From the Innere Medizin III-Kardiologie,
Universität Heidelberg, Bergheimer Strasse 58, D-69115
Heidelberg, the ¶ Institut für Pharmakologie,
Universitätsklinikum Essen, D-45122 Essen, and the
§ Institut für Pharmakologie und Toxikologie,
Fakultät für Klinische Medizin Mannheim, Universität
Heidelberg, D-68169 Mannheim, Germany
Received for publication, October 8, 2002, and in revised form, November 29, 2002
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ABSTRACT |
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Formation of GTP by nucleoside diphosphate
kinase (NDPK) can contribute to G protein activation in
vitro. To study the effect of NDPK on G protein activity in
living cells, the NDPK isoforms A and B were stably expressed in H10
cells, a cell line derived from neonatal rat cardiomyocytes.
Overexpression of either NDPK isoform had no effect on cellular GTP and
ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the
expression of Gs Nucleoside diphosphate kinase
(NDPK)1 catalyzes the
transfer of terminal phosphate groups from 5'-triphosphate to
5'-diphosphate nucleotides. In the cell, the major reaction is the
phosphate transfer from ATP to other NDPs to maintain the levels of
NTPs, especially the relatively high level of GTP. Only a small
fraction of cellular NDPK binds to the plasma membrane, where it may
serve the synthesis of GTP, required for the activation of G proteins (1-3). An activation of G proteins by NDPK has been disputed for more
than 10 years. Although numerous in vitro studies (4-7) have shown G protein activation through the enzymatic activity of NDPK
(synthesis of GTP from a nucleoside triphosphate and GDP), the
specificity of this phenomenon has been questioned (8, 9). Particularly
in the intact cell, where GTP concentrations are in the upper
micromolar range, evidence for a mechanism beyond the sole synthesis of
GTP appears mandatory to support this hypothesis. On the other hand, we
have shown recently (10) that NDPK activates G proteins and regulates
adenylyl cyclase activity in canine cardiac sarcolemmal membranes. This
activation required the catalytic activity of NDPK (synthesis of GTP)
but was clearly distinct from the effect of exogenous GTP, suggesting a
more direct interaction of NDPK and G proteins.
Evaluation of direct G protein activation through phosphotransfer by
NDPK is associated with substantial methodological constraints. Mainly,
GDP is released spontaneously from G proteins and may then serve as a
free substrate for phosphorylation by the NDPK (8). Approaches to
immobilize the bound GDP at the G protein (11) are associated with
protein denaturation, which in turn may lead to unspecific protein
phosphorylation by the NDPK (12). In addition, structural
considerations make an interaction of the NDPK and the guanine
nucleotide-binding G Cell Culture and Preparation of Cell Lysates and
Membranes--
Neonatal rat heart myocytes, immortalized with a
temperature-sensitive SV40 T antigen (H10 cells) (20), were cultured at 33 °C in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 10 µg/ml gentamycin. For
cell lysis, cells were washed twice with ice-cold phosphate-buffered
saline, scraped off in Buffer A (10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride), and homogenized with two 10-s bursts applied by a Polytron (Kinematica) at a setting of 20,000 rpm. Cell lysates were then centrifuged at 100,000 × g for 30 min. Pellets were
resuspended and centrifuged three more times in Buffer A to obtain the
membrane fraction.
Subcloning of Human NDPK A, NDPK B, and NDPK B
H118N--
Original cDNA clones were obtained from Dr. N. Kimura,
Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan and Dr. M.-L. Lacombe, Faculté de Médecine Saint-Antoine, Paris,
France (NDPK B H118N in pcDNA3). After verifying the cDNA
sequences by automated sequencing, specific primers with
5'-EcoRI restriction sites were designed for amplification
of NDPK A and NDPK B coding sequences (NDPK A,
5'-AGGGAATTCCGGAGTTCAAACCTAAGCAG-3' and
5'-ACTGAATTCAAGCAATGTGGTCTGCCCTC-3'; NDPK B,
5'-ACGGAATTCAATCCCTTCTGCTCTCCCA-3' and
5'-ACTGAATTCCTGTTGTGTCCACCTCTTA-3') by PCR. The PCR products were
cleaved with EcoRI, purified by gel extraction, and then
ligated in EcoRI-linearized pIRESpuro vector
(Clontech). The NDPK B H118N cDNA was isolated
from pcDNA3 vector by an EcoRI/XhoI digest.
