From the Metabolic Diseases Branch, NIDDK and § Confocal Microscopy Core Facility, Division of Intramural Research, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, July 20, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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The signal transducing function of
G In eukaryotic cells seven transmembrane-spanning receptors
regulate intracellular processes in response to extracellular signals through their interaction with signal-transducing heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) (1). Complementary DNAs from five G protein G We recently demonstrated the nuclear expression of G Identification of G cDNAs and Mutagenesis of G Cell Culture--
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) (supplemented DMEM). HEK-293 cells
were grown as above except that 10% fetal bovine serum and no horse
serum were used in the medium.
Transient Transfection of PC12 and HEK-293 Cells--
One day
prior to transient transfection, PC12 cells at 90% confluence were
harvested and plated in 2 ml of supplemented DMEM into the chambers of
poly-D-lysine-coated chamber slides. The number of cells
was adjusted to 70% of their density at harvest. After overnight
incubation of the cells, 1.5 µg of plasmid DNA and 4 µl of
LipofectAMINE 2000 reagent (Invitrogen), diluted in 100 µl of
Opti-MEMI medium, were mixed, incubated at room temperature for 20 min,
and added directly to each chamber. After mixing gently, the cultures
were incubated for 24-48 h prior to confocal microscopic analysis.
HEK-293 cells were transfected in 75-cm2 culture flasks
using either LipofectAMINE 2000 or Superfect transfection reagent
(Qiagen) according to the manufacturer's recommendations. Typically 10 µg of total plasmid DNA and 60 µl of LipofectAMINE 2000 reagent or
15 µg of total plasmid DNA and 40 µl of Superfect reagent were used
per 75-cm2 culture flask.
Gel Electrophoresis, Immunoblotting, and
Immunoprecipitation--
Protein samples were separated on 4-20%
gradient (for analysis of G Inositide-specific Phospholipase C Activity
Determination--
To determine the G Subcellular Fractionation of HEK-293 Cells--
48 h following
transfection, HEK-293 cells were harvested from a 75-cm2
flask, split into two fractions, and pelleted by centrifugation at
500 × g for 5 min at 4 °C. One cell pellet was used
to isolate cell nuclei and generate nuclear extracts using a
commercially available cell lysis kit according to the manufacturer's
instructions (NE-PER Reagents, Pierce). The other cell pellet was
resuspended in ice-cold lysis buffer (25 mM HEPES, pH 7.5, 0.3 M NaCl, 0.2 mM EDTA, 1.5 mM
MgCl2, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate containing 1× protease inhibitor mixture (Calbiochem Set
III)). After removal of the insoluble material by centrifugation, the
supernatant fraction was employed as whole cell lysate.
Immunocytochemistry and Confocal Laser Microscopy--
PC12
cells were processed for immunofluorescent staining as described
previously (30). Briefly, PC12 cells were plated onto poly-D-lysine pre-coated covered chamber slides (Lab-Tek
II, Nalge) and grown in supplemented DMEM at 37 °C for 16 h.
The medium was discarded, and the cells were washed and then fixed in
2% (v/v) formaldehyde in Dulbecco's phosphate-buffered saline
(Biofluids, Rockville, MD). The slides were then incubated with one or
more primary antibodies in Dulbecco's phosphate-buffered saline, 10% fetal calf serum (v/v), and 0.075% (w/v) saponin for 1 h, washed, and then incubated with appropriate labeled secondary antibody (fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (H + L)
(Jackson ImmunoResearch, West Grove, PA)) and/or Rhodamine RedTM-X goat anti-mouse IgG (H + L) (Molecular Probes)) in
the same buffer for 45 min. After staining, 1-2 drops of Permount
(Vector Laboratories) was added to the sample surface. Confocal images were collected using a Zeiss LSM 510 laser scanning confocal microscope with a 100×/1.3 N.A. Plan-Neofluar objective. Scans were performed sequentially using 488 and 543 nm excitation and bandpass emission filters of 505-550 and 560-615 nm, respectively, for Rhodamine Red
and fluorescein to eliminate spectral bleed through of fluorescence between the red and green channels. The pinhole in all cases was adjusted to produce a 1.5-µm optical slice thickness. Transmitted light differential interference contrast images were collected simultaneously with the green dye using 488 nm excitation. Final images
were processed using Zeiss LSM software.
