From the
cGMP-dependent protein kinase (cGMP kinase) has been implicated
in the regulation of the cytosolic calcium level
([Ca
Nitric oxide and atrial natriuretic factor activate guanylyl
cyclases and increase cGMP levels. Cyclic GMP decreases
[Ca
Rapoport
(13) suggested that cGMP kinase alters hormone signaling by
interfering with the production of IP
CHO cells contain a
number of hormone receptors that increase
[Ca
CHO cells express a number of PTX-sensitive G proteins including
G
Several
possibilities were tested to explain the observed difference. It is
known that thrombin induces tyrosine phosphorylation
(39, 40, 41) , including phosphorylation and
thereby activation of phospholipase C
IGF-2 superfusion of
the CHO cells resulted in a rapid rise in
[Ca
In contrast to CHO-cGK cells, the IGF-2 response of
CHO-WT cells was not altered by 1 m
M 8-Br-cGMP. Ten n
M IGF-2 increased
[Ca
The IGF-1 response in CHO-WT cells was not affected
by preincubation with 8-Br-cGMP. In the absence of 8-Br-cGMP, IGF-1
increased [Ca
The IGF-1-induced increase in
[Ca
cGMP kinase added at a high but physiological
concentration
(1, 16) phosphorylated a protein of about
40 kDa in preparations of G
A previous study showed that cGMP kinase suppressed
thrombin-stimulated IP
It was intriguing that the signaling of structurally
different hormone receptors, i.e. a seven-membrane-spanning
receptor
(44) , a receptor with a single transmembrane helix
(22) , and a tyrosine kinase-containing receptor
(22) ,
was affected by cGMP kinase. These receptors activated different
cellular targets involving IP
There
was a close correlation between the effects of PTX and cGMP kinase on
the different signal transduction processes, suggesting that both
enzymes modified the same targets, namely PTX-sensitive heterotrimeric
G proteins of the G
G protein preparations containing G
The
primary sequence of the G
An
interaction of cGMP kinase with PTX-sensitive G proteins was already
demonstrated for the renal collecting duct, where cGMP kinase reduced
the open probability of an amiloride-sensitive sodium channel,
involving a PTX-sensitive G protein
(49) . In addition, a few
examples exist for the phosphorylation of G proteins by other protein
kinases. In platelets, the activation of protein kinase C
phosphorylated stoichiometrically G
In conclusion, it has been shown that cGMP kinase
attenuated signal transduction in PTX-sensitive pathways. The involved
receptors and the postulated G protein have growth-promoting effects
(19, 22, 38, 39, 45) . It is
possible that the mechanism interrupted by cGMP kinase in CHO-cGK cells
is responsible for the reported antiproliferative action of cGMP
(53) .
CHO-cGK cells were
preincubated without (-) or with (+) 1 m
M 8-Br-cGMP
for 15 min and then stimulated with 30, 100, or 300 µ
M CCK. Basal values were taken directly before addition of CCK. CCK
values are the peak values reached after CCK superfusion ( n = number of cells). Note that cells not responding to the
particular treatment have not been excluded from the calculations.
CHO-cGK cells were preincubated in
the presence (PTX) and absence (Buffer) of PTX and then stimulated with
CCK or thrombin as described in the legend to Fig. 4. Basal values were
taken directly before addition of the agonists. Thrombin and CCK values
are the peak values reached ( n = number of cells). Note
that cells not responding to the particular treatment have not been
excluded from the calculations.
We are grateful to Dr. K. Spicher for providing the
antisera, to Dr. T. Gudermann for synthesis of GTP-azidoanilide, and to
E. Roller for the graphical work.
e 29, D-80802
München, Federal Republic of Germany and the
Institut
für Pharmakologie, Universitätsklinikum Rudolf Virchow, Freie
Universität Berlin, Thielallee 67-73, D-14195 Berlin,
Federal Republic of Germany
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
]
). In Chinese
hamster ovary (CHO) cells stably transfected with the cGMP kinase
I
(CHO-cGK cells), cGMP kinase suppressed the thrombin-induced
increase in inositol 1,4,5-trisphosphate and
[Ca
]
(Ruth, P., Wang,
G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M.,
Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular
calcium release via a pertussis toxin (PTX)-insensitive pathway in
CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated
[Ca
]
. In contrast,
cGMP kinase suppressed calcium influx stimulated by insulin-like growth
factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The
effects of PTX and cGMP kinase on
[Ca
]
were not
additive. 8-Bromo-cGMP had no effect on
[Ca
]
stimulated by
IGF-1 or IGF-2 in wild type CHO cells. These results suggested that
cGMP kinase inhibited the different signaling pathways by the
phosphorylation of a PTX-sensitive G protein. cGMP kinase
phosphorylated the
subunits of G
, G
,
and G
in vitro. Phosphorylation stoichiometry was
0.4 mol of phosphate/mol of G
after reconstitution of
heterotrimeric G
in phospholipid vesicles. The
subunit of G
was also phosphorylated in vivo.
These results show that cGMP kinase blocks transduction of distinct
hormone pathways that signal via PTX-sensitive G
proteins.
]
(
)
in a number of cells including smooth muscle and platelets
by activation of the cGMP kinase (for extensive discussion see Refs.
1-4). It was postulated that cGMP kinase stimulates
Ca
removal from the cytosol by activation of the
calmodulin-stimulated Ca
-ATPase, by phosphorylation
of phospholamban and stimulation of calcium uptake into the
sarcoplasmic reticulum, or by phosphorylation of the IP
receptor (see Refs. 1-4, 5, and 6 for further references).
