revealed colocalization of proteins as evidenced by the faint orange
coloration of nuclei staining with both antibodies (Fig. 2,
B and C).
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Fig. 2.
Confocal dual immunofluorescence
analysis of stably transfected, NGF-differentiated PC12 cells.
Cells stably transfected with pTRE-HAG 5 encoding HA epitope-tagged
G
5 were induced with doxycycline and analyzed by laser
confocal microscopy after dual staining with affinity-purified ATDG
anti-G
5 antibody (11) (red signal) and
anti-RGS7 (C-19) antibody (green signal) (panels
A-C), or ATDG antibody and the nuclear dye YOYO (18)
(green signal) (panels D-F) as described under
"Experimental Procedures." Immunofluorescence was monitored singly
or in combination (merge) as indicated. The
yellow-orange signal indicates colocalization of
probes.
5 and RGS7 in
Brain--
Mouse brain was studied to probe the generality of the
G
5 expression pattern determined in naïve and transfected
sympathetic neuron-like PC12 cells. Analysis of mouse brain subcellular
fractions was performed by immunoblotting (Fig.
3A). This approach was
preferable over immunocytochemical analysis insofar as a recent
confocal dual immunofluorescence study with antibodies to
G
5 and RGS7 documented their colocalization in many
regions of rat brain but reached no conclusion about their subcellular
distribution (24). As in PC12 cells, G
5 immunoreactivity
at 39 kDa was evident in the membrane, cytosolic, and nuclear fractions
of mouse brain, whereas the G
1 36-kDa immunoreactive band was
confined to the membrane fraction (Fig. 3A). Antibodies to
G
2/
3 reacted strongly only with a ~6.5-kDa band in the membrane
fraction (Fig. 3A). Immunoblotting with two different
antibodies to RGS7, C-19 and R-20, yielded bands in all three
subcellular fractions. In the membrane and cytosolic fractions the
immunoreactivity of the major band at ~55 kDa, but not the reactivity
of a faint upper band in the cytosol, could be blocked by preincubation
with the cognate peptide (Fig. 3A). In the nuclear fraction
a doublet of immunoreactive bands migrating slightly slower than the
~55kDa membrane and cytosolic bands disappeared with peptide
preincubation (Fig. 3A), confirming their identity as
RGS7-related proteins. Interestingly, a recent analysis of the
intracellular distribution of epitope-tagged RGS4 in transfected COS-7
cells also found preferential nuclear expression of a slower migrating
species (25). The nature and significance of the slower migrating forms
of RGS7 in brain nuclei are unclear.
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Fig. 3.
Immunoblot analysis of mouse brain
subcellular fractions and immunoprecipitation analysis of the brain
nuclear fraction. Panel A, mouse brain homogenate was
fractionated into crude membrane (M), cytoplasmic
(C), and nuclear (N) fractions and analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting with the
antibodies shown as described under "Experimental Procedures."
Anti-G 5 blots employed the N-terminally directed
antibody ATDG (11). The RGS7 immunoblots employed either antibody C-19
or R-20 (Santa Cruz) as shown. In the final panel the R-20 antibody was
preadsorbed with the cognate peptide. The relative mobility of the
major immunoreactive bands is given on the right in kDa.
Panel B, mouse brain nuclear extract was incubated with
either goat anti-RGS7 C-terminal antibody C-19 or normal goat IgG, and
after precipitation the washed immunoprecipitates were analyzed for
RGS7 and G
5 immunoreactivity by immunoblotting as
described under "Experimental Procedures." The relative mobility of
the specific immunoreactive bands (in kDa) and the goat immunoglobulin
heavy chain (HC) is indicated on the right.
5 and RGS6 and RGS7
present in brain membranes and cytosol have been demonstrated in
previous studies (10, 11, 24), and the presence of both
G
5 and RGS7 in the brain nuclear fraction raises the
question of their possible interaction in that compartment. To check
for possible complex formation between the RGS7 and G
5
in the brain nuclear fraction, immunoprecipitation with C-terminally
directed RGS7 antibody was performed (Fig. 3B). Both an
RGS7-immunoreactive band at 55 kDa and a
G
5-immunoreactive band at 39 kDa were evident in
anti-RGS7 immunoprecipitates but not in control immunoprecipitates
employing normal goat IgG. These results are consistent with a tight
association between G
5 and RGS7 in the mouse brain
nuclear fraction.