Design and Expression of Putative G
Analysis of the coding sequences of G
Modeling of a G
Both G Interactions of Putative G
The ability of the putative G
The ability of the putative G RGS7 Requirement for Nuclear Expression of G
The expression and subcellular localization of HA epitope-tagged
G Subcellular Localization of G The unique ability of G Several observations in this study suggest G In previous studies of G The studies described herein strongly suggest G Demonstration of a requirement for RGS protein binding is consistent
with several reports demonstrating the nuclear targeting of RGS2
(42-44), RGS10 (42, 45), splice variants of RGS3 (46), and RGS12 (47).
Mutation of a key serine residue in a conserved 14-3-3-binding site in
the core RGS domain of RGS7 (Ser434 5 in brain is unknown. When studied in
vitro G
5 is the only heterotrimeric G
subunit known to interact with both G
subunits and regulators of G protein signaling (RGS) proteins. When tested with G
, G
5
interacts with other classical components of heterotrimeric G protein
signaling pathways such as G
and phospholipase C-
. We recently
demonstrated nuclear expression of G
5 in neurons and
brain (Zhang, J. H., Barr, V. A., Mo, Y., Rojkova, A. M., Liu, S., and Simonds, W. F. (2001) J. Biol.
Chem. 276, 10284-10289). To gain further insight into the
mechanism of G
5 nuclear localization, we generated a G
5 mutant deficient in its ability to interact with RGS7
while retaining its ability to bind G
, and we compared its
properties to the wild-type G
5. In HEK-293 cells
co-transfection of RGS7 but not G
2 supported expression
in the nuclear fraction of transfected wild-type G
5. In
contrast the G
-preferring G
5 mutant was not expressed
in the HEK-293 cell nuclear fraction with either co-transfectant. The
G
-selective G
5 mutant was also excluded from the cell
nucleus of transfected PC12 cells analyzed by laser confocal
microscopy. These results define a requirement for RGS protein binding
for G
5 nuclear expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit genes
(G
1-5) have been identified by molecular cloning. The
G
5 isoform shares much less homology with other isoforms
(~50%) and is preferentially expressed in brain (2). A splice
variant of G
5, G
5-long
(G
5L), is present in retina that contains a 42-amino
acid N-terminal extension (3).
5 is the only G
subunit known with the potential to
assemble with either G
subunits or regulators of G protein signaling (RGS)1 proteins.
G
5 can heterodimerize with G
(2, 4-6) and interact with other classical components of heterotrimeric G proteins signaling pathways such as G
(5, 6) and phospholipase C-
(2, 4, 6-8) when
tested in vitro. Other in vitro studies show that
G
5 and G
5L, but not the other G
isoforms, can form tight heterodimers with RGS proteins 6, 7, and 11, an interaction mediated by a G
-like (GGL) domain present in a
subfamily of RGS proteins (9-11). In native tissues, however,
G
5 has been purified not as a heterodimer with G
but
instead bound to RGS6 (12) and RGS7 (12-15). The failure to
demonstrate native G
5-G
complexes is complicated by
the known instability of such complexes in detergent solution (10, 16).
Thus, purifications employing detergent may fail to identify potential
G
5-G
complexes that could dissociate in the process
of isolation (16). Alternative approaches to assess the biologic
importance of the G
5-G
interaction demonstrated in vitro are needed.
5 in
neurons and brain (17). In this study chimeric protein constructs containing green fluorescent protein (GFP) fused to wild-type G
5 demonstrated nuclear localization in transfected PC12
cells, but not GFP fusions with a mutant G
5 with proline
substitutions in its putative coiled-coil domain. Introduction of
prolines into the putative
-helical N-terminal region of
G
5 would disrupt its ability to form a coiled-coil with
both G
subunits and RGS proteins containing GGL domains. Such a
G
5 mutant therefore fails to provide mechanistic insight
as to which of its two potential heterodimeric conformations is
important for nuclear targeting. We describe here a G
-selective
G
5 mutant that fails to undergo nuclear localization in
transfected PC12 and HEK-293 cells. In the latter cells the nuclear
expression of wild-type G
5 is dependent on RGS7
co-transfection. These results suggest it is interaction with the GGL
domain containing RGS proteins that directs nuclear localization of
G
5. Additional studies of RGS7 do not support a role for
a putative interaction with 14-3-3 proteins in G
5 nuclear localization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5-specific Residues in the
Putative Coiled-coil Domain of G
5--
The Pileup
algorithm of the University of Wisconsin Genetics Computer Group (18)
was used to align the coding sequences of human (GenBankTM
accession number NM_006578) (19), tiger salamander
(GenBankTM accession number AF369757) (20),
Drosophila melanogaster (GenBankTM accession
number AAF46336), and Caenorhabditis elegans (GenBankTM accession number AF291847) (21-23)
G
5 with mammalian G
subunits 1-4. Amino acids in the
line up that were identical among the four G
5 sequences,
but not found in the corresponding positions of G
1-4,
and belonging to a different amino acid homology group from the
corresponding positions of G
1-4 were identified and
considered to be conserved, G
5-specific residues. For
the purposes of this analysis the amino acid homology groups were taken
to be basic (His, Lys, and Arg), acidic (Asp and Glu), polar uncharged (Cys, Gly, Asn, Gln, Ser, Thr, and Tyr), and nonpolar (Ala,
Phe, Ile, Leu, Met, Pro, Val, and Trp). The portion of this analysis
including the N-terminal coiled-coil region of the G
subunits is
shown in Fig. 1.