Cyclic GMP has also been reported to inhibit calcium influx by
modulation of cAMP-hydrolyzing phosphodiesterases
(7, 8) , by phosphorylation of calcium-activated
potassium channels leading to hyperpolarization of the membrane
(9, 10) , or by phosphorylation-dependent inhibition of
L-type calcium channel activity
(11, 12) .
. The latter mechanism
was supported later by other groups
(14, 15) . Recently
we showed that cGMP kinase inhibited thrombin-stimulated IP
synthesis and the rise in
[Ca
]
in CHO-cGK cells
(16) . The general validity of the latter mechanism was unknown
and was investigated further in this study.
]
by different
mechanisms, i.e. a receptor that is activated by high
concentrations of CCK and signals via PTX-insensitive G proteins
(17) and high affinity, low density receptors that are activated
by IGF-1 and IGF-2
(18) . The IGF-1 receptor, a receptor
tyrosine kinase, stimulated calcium influx through a nonspecific cation
ion channel in Balb/c 3T3 cells
(19) . The pathway leading to
channel opening involved a PTX-sensitive G
protein
(20) . Interestingly, IGF-2 receptor activation stimulated
Ca
influx by a similar pathway
(21) . The
IGF-2 receptor has no tyrosine kinase activity
(22) but
contains a single transmembrane helix that has been reported to couple
to several G
proteins
(23) . This IGF-2 receptor may
be identical with the cation-independent mannose 6-phosphate receptor.
,G
, G
, and G
(24, 25) , suggesting that pathways may exist in
CHO cells that are similar to those found in Balb/c 3T3 cells. This
paper shows that cGMP kinase suppressed specific signaling via
PTX-sensitive pathways, probably by phosphorylation of the
subunit of a G
protein.
Stable Transfection of CHO Cells
CHO cells were
transfected by electroporation with the expression vector p91023I
(26) containing the coding sequence of the cGMP kinase I
isozyme and, in a second reading frame, the coding sequence of the
dihydrofolate reductase
(27) . CHO-WT and CHO-cGK cells were
grown for 3-4 days on coverslips in Dulbecco's modified
Eagle's medium supplemented with 10% non-dialyzed and dialyzed
fetal calf serum, respectively.
Measurement of Calcium Transients in CHO
Cells
[Ca]
was
determined by fluorescence measurement of the calcium-sensitive
indicator Fura-2 as described previously
(16) . In brief, cells
were loaded for 1 h with 2.5 µ
M Fura-2/AM at 37 °C.
PTX-pretreated cells were incubated in Dulbecco's modified
Eagle's medium containing 100 ng/ml PTX for 8 h prior to
Fura-2/AM loading. Coverslips were then washed with the NaCl/Hepes
buffer (140 m
M NaCl, 6.6 m
M KCl, 1.18 m
M MgSO
, 2 m
M CaCl
, 10 m
M glucose, 5 m
M Hepes, pH 7.4) and superfused with this
buffer. cGMP kinase stimulation was achieved by superfusion of the
cells for 10 min with 1 m
M 8-Br-cGMP included in the
NaCl/Hepes buffer. The cells were placed in the light path of an
Axiovert-35 microscope (Zeiss) equipped with a xenon light source and a
340/380-nm filter wheel (Luigs & Neumann) alternating at a
frequency of 2 Hz. Fura-2 fluorescence of a single cell was monitored
by a photomultiplier unit (Hamamatsu 928 SF) attached to the
microscope. Changes in the 340/380-nm ratio were analyzed and displayed
on an ATARI-ST computer. The cells were superfused by micropipettes
loaded with the agents to be tested. Superfusion was started by
applying air pressure to the pipette (Lorenz MPCU-3) and lasted, if not
otherwise indicated, for 60 s. Apparent
[Ca
]
values were
calculated according to Ref. 28. All data are expressed as means
± S.E., with the number of cells shown in parentheses.
Purification of G Proteins
PTX-sensitive G
proteins were purified from bovine brain as described
(29) .
Briefly, membranes were prepared and stored in TEM buffer (20 m
M Tris-HCl, pH 8.0, 1 m
M EDTA, 20 m
M -mercaptoethanol). All procedures were carried out at 4
°C. Thawed membranes were extracted for 1 h with TEM containing
0.9% sodium cholate. After centrifugation at 70,000
g,
the clear supernatant was mixed with ethylene glycol to a final
concentration of 30% (v/v), followed by an addition of 10 µ
M GDP. Purified mixtures of G
,
G
/G
, heterotrimeric
G
/G
, and G
were obtained
after sequential chromatography on a 1.5-liter DEAE-Sepharose Fast Flow
column (Pharmacia Biotech Inc.), a 1.2-liter AcA 34 gel filtration
column (Serva), and a 0.65-liter heptylamine-Sepharose column
(30) . Heterotrimeric G proteins containing different subtypes
of G
/G
were separated on a Mono Q column
(Pharmacia), employing a method described by Codina et al. (31) . Repetitive fast protein liquid chromatography runs
resulted in fractions containing G
, G
,
G
, or G
. G
was contaminated with
small amounts of G
, G
with small amounts of
G
, and G
with small amounts of G
as detected by immunoblot analysis. The purified G proteins were
stored in 20 m
M TEM buffer containing 100-300 m
M NaCl, 11 m
M CHAPS, and 0.1% Lubrol.