The cohesive ends were blunted with Klenow fragment and ligated into
the EcoRV-linearized pIRESpuro vector. The constructs were
confirmed by restriction digest analysis and automated DNA sequencing.
Stable Transfection of H10 Cells--
The day before
transfection, H10 cells were seeded at a density of 7 × 105 cells per 60-mm dish in 5 ml of DMEM with 10% fetal
calf serum. For transfection, 8 µg of DNA of pIRES-EGFP
(Clontech) encoding enhanced green fluorescent
protein (EGFP) or the pIRESpuro-NDPK constructs and 50 µl of
Superfect (Qiagen) transfection reagent were used according to the
manufacturer's protocol. 48 h later, cells were diluted 1:15 and
placed in a selective medium containing 2 µg/ml puromycin (Sigma).
Stably transfected cell clones were isolated after 2 weeks, and NDPK
expression was screened by Western blot analysis and NDPK activity assays.
Infection of H10 Cells with Recombinant Gs Co-immunoprecipitation of G Western Blotting--
5 to 20 µg of membrane-enriched or
cytosolic fractions were suspended in SDS sample buffer for SDS-PAGE.
The separated proteins were transferred electrophoretically to
nitrocellulose membranes, and immunodetection was carried out using
antibodies against Gs Measurement of cAMP Levels--
Cells were cultured in 12-well
plates and serum-starved for 4 h. Accumulation of cAMP was assayed
in serum-free medium containing 20 mM HEPES, pH 7.4, 100 µM propranolol, and 1 mM
isobutylmethylxanthine (IBMX) for 30 min at 33 °C. Thereafter, the
medium was removed, and 400 µl of ice-cold 0.1 M HCl were
added. Lysates were centrifuged for 15 min at 4 °C and 20,000 × g, and 100 µl of the supernatant were used for the
competitive enzyme immunoassay for cAMP, according to the
manufacturer's protocol (R&D Systems). The cell pellets were
neutralized with 0.1 M NaOH and used for the determination of protein concentrations with the Bradford Bio-Rad dye-binding assay
and bovine serum albumin as standard.
Enzymatic Activities--
Adenylyl cyclase activity was
determined by measuring the conversion of [
NDPK activity was determined, using [3H]GDP (100 µM, 0.1 Ci/mmol) as substrate, under the conditions used
for measurement of adenylyl cyclase activity, with 0.2-1 µg of
protein and incubation for 10 min at 37 °C. Reactions were stopped
by the addition of 5 µl of 10% (w/v) SDS. Aliquots of 10 µl
(in 2-µl steps) were spotted onto polyethyleneimine cellulose F thin
layer chromatography plates (E. Merck, Darmstadt, Germany). A
mixture of GTP/GDP/GMP (3 mM each, 10 µl) was run in
parallel and used as marker. The nucleotides were identified under UV
light, the polyethyleneimine cellulose was scraped off, and
radioactivity was measured by liquid scintillation counting. The purity
of all nucleotides was analyzed by thin layer chromatography.