5 and
RGS7--
Expression constructs encoding full-length murine
G
5 in pcDNA3 (4) and bovine RGS7 in
pcDNA4/HisMax-C (17), which adds N-terminal His6 and
Xpress epitope tags, have been described previously. N-terminally HA
epitope-tagged G
5 in pcDNA3 (coding sequence inserted between HindIII and BamHI sites) was
created by PCR to give the following sequence,
MAYPYDVPDYAEFKAA ... , in which the starting methionine and the alanine corresponding to position 2 in the
wild-type sequence are underlined, flanking 14 residues of inserted
sequence including the nonapeptide HA epitope (24). The mutants
HA-G
5-QAAC (G
5-Lys25
Gln/Glu29
Ala/Lys32
Ala/Leu33
Cys) and HA-G
5-YMIN
(G
5-Phe93
Tyr/Thr338
Met/Val351
Ile/Ala353
Asn) were
generated by PCR from a template of wild-type HA-G
5 in
pcDNA3 employing the Pwo polymerase (Roche Molecular
Biochemicals) by overlap extension as described previously (25).
N-terminally HA epitope-tagged G
1 in pcDNA3 (coding
sequence inserted between EcoRI and XbaI sites)
was created by PCR to give the following sequence,
MAYPYDVPDYAGS ... , in which the
starting methionine and the serine corresponding to position 2 in the
wild-type sequence are underlined, flanking 11 residues of inserted
sequence including the nonapeptide HA epitope (24). The point mutant
RGS7-Ser434
Ala (S434A) was generated by PCR from a
template of wild-type bovine RGS7 in pcDNA4/HisMax-C (17) employing
the Pwo polymerase by overlap extension (25).
G
2 in pcDNA4/HisMax-C vector (BamHI to
NotI) was generated by Pwo polymerase PCR
using bovine G
2 as a template (26). The DNA sequence of
all inserts was verified by dideoxy sequencing (27).
5, RGS7, or TFIID (TBP)
protein expression) or uniform 14% (for analysis of G
expression)
polyacrylamide slab gels (NOVEX) by SDS-PAGE. Proteins were then
transferred by electrophoresis to 0.45-µm nitrocellulose
membranes (28), and after primary and secondary antibody incubation,
chemiluminescent signal was detected using the SuperSignal® West
Femtostable Peroxide Buffer (Pierce) according to the manufacturer's
instructions. Primary antibodies used are as follows: affinity-purified
rabbit ATDG polyclonal antibody against N terminus of G
5
(12), rabbit polyclonal antibody SGS against the C terminus of
G
5 (4); goat anti-RGS7 C-19 IgG (sc-8139), rabbit
polyclonal antibody against a GST fusion protein containing residues of
312-469 of bovine RGS7 (7RC-1); rabbit anti-TFIID (TBP) N-12 IgG
(Santa Cruz Biotechnology); rabbit anti-G
2 A-16 IgG
(sc-374) (Santa Cruz Biotechnology); and anti-Xpress epitope
(Invitrogen) and HA.11 anti-hemagglutinin (HA) epitope (Covance) mouse
monoclonal antibodies. Secondary antibodies employed were horseradish
peroxidase-coupled donkey anti-rabbit (Amersham Biosciences) to detect
rabbit polyclonal antibodies, horseradish peroxidase-coupled donkey
anti-goat (Santa Cruz Biotechnology) for goat primary antibodies, and
horseradish peroxidase-coupled goat anti-mouse IgG1 (Santa
Cruz Biotechnology) for mouse monoclonal antibodies.