Reconstitution of G Proteins into Phospholipid
Vesicles
Purified heterotrimeric G proteins were reconstituted
into phospholipid vesicles containing azolectin. About 6 µg of
heterotrimeric Gor G
were mixed with
azolectin and sodium cholate and incubated for 1 h. Vesicles containing
G proteins were formed by passing the solution over a gel filtration
column eluted with 20 m
M Hepes, pH 7.4, containing 0.1 m
M EDTA, 1 m
M dithiothreitol, and 100 m
M NaCl. G
proteins reconstituted into phospholipid vesicles eluted in the void
volume and were immediately used for further experiments.
GTP
For quantification
of purified and reconstituted G proteins,
[S Binding to G Proteins
S]GTP
S binding to G proteins was
essentially performed as described
(29) . Aliquots of 3 µl
were incubated with [
S]GTP
S (100,000
cpm/tube; 500 n
M GTP
S) in a total volume of 60
µl/tube for 60 min at 32 °C. Nonspecific binding was estimated
by the addition of 2 m
M GTP.
Phosphorylation of G Proteins
Purified G proteins
(9-20 pmol) were phosphorylated for 10 min at 30 °C in 40
µl of 50 m
M MES buffer, pH 6.9, containing 0.4 m
M EGTA, 1.6 m
M CHAPS, 0.015% Lubrol, 1 m
M MgCl, 10 m
M NaCl, 10 m
M dithiothreitol in the absence and presence of 10 µ
M 8-Br-cGMP, 0.1 m
M [
-
P]ATP
(1500 cpm/pmol), and 780 n
M cGMP kinase holoenzyme.
Reconstituted G proteins (2.5-5 pmol) were phosphorylated for 10
min at 30 °C in 200 µl of 15 m
M Hepes, pH 7.4, 0.075
m
M EDTA, 75 m
M NaCl, 0.75 m
M dithiothreitol,
1 m
M magnesium acetate, 10 µ
M 8-Br-cGMP, 0.1
m
M [
-
P]ATP (1500 cpm/pmol), and
780 n
M cGMP kinase holoenzyme. In both cases, reactions were
stopped with Laemmli's buffer (final concentration: 1% SDS, 5%
mercaptoethanol, 5% glycerol, 0.01% bromphenol blue) followed by
SDS-gel electrophoresis as described below.
Photolabeling and Immunoprecipitation of Membrane
Proteins
Photolabeling of G protein subunits by
[
P]GTP azidoanilide was performed as described
(32) . Each tube contained freshly prepared membranes obtained
from 10
CHO cells. Immunoprecipitations of G proteins were
performed according to Nürnberg et al. (29) and
Harhammer et al. (33) . Essentially, G proteins
reconstituted into phospholipid vesicles and phosphorylated by cGMP
kinase were acetone-precipitated and dissolved in 40 µl of a
solution containing 4% SDS at room temperature. After 5 min, 280 µl
of the immunoprecipitation buffer (10 m
M Tris-HCl, pH 7.4, 150
m
M NaCl, 1 m
M EDTA, 1 m
M dithiothreitol, 1%
(w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 0.3 µ
M aprotinin, and 0.2 µ
M phenylmethanesulfonyl fluoride)
was added, followed by 30 µl of antiserum AS8 or AS6. Peptide
antibodies AS6 (anti-
) and AS8
(anti-
) were characterized previously
(34) . The mixture was gently shaken at 4 °C. After 2 h the
solution was supplemented with 60 µl of a slurry containing 15%
Protein A-Sepharose beads. This mixture was shaken overnight. Protein
A-Sepharose beads were pelleted and washed once with
immunoprecipitation buffer and twice with immunoprecipitation buffer
containing 300 m
M NaCl. Bound proteins were eluted by adding
Laemmli's buffer and loaded onto discontinuous SDS gels. The
separating gels contained 11% (w/v) acrylamide. Dried gels were exposed
to x-ray films. Thereafter, the phosphorylated bands were cut out,
dissolved in scintillation mixture, and counted.
Metabolic Labeling of Cells and Immunoprecipitation of G
Proteins
For detection of cGMP kinase-dependent phosphorylation
of G proteins in CHO-WT and CHO-cGK cells, 10cells/tube
were incubated in a small volume of buffer A (137 m
M NaCl, 1
m
M MgCl
, 2.7 m
M Hepes, pH 7.4, devoid of
phosphate). Subsequently, cells were incubated with
[
P]orthophosphate (0.5 mCi/tube) in buffer A for
2 h at 30 °C. Thereafter, cells were disrupted mechanically in the
presence of 20 m
M
-mercaptoethanol, 25 m
M EDTA,
2 m
M pyrophosphate, 2 m
M vanadate, 0.0005% aprotinin,
and 1 m
M benzamidine. The whole cell homogenate was
centrifuged at 500
g (10 min, 4 °C), and the
supernatant was centrifuged a second time at 30,000
g (10 min, 4 °C). The pellet was solubilized by adding 40 µl
of lysis buffer (1% SDS, 100 m
M phosphate buffer, pH 8, 4
m
M EDTA, 2 m
M pyrophosphate, 2 m
M vanadate,
0.0005% aprotinin, 1 m
M benzamidine) for 15 min at room
temperature. Subsequently, 280 µl of precipitation buffer (50
m
M phosphate buffer, pH 7.2, 150 m
M NaCl, 2 m
M EDTA, 5 m
M sodium fluoride, 1% sodium deoxycholate, 1%
Triton X-100, 0.5% SDS, 2 m
M pyrophosphate, 2 m
M vanadate, 0.0005% aprotinin, 1 m
M benzamidine) was added,
followed by centrifugation at 30,000
g (10 min, 4
°C). The supernatant was transferred to a second tube and incubated
with 20 µl of nonimmune rabbit serum for 30 min at 4 °C.