5--
The colocalization of G
5 with
RGS7 in the nuclei of PC12 cells (Fig. 2) taken together with the
results above demonstrating coimmunoprecipitation of G
5
with RGS7 from brain nuclear extract (Fig. 3B) suggest that
nuclear G
5 is present as a heterodimer with one or more
GGL domain-containing RGS proteins. To check if such heterodimerization
might be required for proper nuclear localization of G
5,
three chimeric protein constructs containing GFP were prepared and
studied in transiently transfected PC12 cells. These chimeras contained
GFP fused in frame to (a) wild-type G
5 (GFP-
G
5), (b) a double point mutant
G
5 in which leucines at positions 22 and 26 were mutated
to prolines (GFP-G
5-L22P,L26P), or (c)
G
1 (GFP-G
1), the last serving as a
negative control. The leucine residues mutated in GFP-
G
5-L22P,L26P were chosen so as to disrupt the postulated
N-terminal coiled-coil region of G
5 comprising the
putative RGS dimerization interface (13, 14) (Fig.
4A).
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Fig. 4.
Analysis of
GFP-G 5,
-G
1, and
-G
5-L22P,L26P fusion protein
expression and RGS protein interaction in transiently transfected PC12
cells. Panel A, diagram of GFP-G
5 fusion
protein construct and position of leucine to proline mutations in
consensus hydrophobic positions within the putative G
5
N-terminal coiled-coil region in the GFP-G
5-L22P,L26P
mutant. Panel B, immunofluorescence analysis of PC12 cells
transiently transfected with the indicated GFP fusion constructs.
Panel C, immunoblotting analysis of PC12 lysates from cells
transiently transfected with empty vector (control) or the
GFP fusion constructs indicated. Antibodies employed for the
immunoblots are shown on the left (SGS antibody, upper
panel; anti-GFP, lower panel), and the positions of the
major immunoreactive bands at ~65 kDa are shown on the
right. For both antibodies, faint nonspecific staining
(present even in control cells) of faster migrating bands is evident in
all lanes. Panel D, PC12 cell lysates prepared 2 days after
transient transfection were divided and incubated with either goat
anti-RGS7 C-terminal antibody C-19 or normal goat IgG. After
precipitation, the washed immunoprecipitates were analyzed for RGS7
(upper panel) and G
5 (SGS antibody,
lower panel) immunoreactivity by immunoblotting as described
under "Experimental Procedures." The relative mobility of the
specific immunoreactive bands (in kDa) and the goat immunoglobulin
heavy chain (HC) is indicated on the right. Cells
were transfected with either vector alone or with cDNAs encoding
GFP-G
5 + RGS7, or GFP-G
5-L22P,L26P + RGS7
as indicated below the lanes.
5 was to disrupt interaction with GGL domain-containing RGS proteins (13,
14), as evidenced by the greatly diminished ability of the
GFP-G
5 mutant to coimmunoprecipitate with RGS7 in
transfected PC12 cells under conditions permissive for the wild-type
GFP-G
5 fusion (Fig. 4D).
5 in naïve and stably transfected PC12
cells analyzed immunocytochemically, the fluorescence of GFP-
G
5 was strong in the nuclei of the majority of
transiently transfected PC12 cells (Fig. 4B, left
panel). In contrast the pattern of signal in GFP-
G
5-L22P,L26P and GFP- G
1 transfected
cells showed only faint fluorescence in the cell nuclei of most labeled
cells (Fig. 4B, right and middle
panels). Such a faint background nuclear signal is often seen with
GFP fusion proteins targeted predominantly to the cytosol and may be
related to the ability of the GFP moiety to enter the nucleus passively
(26). The anomalous cytosolic expression of the GFP-G
1
fusion compared with endogenous G
1 (compare Figs.
1E and 3A) is likely caused by impaired G
binding and/or isoprenylation in the context of GFP-
G
1·G
heterodimers. Cytosolic expression of GFP-G
fusion proteins has been noted previously by other laboratories (27,
28).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 and provides the first example of a
heterotrimeric G
subunit expressed in the neuronal cell nucleus.
G
1 was found in the nucleus of growth factor-stimulated
Swiss 3T3 cells as part of a heterotrimer with Gi
,
although it was absent from the nuclear fraction in quiescent cells
(29). In neurons several G
isoforms have been found to undergo
retrograde axonal transport and localize in the cell nucleus (for
review, see Ref. 30). Whether any functional interactions might exist
between G
5·RGS complexes or perhaps other RGS proteins
(25, 31) and G
subunits within the neuronal nuclear compartment
remains to be shown.