Immunoprecipitation from detergent extracts of transfected HEK-293
cells employed HA.11 monoclonal antibodies and Protein A/G Plus-agarose
beads (Santa Cruz Biotechnology) following the methodology described
previously (12), except that buffer A was used. Buffer A consisted of
0.15% (w/v) polyoxyethylene (10) monolauryl ether (Genapol C-100®)
detergent, 150 mM NaCl, 20 mM Na-HEPES, pH
7.4, 1× protease inhibitor mixture (Calbiochem Set III, number 539134).
-dependent
activation of inositide phospholipid-specific phospholipase C (PLC) by
wild-type and mutant G
5 constructs, HEK-293 cells
metabolically labeled with [3H]inositol and
co-transfected with human PLC-
2 were employed, and the
[3H]inositol phosphates isolated by the method of
Berridge et al. (29) with modifications as described
previously (4). The PLC activity in cells co-transfected with vector
only was compared with that in cells transfected with
G
5 construct without or with G
2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-selective G
5
Mutants--
In order to assess the potential biologic importance of
the G
5-G
interaction for the nuclear localization of
G
5, a G
5 mutant that retained
demonstrable G
interaction with selective loss of RGS protein
binding was desired. By analogy with the crystal structures of G
protein heterotrimers (31, 32) and free
G
1
1 complex (33), it was expected that
the interaction of G
or an RGS protein GGL domain with
G
5 involved many of the interactions observed in
G
1
. These interactions included a two-stranded
parallel coiled-coil involving N-terminal portions of G
5
and G
/GGL domain and hydrophobic interactions between more
C-terminal regions of G
/GGL domain and the
-propeller structure
encoded by WD-40 repeats of the G
(10, 31-33).
5 of mammalian (2,
19), amphibian (20), insect (GenBankTM accession number
AAF46336), or roundworm (21-23) origin in comparison to mammalian G
subunits 1-4 reveals four conserved residues in the putative
coiled-coil region of the G
5s that are G
5-specific (Fig. 1).
These G
5-specific residues occupy the a,
d, and g positions of the G
5
heptad repeat (Fig. 1), positions that may determine the specificity of
coiled-coil dimerization (reviewed in Ref. 34) and that might
contribute to specific interaction of G
5 with RGS
protein GGL domains (9). In order to create a G
-preferring
G
5 mutant, these four residues in HA epitope-tagged
mammalian G
5 were mutated to their counterparts in
G
1 to produce mutant G
5-QAAC
(G
5-K25Q/ E29A/K32A/L33C).
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Fig. 1.
Identification of
G 5-specific residues in the
putative coiled-coil domain of
G
5. An alignment of the
coding sequences of human, salamander (Sal), D. melanogaster (Dm), and C. elegans
(Ce) G
5 with mammalian G
subunits 1-4 was
performed as described under "Experimental Procedures." The portion
of this analysis that includes the N-terminal region coiled-coil region
of the G
subunits is shown. Amino acids that were identical among
the 4 G
5 sequences, but not found in the corresponding
positions of mammalian G
1-4 and belonging to a
different amino acid homology group from the corresponding positions of
G
1-4, were identified and considered to be conserved,
G
5-specific residues (yellow highlights). The
position of the residues of G
1 in the coiled-coil heptad
repeats, taken from the crystal structures of G
1
complexes (31-33), are indicated as a-g above the
G
1 sequence.
5-GGL domain heterodimer by Snow
et al. (10) suggested that G
5
-propeller
residues comprising a hydrophobic pocket able to accommodate a
conserved Trp residue in the GGL domain (corresponding to Phe64 of
G
1) might also contribute to the selectivity of
G
5-RGS protein assembly. Based on the model by Snow
et al. (10), four
-propeller residues forming the
hydrophobic pocket in HA-tagged G
5 were mutated to their
more bulky G
1 counterparts. The aim of this mutagenesis
was to reduce the size of the putative G
5
-propeller
hydrophobic pocket to exclude the conserved Trp residue in the GGL
domain while still accommodating the Phe residue present in G
corresponding to residue 64 of G
1 (10). These alterations produced mutant G
5-YMIN
(G
5-F93Y/T338M/V351I/A353N).