Subsequently, 30 µl of a solution containing 37.5% of Protein A
bound to Sepharose beads were added for another 3.5 h. The slurry was
pelleted, and the supernatant was transferred to a third tube and
incubated with 40 µl of rabbit antiserum AS373 or nonimmune rabbit
serum. AS373 was generated against the peptide ((C)DVIIKNNLKDCGLF)
specific for the C terminus of G
and
G
. The mixture was gently shaken overnight at 4
°C. The solution was finally supplemented with 60 µl of a
suspension containing 12.5% Protein A bound to Sepharose beads. This
mixture was shaken for another 3 h. Protein A-Sepharose beads were
pelleted and washed once with precipitation buffer and twice with
precipitation buffer containing 300 m
M NaCl. Bound proteins
were eluted by adding Laemmli's buffer and loaded onto
discontinuous SDS gels with an 11% separating gel. Dried gels were
exposed to x-ray films.
Materials
The dihydrofolate reductase-deficient
CHO cell line DG44 was kindly provided by L. Chasin, Columbia
University, New York. Dialyzed calf serum was purchased from Life
Technologies, Inc. Dulbecco's modified Eagle's medium was
from Biochrom, Berlin. CCK-8 (sulfated) was obtained from Bachem.
Thrombin, CCK (pancreozymin) from porcine intestine, PTX, azolectin,
and genistein were from Sigma. 8-Br-cGMP was purchased from Boehringer
Mannheim. [-
P]ATP was from Amersham
Buchler.
S-Labeled GTP
S and
[
-
P]GTP were purchased from DuPont NEN.
cGMP kinase was purified as described previously
(35) . All
other reagents were of highest purity commercially available.
Stimulation of Calcium Transients by
CCK
Previously it was shown that thrombin and CCK signal through
different G proteins in CHO cells
(17) . The stimulation of
[Ca]
by thrombin was
completely suppressed by cGMP kinase in CHO-cGK but not in CHO-WT cells
(16) . The general validity of this observation was tested.
Individual CHO-cGK cells were superfused with CCK at various
concentrations (Fig. 1). Superfusion of the cell with 100 n
M CCK elicited a rapid increase in
[Ca
]
(Fig. 1).
At this threshold concentration, 82% of cells responded with an average
increase in [Ca
]
of
155 ± 55 n
M (11 cells) above basal levels. At
concentrations from 100 n
M to 300 µ
M CCK, the
calcium signal typically consisted of an initial transient rise in
[Ca
]
followed by
oscillations superimposed on an elevated plateau (Fig. 1). CCK
stimulation with concentrations above 300 µ
M resulted in a
slightly different response; CCK induced a large peak, which rapidly
decreased to a much lower but still elevated non-oscillating plateau
level. The calcium transients induced by CCK were not affected by
chelating the extracellular Ca
with EGTA
(Fig. 1). Thus, CCK mobilized Ca
from
intracellular stores, supporting the previous notion that CCK activated
IP
synthesis via a G protein-mediated stimulation of
phospholipase C
in CHO cells
(17) .
Figure 1:
CCK stimulates intracellular calcium
release in CHO cells. CHO-cGK cells were superfused with NaCl/Hepes
buffer containing 2 m
M CaCl( upper panel and lower left) or NaCl/Hepes
buffer without CaCl
but containing 5 m
M EGTA
( lower right). CCK addition at the concentrations
indicated is shown by an arrow. Representative experiments of
at least eight individual cells for each condition are
shown.
CCK dose dependently
stimulated [Ca]
up to
9-fold with a half-maximal effective concentration of 120 µ
M in both CHO-WT and CHO-cGK cells (Fig. 2). A similar response was
observed when CHO-cGK cells were superfused with the sulfated CCK-8
octapeptide (data not shown). Similar high concentrations of CCK have
also been used by others to stimulate IP
production in CHO
cells
(17) , suggesting that the properties of this receptor
significantly differ from those of the cloned CCK-A and CCK-B receptors
(36, 37) , but this receptor may share some properties
with the very low affinity CCK receptor postulated for pancreas acinar
cells
(38) .
Activation of cGMP Kinase Prevented Thrombin- but Not
CCK-induced Calcium Transients
The activation of the cGMP kinase
in CHO-cGK cells did not attenuate the Catransients
induced by 100 µ
M CCK (Fig. 3) or that of any other
concentration of CCK tested (). In contrast to CCK, the
thrombin-induced calcium transients were completely suppressed by
activation of cGMP kinase (Fig. 3).
Figure 3:
8-Br-cGMP suppresses thrombin- but not
CCK-stimulated Ca transients in CHO-cGK cells.
Coverslips were pretreated with NaCl/Hepes buffer ( left) or
with NaCl/Hepes buffer containing 1 m
M 8-Br-cGMP
( right). At the arrow, individual cells were
stimulated with 100 µ
M CCK ( upper part)
or 200 n
M thrombin ( lower part) for 60 s.
Recordings are representative for at least eight individual
cells.