Phosphorylation of G Measurement of ATP and GTP Levels--
Cellular ATP and GTP
levels were assessed by HPLC as described (23). Briefly, the cells were
deproteinized by adding 60% (v/v) ice-cold acetonitrile and
subsequently homogenized using a Branson sonifier. Denaturated proteins
were pelleted by centrifugation, and the supernatant was injected into
the chromatograph in an appropriate dilution. The system consisted of
an Amersham Biosciences gradient pump, a Spark autosampler, and
a C18 column (inner diameter 4 mm, length 20 mm; Ziemer, Mannheim,
Germany), which was equipped with a 30-mm guard column of the same
diameter. The UV detector was set to 206 nm. The detected metabolites
were normalized to protein content. The latter was assessed using
standard protocols, following solubilization of the denaturated
proteins in NaOH at 50 °C.
Data Analysis--
All experiments were carried out in
triplicate and were repeated at least three times. Values are given as
means ± S.D. For statistical analysis, one-way analysis of
variance, followed by the Tukey-Kramer post test, was performed
with GraphPad PRISM 3 software.
Characterization of H10 Cells Stably Expressing NDPK
Isoforms--
Untransfected H10 cells and a stable transfectant
expressing EGFP (C-GFP) were compared with stable transfectants,
expressing different levels of NDPK B, its catalytically inactive
mutant, NDPK B-H118N, or NDPK A. The NDPK activity was determined in
cell lysates and membrane fractions. Importantly, differences in total cellular activity were reflected by similar changes in
membrane-associated activity. The factors that determine the
intracellular distribution of NDPK, particularly binding to membranes,
are unknown. The amount and activity of NDPK were increased 1.5-, 2-, and 3-fold in the clones C1, C2, and C3, respectively (Table
I). The
amount of NDPK in cells expressing the
inactive mutant H118N (M118) was also increased 3-fold, whereas
activity remained at the level found in untransfected cells (see Table
I and Fig. 2). In H10 cells, the content of NDPK B (1.1 ng per µg of
total protein) exceeded the content of NDPK A by a factor of 10, as
determined by quantitative immunoblots using isoform-specific
antibodies and recombinant proteins as standard (see Figs. 2 and 3). In
cells expressing NDPK A (CH1), the amount of NDPK A was increased
6-fold, leading to a 1.6-fold increase of combined NDPK A/B (see Fig. 3). This was not associated with an increase in whole cell or membrane-associated NDPK activity. The amount of Gs Isoform-specific Effect of NDPK B on cAMP Synthesis--
As
overexpression of NDPK may lead to activation of both stimulatory
(Gs) and inhibitory (Gi) G proteins, expressed
in H10 cells at a 1:2 ratio (2.0 ng/µg of Gs
Because the stimulatory effect of NDPK B was dependent on
Gs Phosphorylation of G The aim of the present study was to evaluate the possible
interaction of NDPK and G proteins in living cells. Our experimental setting offers two major advantages when compared with prior approaches performed in membranes or reconstituted in vitro systems.
First, we circumvent the technical problems associated with a proof of phosphotransfer to G protein-bound GDP or GTP channeling (9), and
second, we address the issue of a physiologically relevant mechanism by
studying the effects in intact cells. The results of the combined
overexpression of NDPK isoforms and Gs Cellular cAMP is influenced by several factors that had to be
controlled to confine our results to NDPK activity. To block influences
of Evidence of G protein activation by NDPK in cells does not resolve the
problem of its mechanism. Previous studies had demonstrated an
activation of G proteins (13-15) and regulation of adenylyl cyclase
activity (14) by phosphorylated G Assuming that the NDPK B-dependent effects on cAMP
formation are mediated by NDPK B·G and Gi
proteins.
However, co-expression of Gs
led to an increase in cAMP
synthesis that was largely enhanced by the expression of NDPK B, but
not NDPK A, and that was confirmed by direct measurement of adenylyl
cyclase activity. Cells expressing an inactive NDPK B mutant (H118N)
exhibited a decreased cAMP formation in response to Gs
.