5 mutants could be expressed to levels comparable
with wild-type G
5 when transiently transfected into
HEK-293 cells (Fig. 2). The expression of
the G
5-QAAC mutant, like that of wild-type
G
5, was greater in cells co-transfected with RGS7 or G
2 than in cells co-transfected with vector only (Fig.
2A, lanes 2-5, cf. lanes
6 and 7). The ability of G
or GGL domain-containing RGS protein co-expression to stabilize G
5 expression has
been observed previously (15, 35) in several systems. Unlike the wild-type G
5, the expression of the
G
5-YMIN mutant was greater in cells co-transfected with
G
2 than in cells co-transfected with RGS7 (Fig.
2B, lanes 6 and 7, cf.
lanes 2-5). Nevertheless, the expression of the
G
5-YMIN mutant was greater in cells co-transfected with
RGS7 than with vector only (Fig. 2B, cf.
lanes 5 and 7). Also wild-type G
5
but not G
5-YMIN mutant co-transfection enhanced the
expression of RGS7 (Fig. 2B, cf. lanes
2 and 7). As expected the C-terminally directed
G
5 antibody SGS failed to recognize the
G
5-YMIN mutant containing two mutations within the SGS
epitope (4) (Fig. 2B, lane 6) but reacted with
the G
5-QAAC mutant as well as with the wild type (data
not shown).
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Fig. 2.
Expression of wild-type and mutant
G 5 subunits in transfected HEK-293
cells. Immunoblots of lysates from transfected cells treated with
vector (vec), wild-type (wt), or mutant
G
5, RGS7, or G
2 cDNAs were prepared
as described under "Experimental Procedures." The cDNA
combinations used for each transfection are indicated below
the immunoblots. The relative mobility of the immunoreactive bands on
SDS-PAGE (in kDa) is shown to the left of each panel, and
the primary antibodies used for the immunoblots are shown to the
right. A, transfections employing mutant
G
5-QAAC (G
5-K25Q/E29A/K32A/L33C).
B, transfections employing mutant
G
5-YMIN (G
5-F93Y/T338M/V351I/A353N).
Shown are results from a single experiment representative of three
similar experiments with comparable results.
-selective G
5 Mutants
with RGS7, G
, and G
-dependent Effectors--
The
ability of the putative G
-selective G
5 mutants to
bind RGS7 and G
subunits was tested in co-immunoprecipitation
assays. In transiently transfected HEK-293 cells, immunoprecipitation of the G
5-QAAC mutant was able to co-immunoprecipitate
RGS7 from cell lysates as well as wild-type G
5 (Fig.
3A, cf. lanes
2 and 3). In contrast only trace RGS7 was evident in
immunoprecipitates of the G
5-YMIN mutant even though the
mutant G
5 itself was precipitated and wild-type
G
5 was able to fully co-precipitate RGS7 in the same
experiment (Fig. 3B, cf. lanes 2 and
3). However, because the expression of RGS7 when
co-transfected with the G
5-YMIN mutant is poor (Fig.
3B, lane 3 of lysates), the absence of RGS7 in
the G
5-YMIN mutant immunoprecipitates cannot be
interpreted.
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Fig. 3.
Selective co-immunoprecipitation of RGS7 with
HA epitope-tagged wild-type and mutant
G 5 subunits in transfected HEK-293
cells. Cell lysates prepared 2 days after transient transfection
were incubated with anti-HA monoclonal antibody, and after
precipitation, the starting lysates and washed immunoprecipitates
(IP) were both analyzed for RGS7 (upper panels)
and epitope-tagged G
5 (HA antibody, lower
panels) immunoreactivity by immunoblotting as described under
"Experimental Procedures." The relative mobility of the specific
immunoreactive bands (in kDa) is indicated to the left of
each panel. Cells were transfected with either vector alone
(vec), or with cDNAs encoding RGS7 and wild-type
(wt), or mutant HA-G
5 as indicated
below the lanes. A, transfections employing
mutant G
5-QAAC (G
5-K25Q/E29A/K32A/L33C).
B, transfections employing mutant
G
5-YMIN (G
5-F93Y/T338M/V351I/A353N). This
experiment was repeated a total of three times with similar
results.
-selective G
5 mutants to
bind G
subunits was separately tested in co-immunoprecipitation
assays from lysates of transiently transfected HEK-293 cells and
compared with wild-type G
5 and G
1 (Fig.