These results indicated
that CCK and thrombin signal via pathways that are differently affected
by cGMP kinase. The lack of any effect of cGMP kinase activation on the
CCK signaling pathway confirms the previous finding
(16) that
the IP-triggered calcium release channels, the endoplasmic
reticulum Ca
-ATPase, the plasma membrane
Ca
-ATPase, and other calcium removal systems were not
the target for cGMP kinase in CHO cells, as implied for other cell
types (see Refs. 1-4, 5, and 6). On the other hand, this result
did not support the general validity of the previous findings that
active cGMP kinase prevents hormone-triggered IP
synthesis,
since activation of both the thrombin and the CCK receptor increased
IP
levels in CHO cells
(16, 17) .
(40) . Phospholipase
C
is phosphorylated in vitro by cAMP kinase
(42) ,
suggesting that it is also a potential target for cGMP kinase. In
addition, inhibition of tyrosine phosphorylation blocked the mitogenic
action of thrombin without affecting the Ca
release
from internal stores
(40) . It was reported that CCK exerted
some of its effects by tyrosine phosphorylation in pancreas acinar
cells
(41) . However, CCK-induced amylase release was inhibited
by genistein distal of Ca
mobilization
(43) .
Hence, although unlikely, we tested the possibility whether
phospholipase C
activation or tyrosine phosphorylation of other
proteins contributed to the increase of
[Ca
]
by thrombin and
CCK in CHO cells. CHO-cGK cells were preincubated with 200 µ
M genistein. The cells were then superfused with 200 n
M thrombin or 100 µ
M CCK, hormones that stimulated
[Ca
]
to mean peak
levels of 618 ± 155 n
M (9 cells) or 800 ± 102
n
M (5 cells). Both values are similar to the peak
Ca
values in the absence of genistein (compare with
Tables I and II). These results indicated that tyrosine phosphorylation
was not involved in the signal transduction pathways or in the
calcium-releasing mechanisms stimulated by thrombin or CCK in CHO
cells.
PTX Pretreatment Suppressed Thrombin-induced but Not
CCK-induced Calcium Transients
Another possible cause for the
distinct effects of cGMP kinase was that thrombin and CCK signal via
different G proteins and phospholipase Cs. Preincubation of CHO-cGK
cells with 100 ng/ml PTX for 8 h completely prevented the
thrombin-stimulated calcium transient ( Fig. 4and ).
Thrombin stimulated
[Ca]
in only 1 out of
18 PTX-pretreated cells. In contrast, PTX preincubation did not
suppress the increase in
[Ca
]
stimulated by
CCK. Superfusion of a PTX-pretreated CHO cell with 300 µ
M CCK stimulated
[Ca
]
to a similar peak
value as in the control cell (Fig. 4). Furthermore, PTX
pretreatment did not affect
[Ca
]
stimulated by 100
µ
M CCK (). Statistical evaluation of control
CHO-cGK cells and CHO-cGK cells pretreated with PTX did not result in a
significant difference between both cell populations and, therefore,
confirmed that PTX pretreatment did not affect the CCK-activated
[Ca
]
.
Figure 4:
Pretreatment with PTX prevents thrombin-
but not CCK-induced Ca transients in CHO-cGK cells.
The cells were incubated with buffer ( left) or 100 ng/ml PTX
( right) for 8 h. Thereafter, the cells were superfused with
300 µ
M CCK ( upper part) or 200 n
M thrombin ( lower part) for 60
s.
It was
possible that the CHO-cGK cells were composed of two subpopulations in
which the hormone-stimulated
[Ca]
was differently
affected by cGMP kinase and PTX. To exclude this unlikely possibility,
individual CHO-cGK cells were superfused consecutively with either
agonist. The CHO-cGK cells were first superfused with thrombin and
then, after a short wash out, with CCK. The thrombin-induced
Ca
transients were suppressed by both cGMP kinase and
PTX pretreatment (Fig. 5). In contrast, neither PTX nor 8-Br-cGMP
affected the CCK response in the same cell.
[Ca
]
values in the
presence of 300 µ
M CCK were increased from 0.17 ±
0.02 to 1.1 ± 0.2 µ
M (5 cells) after 8-Br-cGMP and
from 0.14 ± 0.02 to 1.0 ± 0.2 µ
M (8 cells)
after PTX pretreatment. These values are similar to those obtained with
CCK in the absence of any pretreatment (compare with ).
Figure 5:
Ca transients in CHO-cGK
cells consecutively stimulated by thrombin and CCK. Two individual
CHO-cGK cells were preincubated with 1 m
M 8-Br-cGMP for 15 min
( left) or 100 ng/ml PTX for 8 h ( right). The cells
were superfused consecutively with 200 n
M thrombin followed by
a short washout with NaCl/Hepes buffer without agonist and then with
300 µ
M CCK. The arrows indicate the start of the
individual superfusions with hormone, which lasted for 60 s
each.
The results indicated that in CHO cells thrombin and CCK receptors
couple to phospholipase C via a PTX-sensitive and a PTX-insensitive G
protein, respectively. cGMP kinase interfered selectively with the
signaling of thrombin. The coupling to different G proteins suggested
that the involvement of a PTX-sensitive G protein might be required for
the suppressive action of cGMP kinase on agonist-induced Catransients.
IGF-2 and IGF-1 Stimulated Ca
In order to test whether the
susceptibility to the inhibitory action of cGMP kinase could in fact be
attributed to the type of G protein involved or was rather an
individual characteristic of the thrombin receptor or the phospholipase
C coupled to this receptor, we tested the effect of cGMP kinase on
other receptors involving GInflux in CHO Cells
proteins. CHO cells
endogenously express receptors that are activated by IGF-1 and IGF-2
(18) . IGF-1 and IGF-2 activate a calcium-permeable cation
channel via a PTX-sensitive G protein in Balb/c 3T3 cells
(20, 21) , thereby increasing
[Ca
]
. Both receptors
differ in their structure from each other and are quite different from
the thrombin receptor
(22, 44) .