5 in
neurons and brain is unclear. The primary sequence of G
5
lacks an obvious consensus nuclear localization signal (NLS) (32).
Furthermore, its sequence is wholly contained within the retinal splice
variant G
5L, which is strongly membrane-anchored (4, 12)
and is therefore unlikely to contain a functional NLS.
5, which blocks its ability to form tight complexes with the GGL domain-containing RGS7, also prevents nuclear expression of the GFP fusion protein suggests that RGS7 (or other G
5-binding RGS proteins (14)) may contain a NLS. Recent
evidence that other RGS proteins including RGS2, RGS10 (25), and a
truncated version of RGS3 (RGS3T) (31) exhibit nuclear localization and contain putative NLS sequences makes this possibility more likely. Neither RGS6 nor RGS7 contains the Arg-Lys-Arg-Lys and Arg-Arg-Arg NLS
sequence postulated for RGS3T (31) nor the Lys-Lys-Xaa-Lys/Arg putative
NLS motif present in RGS2, RGS4, and RGS16 (25). Nevertheless, RGS6 and
RGS7 contain other sequence patterns suggestive of NLS (32) including
Lys454-Lys-Lys-Pro and Lys370-Lys/Arg-Arg-Pro
present in the long splice variant of RGS6 (15) and RGS7 (33),
respectively. Whether these or other candidate sequences in RGS6 and
RGS7 actually function as NLS remains to be tested experimentally, however.
5 nuclear staining. This result might be expected because RGS7 is only one of several G
5-binding RGS partners present in PC12 cells. Indeed,
preliminary analysis of PC12 cells and mouse brain with anti-RGS6
antibody demonstrates nuclear expression similar to that of RGS7 (not
shown). Other GGL domain-containing RGS proteins such as RGS9 and 11 expressed in brain may also be present with G
5 in the nucleus.
5 and RGS7 to the nuclear
compartment regulated or constitutive? Recently a
phosphorylation-dependent interaction of RGS7 with 14-3-3 proteins was demonstrated by Benzing et al. (34).
Interaction with 14-3-3 proteins is known to govern the nuclear
localization of the Cdc25 phosphatase (35) and certain transcription
factors (36) and might possibly regulate the nuclear targeting of
G
5 and RGS7. Preliminary experiments testing the influence of various ligands for endogenous receptors in PC12 cells on
the subcellular distribution of transfected GFP-G
5
fusion protein in living cells revealed no evident alterations at the light microscopic level (not shown). Nevertheless the question of
possible cell surface receptor regulation of G
5 and RGS7
nuclear targeting will require more thorough investigation.
5 and RGS7 in the nucleus remains
unknown. It is conceivable that G
5 and/or RGS7 may
directly or indirectly regulate nuclear transcription. If this were
true, how would such a function relate to the well documented ability
of domains within G
5·RGS complexes to interact with
classical heterotrimeric G protein subunits and/or their effectors when
tested in vitro? The distribution of G
5·RGS
heterodimers (and likley other RGS proteins (25, 31)) among membrane,
cytoplasmic, and nuclear compartments might allow for information
transfer between signaling elements at the plasma membrane and protein
targets in the cell nucleus. Thus as has been shown for RGS proteins
such as p115 RhoGEF (37, 38) and PDZ-RhoGEF (39) perhaps
G
5·RGS heterodimers function as intracellular signal transducers.
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ACKNOWLEDGEMENTS |
---|
We thank George Poy for oligonucleotide synthesis and DNA sequencing, Dr. Carolyn Smith for sharing her expertise in confocal imaging, Dr. Vladlen Slepak for the RGS7 cDNA, and Dr. Allen Spiegel for continued support and encouragement.
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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: Clinical Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892.
Present address: Dept. of Biochemistry and Cell Biology, State
University of New York at Stony Brook, Stony Brook, NY 11794.
** To whom correspondence should be addressed: Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bldg. 10, Rm. 8C-101, 10 Center Dr., MSC 1752, Bethesda, MD 20892-1752. Tel.: 301-496-9299; Fax: 301-402-0374, E-mail: wfs@helix.nih.gov.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009247200
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ABBREVIATIONS |
---|
The abbreviations used are:
RGS, regulators of G
protein signaling;
G-like, GGL;
DMEM, Dulbecco's modified Eagle's
medium;
NGF, nerve growth factor;
AEBST, 4-(2-aminoethyl)benzenesulfonyl fluoride;
GFP, green fluorescent
protein;
HA, hemagglutinin;
NLS, nuclear localization signal.
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