4). Analysis of the lysates demonstrated the co-expression of all transfected G
constructs with
G
2 (Fig. 4A). Neither the wild-type
G
5 nor the G
5-QAAC mutant was able to
co-immunoprecipitate G
2 from cell lysates, however (Fig.
4B, lanes 2 and 4), a finding
consistent with the known instability of
G
5-G
2 binding in detergent solution (10,
16). Unlike wild-type G
5 and G
5-QAAC,
however, both the G
5-YMIN mutant and G
1
clearly co-immunoprecipitated with G
2 under the same conditions (Fig. 4B, lanes 3 and
5).
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Fig. 4.
Expression and selective
co-immunoprecipitation of G 2 with
HA epitope-tagged wild-type or mutant G
subunits in transfected HEK-293 cells. Cell lysates prepared
2 days after transient transfection were incubated with anti-HA
monoclonal antibody, and after precipitation, the starting lysates
(A) and washed immunoprecipitates (IP)
(B) were analyzed in parallel for epitope-tagged G
(HA
antibody, upper panels) and G
2 (lower
panels) immunoreactivity by immunoblotting as described under
"Experimental Procedures." The relative mobility of the specific
immunoreactive bands (in kDa) is indicated to the right of
each panel as is the mobility of the immunoglobulin G heavy chain
(HC) in B. Cells were transfected with either
vector alone (vec) or with cDNAs encoding
G
2, wild-type (wt), mutant
HA-G
5, or wild-type HA-G
1 as indicated
below the lanes. The G
5 mutants employed were
G
5-QAAC (G
5-K25Q/E29A/K32A/L33C) and
G
5-YMIN (G
5-F93Y/T338M/V351I/A353N). A
representative result is shown in an experiment repeated two more times
with similar findings.
-selective G
5 mutants to
activate inositide-specific PLC-
in co-transfected HEK-293 cells in
a G
-dependent fashion was also tested. Wild-type
G
5 activated PLC-
2 in a
G
-dependent fashion in COS-7 cells as described
previously (2, 4). Both mutants G
5-QAAC and
G
5-YMIN activated PLC-
2 in a
G
-dependent fashion indistinguishable from the wild-type G
5 (data not shown).
5 in
HEK-293 Cells--
Because neither endogenous G
5 (36)
nor GGL domain-containing RGS proteins such as RGS7 is expressed in
HEK-293 cells (Fig. 5, A and
B, lane 1), they offer a useful model system to
study the requirements for the expression and subcellular localization of these subunits upon transfection. When transfected alone, wild-type G
5 can be detected in the whole cell lysate but not in
the 293 cell nuclear extract (Fig. 5A, lane 2).
Co-transfection of G
5 with either RGS7 or
G
2 greatly enhances the G
5 signal in the cell lysate (Fig. 5A, lanes 3 and 4,
cf. lane 2), presumably due to subunit
stabilization by heterodimer formation (15, 35), but only RGS7
co-transfection supported expression of G
5 in the nuclear extract (Fig. 5A, lane 3).
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Fig. 5.
Expression and nuclear targeting of wild-type
and mutant G subunits in transfected HEK-293
cells. Cell harvested 2 days after transient transfection with
either wild-type G
5 (A) or HA epitope-tagged
G
5 and G
5 mutants (B) were
processed for either nuclear isolation and extraction (upper
panels) or whole cell lysates (lower panels) as
described under "Experimental Procedures." The relative mobility of
the specific immunoreactive bands (in kDa), including that of TBP, is
indicated to the right of each panel. The antibody used for
immunodetection is indicated to the left of each panel.
Cells were transfected with either vector alone (vec) or
with cDNAs encoding RGS7, G
2, wild-type untagged, or
HA epitope-tagged G
5 or mutant HA-G
5, as
indicated below the lanes. A representative result is shown
in an experiment performed three times with similar findings.
5, G
5-QAAC, and G
5-YMIN
were also studied in transfected HEK-293 cells in similar experiments
(Fig. 5B). As expected HA epitope-tagged wild-type
G
5 was expressed in nuclear extracts upon
co-transfection with RGS7 (Fig. 5B, lane 2) but
not with G
2 (data not shown). Like wild-type
G
5, mutant G
5-QAAC also demonstrated RGS7
dependence for nuclear localization (Fig. 5B, cf.
lanes 3 and 4). In contrast the G
-selective
mutant G
5-YMIN failed to appear in the nuclear extract
with either RGS7 or G
2 co-transfection (Fig.