]
followed by a
sustained plateau superimposed by oscillations (Fig. 6). The long
lasting stimulation of
[Ca
]
was reversed upon
removal of the hormone. Chelation of the extracellular calcium
prevented the IGF-2-induced calcium transient (Fig. 6). Ten
n
M IGF-2 elicited calcium transients in 94% of the CHO-cGK
cells, with an average peak value of 439 n
M above basal values
of [Ca
]
(Fig. 7).
Figure 6:
IGF-2
and IGF-1 stimulate calcium influx in CHO-cGK cells. Individual CHO-cGK
cells were superfused with 10 n
M IGF-2 ( left panel) or 50 n
M IGF-1 ( right panel) in the presence of NaCl/Hepes buffer containing 2
m
M CaCl( upper part) or in the
presence of NaCl/Hepes buffer without CaCl
but containing 5
m
M EGTA ( lower part). The superfusion with
IGF-2 and IGF-1 started at the arrows and lasted, if not
otherwise indicated, until the end of the shown recording. wash indicates superfusion with NaCl/Hepes buffer of the cell in the
absence of IGF-2. Note the different time scales in the left and right panels. Calcium transients activated by IGF-2
( left) and IGF-1 ( right) are representative for at
least 10 cells.
Figure 7:
Inhibition of IGF-2- and IGF-1-induced
calcium influx by 8-Br-cGMP and PTX. CHO-cGK cells were incubated in
the absence ( con) or presence of 1 m
M 8-Br-cGMP
( 8-cG) or 100 ng/ml PTX. The mean peak values (10-27
cells/condition) of the calcium transients ( open bars) are superimposed on the corresponding basal
[Ca] levels ( shaded bars). Basal values were recorded directly before
stimulation with 10 n
M IGF-2 ( left) or 50 n
M IGF-1 ( right). Please note that cells not responding to
the particular treatment have not been excluded from the
calculations.
In contrast to IGF-2, CHO-cGK cells responded to
IGF-1 with a slow and long lasting rise in
[Ca]
(Fig. 6).
IGF-1 at 50 n
M stimulated
[Ca
]
from 112 ±
13 to 322 ± 25 n
M (29 cells) in CHO-cGK cells (Fig. 7).
Only 3 out of 29 cells showed no response to IGF-1 superfusion. Removal
of extracellular Ca
by EGTA completely abolished the
IGF-1-induced increase in
[Ca
]
(Fig. 6). These
results suggested that both IGF-2 and IGF-1 activated a
Ca
-permeable cation channel in CHO-cGK cells. Similar
results were obtained in CHO-WT cells.
Inhibition of IGF-2-induced Calcium Influx by cGMP Kinase
and PTX
Stimulation of the cGMP kinase in CHO-cGK cells with
8-Br-cGMP substantially reduced the IGF-2-activated calcium influx.
Incubation with 8-Br-cGMP decreased IGF-2-stimulated calcium transients
by 54% (Fig. 7). An almost identical reduction of the IGF-2
response, namely by 58%, was observed after pretreatment of CHO-cGK
cells with 100 ng/ml PTX (Fig. 7). To evaluate whether the
partial suppression by cGMP kinase and PTX was mediated by interference
with different targets, we tested whether the effects of cGMP kinase
and PTX were additive. Treatment of CHO-cGK cells for 8 h with 100
ng/ml PTX, followed by incubation with 1 m
M 8-Br-cGMP in the
presence of PTX, did not result in a more pronounced inhibition of the
IGF-2 response. The simultaneous treatment of 10 cells with 8-Br-cGMP
and PTX resulted in a 62% reduction of IGF-2-induced calcium
transients.
]
from 70 ±
29 to 430 ± 94 n
M (8 cells) in the absence of 8-Br-cGMP
and from 84 ± 22 to 428 ± 69 n
M (7 cells) in the
presence of 8-Br-cGMP. This indicated that the partial suppression of
the IGF-2-stimulated Ca
transients in CHO-cGK cells
with 8-Br-cGMP was mediated by cGMP kinase and not by other
cGMP-regulated proteins.
Inhibition of IGF-1-induced Calcium Influx by cGMP Kinase
and PTX
The IGF-1-induced rise in
[Ca]
was also
inhibited by activation of cGMP kinase. In the absence of 8-Br-cGMP,
92% of the cells responded to IGF-1. After pretreatment with 8-Br-cGMP,
[Ca
]
did not increase
in 79% of the cells after application of 50 n
M IGF-1, and only
21% of the cells responded to 50 n
M IGF-1 with a reduced rise
in [Ca
]
. Overall, the
treatment with 1 m
M 8-Br-cGMP caused a reduction by 75% of the
IGF-1 response in the CHO-cGK cells (Fig. 7). The IGF-1-induced
Ca
transients were also suppressed by PTX. PTX
pretreatment of CHO-cGK cells abolished the response to IGF-1 in 10 of
13 cells. Overall, PTX inhibited the IGF-1 response by 71%
(Fig. 7).
]
from
109 ± 20 to 280 ± 29 n
M (7 cells). In the
presence of 8-Br-cGMP, IGF-1 stimulated
[Ca
]
from 135 ±
17 to 293 ± 35 n
M (6 cells), demonstrating that
inhibition of the Ca
transients required activation
of cGMP kinase.