Co-immunoprecipitation studies demonstrated a complex formation of the
NDPK with G
dimers. The overexpression of NDPK B, but not its
inactive mutant or NDPK A, increased the phosphorylation of G
subunits. In summary, our data demonstrate a specific NDPK B-mediated
activation of a G protein in intact cells, which is apparently caused
by formation of NDPK B·G
complexes and which appears to
contribute to the receptor-independent activation of heterotrimeric G proteins.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit unlikely. Access of the NDPK to the
G
subunit would require dramatic conformational changes, including
the release of G
dimers prior to the phosphorylation step. On the
other hand, GTP formation by a phosphate transfer via intermediately
phosphorylated G
subunits has been observed in several tissues
(13-18), and a complex formation of NDPK B with G
is reported in
the accompanying paper (19). Beyond the molecular mechanisms, the
important question of whether NDPK can activate G proteins in intact
cells has not been addressed, most likely because of the lack of
specific activators and inhibitors of NDPK. To circumvent these
problems, we took a pragmatic approach by co-expressing the human NDPK
isoforms A (nm23-H1) and B (nm23-H2) and the
subunit of the
Gs protein (Gs
) and measured the effect on
cAMP synthesis and phosphorylation of G
. We report that the Gs
-mediated stimulation of cAMP formation increased with
the expression level of NDPK B but not NDPK A. Moreover, evidence is
provided that the increase in cAMP formation is dependent on the
catalytic activity of the NDPK B isoform and a complex formation with
G
.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Adenovirus--
Recombinant adenovirus (type 5) encoding for the long
form of rat Gs
was a kind donation of Dr. T. Eschenhagen, Institut für Klinische Pharmakologie,
Universität Erlangen, Erlangen, Germany. One day before
infection, untransfected H10 cells and cells stably transfected with
NDPK isoforms were seeded at a density of 105 cells per
well in 12-well tissue culture dishes. Cells were washed with
serum-free DMEM and then incubated with different amounts of
recombinant Gs
-encoding adenovirus in 300 µl of
serum-free DMEM for 20 min at 33 °C. Then, 700 µl of DMEM
containing 4% fetal calf serum were added. Cells were used in the
assays 48 h after infection.
with Gs
and
NDPK--
For immunoprecipitation of Gs
and NDPK from
H10 cell membranes, 200 µg of protein were solubilized in 400 µl of
Tris-buffered saline containing 0.1% Triton X-100 (Gs
)
or 0.05% Lubrol PX (NDPK) and 1 µM phenylmethylsulfonyl
fluoride. After incubation on ice for 20 min and centrifugation
(26,000 × g for 15 min), 4 µg of anti-Gs
antibody (RM/1; PerkinElmer Life
Sciences) or anti-NDPK antibody (C-20; Santa Cruz Biotechnology,
Inc.) were added to the clear supernatant and incubated for 2 h at
4 °C. After addition of 20 mg of protein G-Sepharose beads, the
mixture was gently shaken for 4 h at 4 °C. Beads were washed
three times with Tris-buffered saline, 0.1% Triton X-100, and 1 µM phenylmethylsulfonyl fluoride, and bound proteins were
eluted with 30 µl of sample buffer for 5 min at 95 °C.
Precipitated proteins were loaded onto a 10% polyacrylamide gel. After
SDS-PAGE and transfer to nitrocellulose membranes, proteins were
detected by immunoblotting with anti-Gs
and anti-G
(T-20; Santa Cruz Biotechnology, Inc.) antibodies.
(RM/1; PerkinElmer Life Sciences),
Gi
, G
, and NDPK (C-10, M-14, and C-20, respectively;
Santa Cruz Biotechnology, Inc.), and
-actin (Sigma). Specific
antibodies to the NDPK isoforms A and B were kind gifts of Dr. Ioan
Lascu, Université de Bordeaux, Bordeaux, France. Binding of the
primary antibody was visualized using a horseradish peroxidase-labeled
anti-rabbit IgG secondary antibody and Lumilight plus (Roche Molecular
Biochemicals). Chemiluminescence was quantified with a
FluorS-MultImager (Bio-Rad).