5B, lanes 5 and 6). The expression of
RGS7 in G
5-YMIN co-transfected cells could be
demonstrated in whole cell lysates with immunoblots employing the
polyclonal antibody 7RC-1 such as that shown in Fig. 5B
(anti-RGS7 panel). Nevertheless, the expression of RGS7 in
lysates of G
5-YMIN co-transfected cells was much lower
than in wild-type G
5 and G
5-QAAC
co-transfected cells (Fig. 5B, lane 6, cf.
lanes 2 and 4). Indeed, blots of lysates from
RGS7 and G
5-YMIN co-transfected HEK-293 cells employing antibodies of lesser avidity, such as those in Figs. 2B
(lane 7 of Xpress panel) and 3B
(lane 3 of lysate RGS7 (C-19)
panel), fail to show an RGS7 band at all. The relative
instability of RGS7 when not partnered with G
5 as a
heterodimeric complex has been well documented in transfected COS-7
cells (15).
5 Mutants in
Transfected PC12 Cells--
PC12 cells, unlike HEK-293 cells, express
endogenous G
5 (36). Recent laser confocal microscopic
analysis of naive and transfected PC12 cells demonstrated
G
5 in the cell nuclei and cytosol (17). The subcellular
localization of the G
5-QAAC and G
5-YMIN
mutants in transfected PC12 cells was therefore examined by antibody
staining and confocal microscopy and compared with wild-type
G
5 (Fig. 6). In these
experiments no GGL domain containing RGS proteins or G
subunits were
co-transfected in order to allow the transfected G
5
constructs to "choose" binding partners from the endogenous pool of
RGS proteins and G
subunits present in PC12 cells (17, 37).
Antibodies to the G
5 N terminus and the HA epitope tag both demonstrated the distribution of transfected wild-type
G
5 throughout the PC12 cell including the cell nucleus
(Fig. 6) as described previously (17). The subcellular localization of
the G
5-QAAC mutant closely resembled that of wild-type
G
5 (Fig. 6). In contrast the G
5-YMIN
mutant was excluded from the PC12 cell nucleus when probed by either
antibodies to the G
5 N terminus or to the HA epitope
tag. The G
5-YMIN mutant-transfected cells were still
capable of G
5 nuclear localization, because when
non-epitope-tagged G
5 was co-transfected with the
G
5-YMIN mutant, nuclear localization of non-tagged
G
5 but not the HA-tagged mutant was evident in the same
cells (Fig. 6).
View larger version (64K):
[in a new window]
Fig. 6.
Confocal dual immunofluorescence analysis of
PC-12 cells transiently transfected with HA epitope-tagged and/or
wild-type G 5. Two days
following transfection with the cDNAs indicated above
the columns, cells were analyzed by laser confocal microscopy after
dual staining with affinity-purified ATDG anti-G
5
antibody (green signal) and anti-HA antibody (red
signal) as described under "Experimental Procedures." Images
of the cells illuminated by transmitted light only and the
corresponding immunofluorescence signal (monitored singly or in
combination (merge)) are indicated. Yellow-orange
signal indicates co-localization of probes. Results of one
experiment are shown as representative of four independent experiments
with similar findings.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 to interact selectively
with G
- and G
-regulated effectors as a G
5-G
complex and with RGS proteins 6, 7, 9, and 11 as a
G
5-RGS heterodimer may be critical to the role of
G
5 in signal transduction. New approaches are needed to
assess the biological importance of the G
5-G
interactions so well documented in vitro since
detergent-based purifications might miss or underestimate the presence
of native G
5-G
complexes. Besides the mutational
strategy employed here, other potential analytical tools such as
conformation-dependent antibodies or selective proteolysis
specific for the G
-bound conformation of G
5 would be
valuable in this regard. Although well documented purifications from
defined subcellular fractions of adult tissues have demonstrated only
the presence of G
5-RGS complexes and not G
5-G
complexes (15), the potential importance of
G
5-G
complex formation during ontogeny or in highly
localized brain regions remains to be addressed.