Effect of Genistein on IGF-2- and IGF-1-induced Calcium
Transients
In order to test whether the remaining Cainflux, which was not suppressed by 8-Br-cGMP, resulted from the
activation of a pathway involving tyrosine phosphorylation, we studied
the effect of genistein on IGF-2- and IGF-1-induced Ca
transients. Incubation of CHO-cGK cells with 100 µ
M genistein for 15 min did not alter the IGF-2-induced calcium
influx; in the presence of genistein (100 µ
M), IGF-2
elicited a rise in
[Ca
]
from 110 ±
36 n
M up to a mean peak value of 504 ± 41 n
M (9 cells), a value similar to that obtained in the absence of
genistein (Fig. 7).
]
was also not
affected by genistein. At concentrations up to 200 µ
M,
genistein did not inhibit the IGF-1-induced rise of
[Ca
]
in CHO-cGK cells.
The basal and peak values of
[Ca
]
after IGF-1
stimulation were 173 ± 19 and 349 ± 34 n
M (10
cells), respectively, corresponding to the increase observed in the
absence of genistein. These results suggested that either tyrosine
phosphorylation did not contribute to the calcium influx, triggered by
IGF-1 and IGF-2 in CHO cells, or that genistein did not inhibit the
tyrosine phosphorylation step involved in the calcium influx pathway.
Phosphorylation of G Proteins by cGMP Kinase
The
results presented so far demonstrate that the signaling pathways
affected by cGMP kinase involved PTX-sensitive G proteins. CHO cells
are known to express the PTX-sensitive G proteins G,
G
, G
, and G
(24, 25) . These observations raised the
possibility that cGMP kinase affected the various signal transduction
pathways through the modification of a G protein. To examine the
potential phosphorylation of G proteins by cGMP kinase, purified
heterotrimeric G proteins were tested as in vitro substrates
for cGMP kinase.
, G
, and G
(Fig. 8 A). The approximate molecular mass of 40 kDa was
identical with that of the
subunits of G
/G
and indicated that the
subunits were phosphorylated.
However, the phosphorylation was less than stoichiometric as estimated
from counting the radioactive band after dissolving the gel and from
the amount of G protein added to the incubation. The substoichiometric
phosphorylation was presumably due to the presence of detergents in the
reaction mixture carried over with the G protein storage buffer. The
detergents may have altered the G protein structurally in such a way
that a potential phosphorylation site became more or less accessible
for cGMP kinase. To test this possibility, the heterotrimeric G
complex was reconstituted in phospholipid vesicles prior to
phosphorylation. cGMP kinase phosphorylated the reconstituted
G
subunit at an estimated stoichiometry of about 0.4
mol of phosphate/mol of G
(Fig. 8 B).
The identity of the phosphorylated band with G
was
ensured by immunoprecipitation of the subunit with the antibody AS8
recognizing all PTX-sensitive G subunits
(34) . A phosphorylated
immunoprecipitate was not obtained when the antibody AS6 recognizing
specifically G
isoforms
(34) was used
(Fig. 8 B). In contrast to G
, a preparation
containing G
was not phosphorylated by cGMP kinase (not
shown), suggesting that cGMP kinase phosphorylated preferentially the
subunits of the G
subfamily.
Figure 8:
Phosphorylation of G proteins
by cGMP kinase. A, G
, G
, and
G
( lanes 1-3) were phosphorylated
by cGMP kinase and thereafter applied to an SDS gel. The amounts of
G
, G
, and G
in the
phosphorylation assays were 12, 9, and 20.4 pmol. The phosphorylated
80-kDa protein corresponds to autophosphorylated cGMP kinase.
B, the reconstituted G
protein (5 pmol) was
phosphorylated by cGMP kinase and was then applied directly ( lane 4) or first immunoprecipitated with the
( lane 5) or with the
antibody ( lane 6) as described under
``Experimental Procedures.'' Two different autoradiographs of
phosphorylated G
proteins are
shown.
We tested, therefore,
whether or not the subunit of a G
protein was
phosphorylated in vivo in CHO cells. CHO-cGK cells were
prelabeled with
PO
and then stimulated with
8-Br-cGMP. Separation of the cell extract on an SDS gel yielded a
phosphorylated band at 80 kDa, which was immunoprecipitated with
specific cGMP kinase antibodies. Separation of a crude membrane
fraction on an SDS gel yielded no pronounced phosphorylated band in the
40-kDa range where the
subunit of heterotrimeric G proteins
should be localized. A positive result was obtained after
solubilization and immunoprecipitation of the membrane fraction with
the antibody AS372, which recognizes specifically the
and
protein (Fig. 9). Precipitation of a
membrane preparation prelabeled with azido-GTP with nonimmune serum
yielded no labeled band (Fig. 9, lane 1),
whereas precipitation of the membrane fraction with the specific
antibody resulted in a strong signal at a M
of
41,000 (Fig. 9, lane 2). The same protein band
contained radioactive phosphate. Phosphor imaging analysis indicated
that the
P content of this band was 1.7-fold higher in the
protein isolated from CHO-cGK cells than in that from CHO-WT cells. The
three characteristics of the phosphorylated band, i.e. the
immunoprecipitation of the protein by the G
-specific
antibody, its labeling by azido-GTP, and its M
of
41,000, strongly supported the notion that the phosphorylated band was
the
subunit of a G
protein.