-32P]ATP to
[32P]cAMP (21). The assay volume was 100 µl, containing
0.1 mM ATP with 0.5-5 × 106 cpm
[
-32P]ATP (3000 Ci/mmol), 3 mM
MgCl2 or 11 mM MnCl2, 0.1 mM cAMP, 1 mM IBMX, 1 mM EDTA, 0.5 mM dithiothreitol, and 75 mM triethanolamine hydrochloride, pH 7.6. The membranes (10-25 µg of protein) were pre-incubated with alamethicin for 20 min at 4 °C at a 1:1 ratio (w/w) to unmask latent adenylyl cyclase activity. This peptide ionophore increases the accessibility of substrates to the adenylyl cyclase in sealed membrane vesicles without affecting the functional coupling to receptors (22). The adenylyl cyclase reaction was started
by the addition of membrane protein and conducted for 10 min at
37 °C. Under these conditions, enzyme activity was stable during the
entire incubation period.
and NDPK in H10 Cell Membranes--
The
indicated amounts of H10 cell membranes were phosphorylated with 10 nM [
-32P]GTP (PerkinElmer Life Sciences)
for the indicated periods of time at 30 °C in a reaction buffer
containing 50 mM triethanolamine hydrochloride, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol in a total
volume of 20 µl. The reaction was terminated by the addition of 10 µl of 3-fold concentrated sample buffer, followed by incubation at
room temperature for 1 h. Proteins were separated by discontinuous
SDS-PAGE on gels containing 10-12% (w/v) acrylamide and
autoradiographed. Immunoprecipitation of G
from 200 µg of
phosphorylated H10 cell membranes was performed as described (15),
using 0.6 µg of anti-G
antibody (T-20).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
Gi
subunits, as well as basal, G protein-uncoupled
adenylyl cyclase activity (with 10 mM free Mn2+
as co-substrate), and cAMP content were not affected by overexpression of the NDPK isoforms (Table I). An increase of NDPK activity could
change cellular levels of ATP and GTP, which might cause direct or G
protein-mediated effects on adenylyl cyclase activity. However, we
could not detect any significant changes in the ATP and GTP contents or
in the ATP/GTP ratio in cells transfected with the NDPK isoforms (Table
I).
Characterization of H10 cells and stably transfected cell clones
versus 4.0 ng/µg of Gi
), their effects on
cAMP synthesis may be neutralized. We therefore co-expressed
Gs
in NDPK transfectants with a recombinant adenovirus (Fig. 1). The functional integration of
overexpressed Gs
into trimeric G proteins was assessed
by co-immunoprecipitation of G
dimers. Immunoprecipitation of
Gs
from cells with increasing levels of expression was
associated with a parallel increased co-precipitation of G
(Fig.
1B). Overexpression of Gs
, up to 10-fold
above the endogenous protein content, led to a linear increase in cAMP
accumulation, measured in the presence of the phosphodiesterase
inhibitor, IBMX, and the inverse
-adrenoreceptor agonist,
propranolol (24). Most important, the increase in cAMP accumulation
induced by overexpression of Gs
was strongly enhanced in
cells overexpressing the NDPK B isoform. In cells with an ~3-fold higher expression of NDPK B (C3), the increases in cAMP in content in
response to Gs
were ~4-fold higher at each level of
Gs
compared with H10 cells stably expressing EGFP
(C-GFP; see Fig. 1A) or untransfected H10 cells (H10; see
Fig. 2A). In cells with lower expression of NDPK B (C1 and C2), intermediate levels of cAMP accumulation were observed. In contrast, cells expressing the same
amount of the inactive mutant of NDPK B, H118N (M118), exhibited a
lower cAMP content than untransfected H10 cells at any
Gs
level (Fig. 2). Furthermore, in cells with a 6-fold
overexpression of NDPK A (CH1), the cAMP content was not altered
compared with untransfected H10 cells at any level of Gs
(Fig. 3).