5 and its
mutants are capable of a wider range of interactions with
G
2 and/or RGS7 than the most stringent assays, such as
co-immunoprecipitation, would seem to suggest. Both wild-type
G
5 and the G
5-QAAC mutant stimulated
PLC-
in a G
2-dependent fashion, although
neither co-immunoprecipitated G
2. Co-transfection of
RGS7 was observed to enhance the expression of the
G
5-YMIN mutant even though the pair could not be
immunoprecipitated. Several laboratories (15, 35) have documented the
ability of G
5-RGS and G
5-G
protein interaction to mutually stabilize the heterodimeric subunits. Since the
G
5-YMIN mutant was designed to impair interaction
between a hydrophobic pocket on the side of the G
5
-propeller and a critical Trp residue in the RGS protein GGL domain
(10), the ability of RGS7 to partially stabilize the
G
5-YMIN mutant may reflect an RGS7-G
5
interaction outside this vicinity. The potential for such an
interaction was recently demonstrated in C. elegans between
an isolated N-terminal fragment of the RGS7 homolog EGL-10 and the
G
5 homolog GPB-2 (38). The finding that only the
G
5-YMIN mutant and G
1 were able to
co-immunoprecipitate G
2 from detergent lysates
complements an experiment performed by Snow et al. (10) in
which the strength of G
5-G
2 interaction
in detergent solution was greatly enhanced by replacing the conserved
Phe-61 in G
2 with a corresponding Trp residue
characteristic of GGL domains, and further supports their model of the
G
5-GGL/G
interface.
5 nuclear localization the
possible requirement for G
5-G
interaction for nuclear
targeting was not fully assessed (17). Nuclear localization of G
protein heterotrimers including apparent G
complexes has been
reported in rat liver (39) and thrombin- and phorbol ester-treated
Swiss 3T3 cells (40). In the previous study of G
5, a
mutant GFP-G
5 fusion protein that failed to undergo
nuclear localization was likely deficient in both G
and GGL domain
interaction due to double proline insertions in the G
5
-helical N-terminal region (17). This approach thus provided little
insight into the heterodimerization requirements for G
5
nuclear targeting.
interaction is
insufficient for G
5 nuclear targeting and that
heterodimerization with a GGL domain-containing RGS protein such as
RGS6 or -7 is critical. In HEK-293 cells, which do not express
endogenous G
5 or RGS7, the nuclear targeting of
wild-type G
5 (or the RGS7-interacting G
5-QAAC mutant) could only be reconstituted by RGS7 and
not G
2 co-transfection. Next, because the
G
5-YMIN mutant failed to localize to the nucleus in
either 293 or PC12 cells, despite its tight binding to G
demonstrated in vitro, the key molecular determinants of
nuclear targeting appear to be intrinsic to the RGS protein, not to the
G
5 or G
subunits. However, the approach utilized here
cannot exclude that mutation of the 4 residues in the
G
5-YMIN mutant abrogated a nuclear localization signal
(NLS), but this is unlikely for two reasons. No recognizable NLS motifs
could be discerned in the native G
5 sequence (41).
Furthermore, when tested in HEK-293 cells neither untagged or HA
epitope-tagged wild-type G
5 demonstrated nuclear
expression when co-transfected with G
2 making the
presence of an occult NLS on G
5 unlikely.
Ala), a site
present in many RGS proteins including RGS2 and RGS3 (48), made no
difference in the steady-state distribution of RGS7 in resting PC12
cells (data not shown). This would seem to argue against a major role
for 14-3-3 binding in the regulation of nucleocytoplasmic distribution
of G
5-RGS complexes in resting cells. Whether
phosphorylation or other post-translational modification of
G
5-RGS complexes might govern their nuclear localization
under stimulated conditions, and how potential nucleocytoplasmic
shuttling of G
5-RGS heterodimers might mediate
information transfer in the cell in response to extracellular signals
remain two central questions for future study.
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FOOTNOTES |
---|
* 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: Medical College of Georgia, School of Medicine,
1120 15th St., Augusta, GA 30912.
¶ To whom correspondence should be addressed: NIDDK, Metabolic Diseases Branch, Bldg. 10, Rm. 8C-101, 10 Center Dr., MSC 1752, National Institutes of Health, Bethesda, MD 20892-1752. Tel.: 301-496-9299; Fax: 301-402-0374; E-mail: wfs@helix.nih.gov.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M207302200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RGS, regulators of G
protein signaling;
GFP, green fluorescent protein;
GGL, G-like;
GCG, Genetics Computer Group;
DMEM, Dulbecco's modified Eagle's medium;
HEK, human embryonic kidney;
TBP, TATA-binding protein;
PLC, phospholipase C;
NLS, nuclear localization signal;
HA, hemagglutinin.
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