From the Department of Molecular Cardiology and
¶ Department of Anesthesiology, Lerner Research Institute,
Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, September 8, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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Effectors involved in G protein-coupled receptor
signaling modulate activity of GTPases through GTPase-activating
protein or guanine nucleotide exchanging factor (GEF). Phospholipase
C Phospholipase C (PLC)1
PLC All known GTP binding subunits (G To date, none of the known heterotrimeric G proteins stimulates PLC Materials--
Fura 2-AM was obtained from Texas Fluorescence
Laboratories (Austin, TX), and G418 was obtained from Life
Technologies, Inc. Radioactive materials including
[ Purification of Proteins--
PLC Reconstitution--
Throughout the study, the proteins were
reconstituted in phospholipid vesicles by a dilution method (31).
Briefly, an appropriate amount of proteins was mixed with 0.2 mg of a
clear phospholipid suspension (8 mg/ml in 0.2% SM solution) consisting
of phosphatidylcholine, phosphatidylethanolamine, and
phosphatidylserine (3:1:1, w/w/w). The concentrations of phospholipid
mixture were 20-30 µg (final), and SM concentrations were ~0.008%
(final). The reconstitution mixtures in the assay buffer were
preincubated in an ice bath for 40 min throughout the study. For the
studies involving Preparation of Radiolabeled Guanine Nucleotide-bound TGII--
A
complex of [ Measurement of GTP Hydrolysis--
Since we have found that GTP
hydrolysis by TGII is temperature-sensitive, the reaction was performed
at room temperature. Single turnover GTP hydrolysis was determined with
the [ Determination of [3H]GDP Release and GTP Measurement of TGase Activity--
After preincubation of TGII
(4 nM) with various concentrations (0-10 nM)
of PLC Transfection and Cell Culture--
DNAs (10 µg/dish) of TGII
and its mutants inserted into a neomycin-resistant selection vector
pcDNA3.1-Neo (Invitrogene) were transfected to hamster
leiomyosarcoma (DDT1-MF2) using LipofectAMINE method provided by the
manufacturer (Life Technologies, Inc.). DDT1-MF2 cell has been shown to
express the Measurement of [Ca2+]i--
Cytosolic
free Ca2+ concentration
([Ca2+]i) was determined using the
fluorescent Ca2+ indicator Fura 2-AM as described by Xu
et al. (33). DDT1-MF2 cells (1 × 104
cells) were trypsinized and seeded on 35-mm glass culture dishes designed for fluorescence microscopy (Bioptech, Butler, PA).
After the cells were incubated overnight in DMEM containing 10%
heat-inactivated, the cells were further incubated in a serum-free DMEM
for 24 h. The cells were then loaded with Fura 2-AM (2 µM) at room temperature for 40 min in Krebs Ringer (KR)
buffer (25 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose) containing
0.2% bovine serum albumin. After washing cells three times with KR
buffer, the cells were kept in the tissue culture incubator until use.
Before measurement of [Ca2+]i, the
cells were washed three times with Ca2+-free KR buffer
containing 1 × 10 Effects of PLC
To determine whether PLC
GEF action of PLC
It has also been shown that conformational changes in TGII modulate
TGII activity, which are induced by the activators (35, 36). Thus,
Ca2+-bound TGII can not function as GTPase, and GTP-bound
TGII does not exhibit TGase activity. To determine whether PLC PLC
To understand the mechanism of GEF activity of the Overexpression of PLC
To date, an effector protein acting as both GEF and GHIF for a GTPase
has not been described in either a heterotrimeric or a monomeric GTPase
signaling system. Our studies on the roles of PLC
The observation that overexpression of PLC
Effectors such as PLC1 (PLC
1) is an effector in tissue transglutaminase
(TGII)-mediated
1B-adrenoreceptor
(
1BAR) signaling. We investigated whether PLC
1
modulates TGII activity. PLC
1 stimulated GDP release from TGII in a
concentration-dependent manner, resulting in an increase in
GTP
S binding to TGII. PLC
1 also inhibited GTP hydrolysis by TGII
that was independent from the
1BAR. These results
indicate that PLC
1 is GEF for TGII and stabilizes the
GTP·TGII complex. When GEF function of PLC
1 was compared
with that of the
1BAR, the
1BAR-mediated
GTP
S binding to TGII was greater than PLC
1-mediated binding and
was accelerated in the presence of PLC
1. Thus, the
1BAR is the prime GEF for TGII, and GEF activity of
PLC
1 promotes coupling efficacy of this signaling system.