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Fig. 1.
Overexpression of NDPK B enhances
Gs -mediated cAMP accumulation in H10 cells.
A, the cAMP content was determined in H10 cell clones stably
expressing EGFP (C-GFP) or NDPK B at different expression
levels and increasing expression levels of Gs
.
C1, C2, and C3 represent cell clones
with a 1.5-, 2.1-, and 2.7-fold overexpression of NDPK B, respectively.
Representative immunoblots of NDPK B content are shown in the
insets. Expression of Gs
achieved by
infection with a recombinant adenovirus at different multiplicities of
infection (MOI) was quantified by immunoblotting. A
representative immunoblot is shown. B, integration of
overexpressed Gs
into heterotrimeric G proteins.
Gs
was immunoprecipitated with a specific antibody and
protein G-Sepharose from lysates of infected cells. Immunoprecipitates
were subjected to SDS-PAGE, transferred to nitrocellulose, and
immunostained with antibodies against Gs
and
G
.
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Fig. 2.
Catalytic activity of NDPK B is required for
its stimulatory effect on cAMP levels. A, the cAMP content
was determined in untransfected H10 cells, cell clones 3-fold
overexpressing NDPK B (C3) or its catalytically inactive mutant, H118N
(M118), and increasing expression levels of Gs , induced
by adenoviral infection at different multiplicities of infection
(MOI). B, the NDPK activity was quantified as
formation of [3H]GTP from [3H]GDP and ATP
in membranes of untransfected H10 cells, the C3 clone, and the H118
clone as described under "Experimental Procedures." C,
immunoblot analysis with a specific anti-NDPK B antibody of H10 cells,
the C3 clone, and H118 clone. The indicated amounts of recombinant NDPK
B were used as standard for quantification.
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Fig. 3.
Lack of effect of NDPK A on
Gs -mediated cAMP
accumulation. A, the cAMP content was determined in
untransfected H10 cells and a cell clone with a stable, 6-fold
overexpression of NDPK A (CH1) and increasing expression
levels of Gs
, induced by adenoviral infection at
different multiplicities of infection. B, the amount of
expressed NDPK A was quantified by immunoblotting with an
isoform-specific antibody. The recombinant human NDPK A migrates at an
apparent molecular mass of 21 kDa, whereas endogenous rat NDPK A is
detected at 17 kDa. The indicated amounts of recombinant NDPK A were
used as standard. C, membranes of untransfected H10 cells
and of the CH1 clone were phosphorylated with
[
-32P]GTP (10 nM). Autophosphorylated NDPK
A and NDPK B are indicated.
, stimulation of adenylyl cyclase is the most likely
mechanism for the increase in cAMP content observed in the NDPK B
transfectants. This was supported by direct measurements of adenylyl
cyclase activity in membranes of cells with increased expression levels of NDPK B and Gs
. Whereas activity of the adenylyl
cyclase uncoupled from regulatory influences, measured with 10 mM Mn2+ as co-substrate, was not altered by
overexpression of NDPK B (Table I) in the presence of Mg2+
and specifically upon the addition of GDP, adenylyl cyclase activity was increased in membranes of C3 cells, and this increase was enhanced
largely by overexpression of Gs
(Fig.
4). In contrast, when GDP was replaced by
its analog, guanosine 5'-O-(2-thio)diphosphate (GDP
S), which, similar to GDP, binds to G proteins but is a
poor substrate for NDPK, the NDPK B-induced increase in adenylyl
cyclase activity was diminished.
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Fig. 4.
Stimulatory effect of NDPK B on adenylyl
cyclase activity. Adenylyl cyclase activity was determined in
membranes from untransfected H10 cells (empty bars) and from
cells with a 3-fold overexpression of NDPK B (C3;
filled bars) as described under "Experimental
Procedures." Activity was determined with 3 mM
Mg2+ as co-substrate, and, as shown from left to
right, in the absence (C) and presence of 100 µM GDP or 50 µM GDP S (G
S).