Figure 9:
[P]GTP azidoanilide
and 8-Br-cGMP induced [
P]orthophosphate labeling
of G proteins in CHO cells. Lanes 1 and 2,
membranes obtained from 10
cells were subjected to
[
P]GTP azidoanilide labeling and
immunoprecipitation by nonimmune serum ( lane 1) or
AS373 ( lane 2) followed by separation on
SDS-polyacrylamide gel electrophoresis. Lanes 3 and
4, 10
CHO-WT cells ( lane 3) or
10
CHO-cGK cells ( lane 4) were
metabolically labeled with induced
[
P]orthophosphoric acid and subsequently
incubated with 8-Br-cGMP. Thereafter, cell membranes were prepared and
subjected to immune precipitation employing AS373 followed by
separation on SDS-polyacrylamide gel electrophoresis. Radiolabeled
proteins were visualized by autoradiography.
production and
[Ca
]
elevation in
CHO-cGK cells
(16) , suggesting that cGMP kinase may interfere
with the generation of second messengers by specific hormones. The
general applicability of this hypothesis was tested in the present
experiments, which investigated the effects of cGMP kinase on
thrombin-, CCK-, IGF-1-, and IGF-2-stimulated Ca
transients. cGMP kinase suppressed Ca
transients triggered by thrombin, IGF-1, and IGF-2 via
PTX-sensitive G proteins. These effects were specific for cGMP kinase
and were not observed in CHO-WT cells. In contrast, Ca
transients induced by CCK, which was signaling via an
unidentified low affinity receptor and a PTX-insensitive G protein,
were not affected by cGMP kinase. These different responses toward cGMP
kinase activation ruled out the possibility that cGMP kinase
phosphorylated and affected the function of plasma membrane or
endoplasmic reticulum Ca
-ATPases, the IP
release channel, or other common calcium-lowering mechanisms in
CHO-cGK cells.
production and opening of a
cation channel. The diversity of receptors and targets led to the
conclusion that it was unlikely that either of them was phosphorylated
by cGMP kinase. In agreement with other groups
(40, 43) , the experiments with genistein excluded the
possibility that these receptors stimulated
[Ca
]
by a tyrosine
phosphorylation step or that cGMP kinase affected such a step.
/G
subfamily. CHO cells
predominantly express G
and G
and at lower
concentrations G
and G
(24, 25) . The thrombin receptor couples to
several G proteins including PTX-sensitive G proteins
(17, 32, 45, 46) . In Balb/c 3T3 cells,
a PTX-sensitive G
protein mediated the IGF-1- and
IGF-2-stimulated calcium influx
(20, 21, 23) .
cGMP kinase inhibited the thrombin-, IGF-1-, and IGF-2-stimulated
Ca
transients to the same extent as PTX. This
apparent identity suggested that the signal transduction of the
thrombin receptor and the IGF-1 and IGF-2 receptors involved a similar
or identical G protein in CHO cells, presumably a member of the G
subfamily. Since these G proteins are the only link involved in
both the thrombin and IGF-1/IGF-2 signal transduction pathways
(17, 20, 23, 45, 46) , they
could be the candidates for cGMP-dependent phosphorylation (see Fig.
10).
,
G
, and G
contaminated with small amounts of
G
were phosphorylated by cGMP kinase. Immunoprecipitation
of reconstituted G
confirmed that the
subunit was
phosphorylated by cGMP kinase. In agreement with the in vitro data cGMP kinase phosphorylated a G
subunit
in vivo. Such a phosphorylation has not been detected
previously
(47) , presumably since at that time the
heterogeneity of the available G protein preparations was not
appreciated. G
proteins were not phosphorylated by cGMP
kinase, suggesting specificity of the phosphorylation reaction.
subunit contains a
potential cGMP kinase phosphorylation site (Arg-Lys-Asp-Thr-Lys) in the
carboxyl-terminal effector region
(48) . The same or similar
phosphorylation sites are present in the sequences of the
subunits of G
, G
, G
,
G
, and G
but not in G
,
G
, G
, G
, G
,
G
, G
, and G
. This analysis
supports the experimental findings and strengthens further the
hypothesis that cGMP kinase attenuated the calcium transients by the
phosphorylation of the subunit of a G
protein.
in situ (50) . In S49 lymphoma cells, phosphorylation of a G
protein by protein kinase C prevented somatostatin-induced
inhibition of adenylyl cyclase
(51) . In neuroblastoma
Glioma hybrid cells G
is phosphorylated by protein
kinase C
(52) . This reaction attenuates the inhibitory adenylyl
cyclase pathway. Therefore, it is not too speculative to assume that
cGMP-dependent phosphorylation of a G
protein interrupted
the coupling of the active G
subunit with its effector in
the CHO-cGK cells.
Table: Activation of cGMP kinase does not affect
CCK-induced Catransients
Table: Effect of PTX on CCK- and thrombin-induced
Catransients
], cytosolic calcium concentration; cGMP
kinase, cGMP-dependent protein kinase; CHO-WT, CHO wild type cells;
CHO-cGK, CHO cells expressing cGMP kinase I
; IGF, insulin-like
growth factor; CCK, cholecystokinin; G protein, guanine
nucleotide-binding protein; GTP
S, guanosine
5`-(3- O-thio)triphosphate; PTX, pertussis toxin; 8-Br-cGMP,
8-bromo-cGMP; IP
, inositol 1,4,5-trisphosphate; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid; MES, 4-morpholineethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.