Overexpression of TGII and its mutants with and without PLC
1
resulted in an increase in
1BAR-stimulated
Ca2+ release from intracellular stores in a TGII-specific
manner. We conclude that PLC
1 assists the
1BAR
function through its GEF action and is primarily activated by the
coupling of TGII to the cognate receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 is a member of the PLC family that produces two second messengers,
inositol 1,4,5-triphosphate (IP3) and diacylglycerol by
hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) (1). Among PLCs, PLC
isozymes are stimulated by
guanine nucleotide-binding proteins (G protein) Gq and its
family of proteins in response to activation of cell surface receptors.
PLC
isozymes are activated by phosphorylation through growth factor
receptors. A number of laboratories have reported that PLC
1 is
stimulated by a unique GTP-binding protein known as tissue
transglutaminase (TGII, G
h) (2-5). TGII is a
bifunctional enzyme, having GTPase and transglutaminase (TGase)
activity (6, 7) and is present in the plasma membrane, cytosol, and
nucleus in a variety of tissues and cells (6). Exchange of GDP to GTP
by TGII is facilitated by activation of the cell surface receptors
(4-9). These receptors include the
1B-adrenoreceptor
(AR) (5, 7, 8),
1DAR (8),
-thromboxane receptor (9),
and oxytocin receptor (4). The coupling of TGII with these receptors
appears to be receptor subtype-specific (8, 9).
1 is widely distributed and expressed highly in some tissues such
as mouse heart (1, 10). Stimulation of the enzyme by TGII involving
1BAR is modulated in a concerted fashion within the
system. Thus, bimodal regulation of PLC
1 activity has been observed
depending on the Ca2+ levels and occupancy of guanine
nucleotide by TGII (3, 11, 12). PLC
1 is stimulated with low
concentrations of Ca2+ by GTP
S·TGII (11). However,
activity of the enzyme is subsequently inhibited by increasing
Ca2+ concentrations where PLC
1 is stimulated in the
presence or absence of GDP. Similarly, Murthy et al. (3)
have reported that GTP·TGII inhibits PLC
1, while GDP·TGII
stimulates the enzyme. The Ca2+ dependence is not clearly
defined in this study. The TGII-mediated PLC stimulation is also
modulated by the level of TGII expression (12). At low levels of TGII
expression, the
1BAR-stimulated PLC activity is
increased, whereas the receptor-mediated PLC stimulation is inhibited
when TGII is highly expressed. The PLC
1 activity is also inhibited
by IP3, competing with its substrate PIP2 for a
binding site known as the pleckstrin homology domain (13-15). Studies
have also demonstrated that an increase in the intracellular concentration of Ca2+ activates PLC
1 (13, 16, 17),
indicating that activation of PLC
1 occurs secondarily in response to
the receptor-mediated activation of other PLCs or Ca2+
channels. A GTPase activating-protein (GAP) for the small GTPase RhoA
(RhoGAP) also activates PLC
1 by direct association (18). All of
these observations suggest that the PLC
1 activity is regulated by
multiple factors.
) of G proteins are GTPases, which
hydrolyze GTP to GDP and orthophosphate (Pi). It is now recognized that a large number of regulators of G protein signaling (RGS) facilitate GTP hydrolysis by G
proteins (19-21). Independent from the actions of these RGS proteins, certain effectors in the G
protein-coupled receptor system modulate GTP hydrolysis by G
proteins acting as GAP or guanine nucleotide exchanging factor (GEF)
(22-26). For example, PLC
1(22-24) and the
subunit of cGMP phosphodiesterase (25) directly accelerates GTP hydrolysis by G
q and G
t, respectively. A recent study
by Scholich et al. (26) has shown that adenylyl cyclase
facilitates GTP binding to Gs, thereby functioning as both
GEF and GAP. These findings indicate that the effector molecules
modulate their cognate GTPases to terminate or facilitate the signals.