* denotes measurements in membranes from cells overexpressing equal
amounts of Gs
as determined by immunoblot (see
inset).
by NDPK B in H10 Cell Membranes--
In
the accompanying paper (19), a complex formation of NDPK B with G
has been described. Therefore, we studied whether NDPK B forms
complexes with G
in H10 cells, as well. Membranes of the C3 clone
with 3-fold overexpression of NDPK B were solubilized at low detergent
and subjected to immunoprecipitation with the anti-NDPK antiserum
(C-20). As shown in Fig. 5A,
co-precipitated G
was detected by Western blot analysis. Next, we
studied whether phosphorylation of G
occurs in H10 cells.
Phosphorylation of H10 cell membranes with [
-32P]GTP
disclosed the presence of a 36-kDa phosphoprotein that could be
immunoprecipitated with the G
-specific antiserum (Fig.
5B). We therefore analyzed whether the overexpression of
NDPK isoforms alters the phosphorylation of G
in H10 cells. An ~2-
and 3-fold increase in G
phosphorylation was observed in membranes
of H10 cells with 1.5-fold (C1) and 3-fold (C3) overexpression of NDPK B, respectively. Concurrently, the overexpression of NDPK B could be
detected by its autophosphorylation (Fig. 5, C and
D). In contrast, no increase in G
phosphorylation was
detected in membranes from cells 3-fold overexpressing the catalytic
inactive NDPK B mutant, H118N (M118) (Fig. 5C), or 6-fold
overexpressing NDPK A (CH1; see Fig. 5D).
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Fig. 5.
Phosphorylation of G
by NDPK and formation of
G
-NDPK complexes.
A, membranes (200 µg of protein) of H10 cells 3-fold
overexpressing human NDPK B (C3) were subjected to immunoprecipitation
with an anti-NDPK antiserum or a nonspecific IgG and subjected to
SDS-PAGE. Precipitated G
subunits were detected by Western blotting.
B, H10 cell membranes (200 µg of protein) were
phosphorylated with [
-32P]GTP and subjected to
immunoprecipitation with the anti-G
antibody (T-20). Purified IgG
was used as a control. C, Membranes (5 µg of protein) of
non-transfected H10 cells (H10) or H10 cells 3-fold
overexpressing wild-type NDPK B (C3) or its catalytically
inactive mutant (M118) were phosphorylated with
[
-32P]GTP for 1 min at 30 °C. D,
membranes (2.5 µg of protein) of non-transfected H10 cells
(H10) and H10 cells 1.5-fold overexpressing wild-type NDPK B
(C1) or 6-fold NDPK A (CH1) were phosphorylated
with [
-32P]GTP for 5 min at 30 °C. Autoradiographs
after SDS-PAGE are shown.
DISCUSSION
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ABSTRACT
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RESULTS
DISCUSSION
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in H10 cells can
be summarized as follows. The stimulation of cAMP synthesis by NDPK
required Gs
, and much of the effect of Gs
was dependent on NDPK activity. The effects of both NDPK and
Gs
were dependent on and linear with the expression of
the respective proteins. The activation of Gs
by NDPK
required the catalytic activity of the enzyme. It was not seen with its
inactive mutant. Furthermore, the effect was specific for the NDPK B
isoform. Orlov et al. (25) recently found a
transducin-mediated isoform-specific binding of NDPK to rod outer
segment membranes. In their study, the transducin-mediated binding of
NDPK B to the membrane exceeded that for the A isoform 100-fold,
indicating a specific interaction of the G protein with NDPK B.
-adrenoceptors and phosphodiesterases, cAMP assays were performed
in the presence of propranolol and IBMX, respectively. Furthermore,
expression of NDPK had no influence on the expression levels of the
adenylyl cyclase-regulating G proteins, the basal adenylyl cyclase
activity, or cellular ATP and GTP contents. Finally, direct
measurements of adenylyl cyclase activity in vitro exhibited an NDPK B- and Gs
-dependent activation in
the presence of GDP and ATP.
subunits. In H10 cell membranes,
the extent of G
phosphorylation was dependent upon the level,
activity, and isoform of NDPK. In addition, G
subunits
co-precipitated with an antibody against NDPK. Furthermore, the
accompanying paper (19) shows a highly selective co-purification of
NDPK B and G
dimers with different isolation protocols. A reciprocal co-immunoprecipitation of NDPK with transducin G
further confirms a complex formation. Most important, the
phosphorylation of G
subunits could be reconstituted by the addition
of NDPK-enriched co-factor fractions to purified G
dimers of
different origin. The identification of a phosphorylated histidine
residue (His-266 in G
1) that is exposed at the surface
of the G
molecule raises the possibility of a phosphate transfer
from His-118 of NDPK B to His-266 in G
(19). The enhanced
phosphorylation of G
in membranes of cells overexpressing NDPK B and
the lack of such an effect in membranes of cells overexpressing its
catalytic inactive mutant H118N (Fig. 5) substantiates this hypothesis.
complexes, our data suggest
that the activation of G proteins by GTP formation via intermediately
phosphorylated NDPK B·G
complexes is a mechanism to regulate
the basal, receptor-independent activation of heterotrimeric G proteins
(Fig. 6). In line with this hypothesis,
an at least 3-fold increase of the NDPK content and activity in
sarcolemmal plasma membranes from failing human myocardium was
accompanied by a higher basal activation of Gi proteins
(26). As Gi protein expression is also increased in heart
failure (27, 28), the higher NDPK and Gi content may contribute to the well known decreased response to
-adrenoreceptor agonists (29) and diminished basal cAMP formation (30).
View larger version (23K):
[in a new window]
Fig. 6.
Hypothetical model of
receptor-dependent and NDPK B-dependent G
protein activation. The agonist-activated G protein-coupled
receptor (GPCR) stimulates the GDP/GTP exchange at the G
protein subunit, thereby leading to G protein activation and
effector regulation. In a heterotrimeric G protein complexed with NDPK
B, a nucleoside triphosphate (NTP) is used to phosphorylate
His-118 in the NDPK. This phosphate is transferred onto His-266 in
G
. Out of the phosphoamidate bond, the phosphate is transferred onto
GDP, and the formed GTP leads to receptor-independent G protein
activation.
If NDPK B is a regulator of G protein activity, an important question
is whether its contribution is regulated in cells. Recently, we found a
3-fold elevated plasma membrane-associated NDPK in hearts from patients
with severe congestive heart failure (26). This elevation was
diminished in patients treated with a -adrenoreceptor antagonist.
Furthermore, chronic treatment of rats with the
-adrenoreceptor agonist, isoproterenol, induced an increase in plasma membrane-bound NDPK (31), suggesting chronic
-adrenergic activation as a mechanism for regulation of the NDPK membrane content. Therefore, it is a
reasonable assumption that an NDPK B·G
complex, as a
receptor-independent activator of G proteins, may have the potential to
regulate a broad spectrum of cellular functions. Studies are in
progress to further substantiate this hypothesis.
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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.
To whom correspondence should be addressed. Tel.:
49-6221-568611; Fax: 49-6221-565515; E-mail:
feraydoon_niroomand@med. uni-heidelberg.de.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M210305200
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ABBREVIATIONS |
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The abbreviations used are:
NDPK, nucleoside
diphosphate kinase;
EGFP, enhanced green fluorescent protein;
IBMX, isobutylmethylxanthine;
GDPS, guanosine
5'-O-(2-thio)diphosphate;
DMEM, Dulbecco's modified
Eagle's medium.
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