1
(1, 27), and the mechanisms that regulate PLC
1 activity remain
complex and unclear. To further understand the characteristics and the
interaction of PLC
1 with TGII and its activation by TGII and the
1BAR, we investigated the roles of PLC
1 in the
modulation of TGII activities, including the
1BAR. Here,
we report a distinct role of PLC
1 in a coupling system involving the
1BAR and TGII. PLC
1 displays two regulatory functions for TGII. One is a GEF function, and the other is the inhibition of GTP
hydrolysis by TGII. The GEF function of PLC
1 promotes the
1BAR-mediated GTP binding by TGII. Furthermore, our
results also reveal that PLC
1 is primarily activated by the
activation of the
1BAR through TGII, resulting in
Ca2+ release from intracellular stores.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]GTP (3000 Ci/mmol), [
-32P]GTP
(3000 Ci/mmol), [35S]GTP
S (~1300 Ci/mmol),
[3H]GDP (25-50 Ci/mmol), [3H]prazosin
(79.8 Ci/mmol), and [3H]putrescine (37.5 Ci/mmol) were
purchased from PerkinElmer Life Sciences. Heparin-agarose, wheat
germ agglutinin-agarose, GTP-agarose, guinea pig liver TGase,
hygromycin, and N,N'-dimethylcasein,
phospholipids were purchased from Sigma. A monoclonal TGII antibody
CUB7402 was obtained from NeoMarkers (Freemont, CA). A monoclonal
PLC
1 antibody was purchased from Upstate Biotechnology Inc. (Lake
Placid, NY), and a polyclonal Gq/11
antibody was from
BIOMOL (Plymouth Meeting, PA). Fast Mono-Q Sepharose was obtained from
Amersham Pharmacia Biotech. Nitrocellulose membrane BA85 was from
Schleicher & Schuell. Norit A charcoal was from Serva (New York, NY).
1 was expressed in DH
5
cells and purified as described (2). The purity of the PLC
1
preparation was
90%, as judged by silver staining, and neither
GTP
S binding and TGase activity were observed. Guinea pig liver TGII
was further purified using GTP-agarose as described (7, 28). The purity
of the TGII preparation was
95% as judged by silver staining, and
PLC activity was not found in the TGII preparation as determined by
measurement of PIP2 hydrolysis (2). It should be noted that
purified TGII was stable for
3-4 weeks in the presence of 10%
glycerol at
80 °C. The
1BAR was expressed in COS-1
cell (5) and partially purified in the presence of phentolamine by
chromatography using heparin-agarose and wheat germ agglutinin-agarose
as described (29). Contamination of TGII as well as other GTP-binding
proteins was determined by measurement of TGase activity, direct
photolabeling of GTPases with [
-32P]GTP (28), and
immunoblotting with antibodies against TGII and Gq/11
(5). G
q (the virus was kindly provided by Dr. Elliott
Ross at Northern Texas University, Dallas, TX) was expressed in
Sf9 cells. Since we have found that Sf9 cells do not
express TGII as judged by immunoblotting and measurement of TGase
activity, G
q was partially purified by one-step
chromatography using Mono Q-Sepharose. Membrane (5 mg/ml) prepared from
G
q-expressed Sf9 cells was extracted with 1%
sodium cholate in 50 mM HEPES, pH 7.4, 50 mM
NaCl, 3 mM 1,4-dithiothreitol, 3 mM EGTA, 1 mM EDTA, 5 mM MgCl2, 10 mM NaF, 30 µM AlCl3 as described
(24, 27). The extract was diluted 10-fold with the same buffer
containing 0.02% sucrose monolaurate (SM) and loaded onto Mono
Q-Sepharose, which was pre-equilibrated with the same buffer containing
0.02% SM. The column was washed with the buffer and eluted with 300 mM NaCl and 10% glycerol in the same buffer. The amount of
G
q was determined by immunoblotting with
Gq/11
antibody. A single band with molecular mass of 42 kDa was detected with the antibody. Known concentrations of TGII were
simultaneously immunoblotted to estimate the amounts of
G
q. The protein preparations were aliquoted and stored
at
80 °C until use. For each experiment, the protein preparations
in the elution buffer was loaded onto a dried Sephadex G-25 column (3 ml) to remove the salts (29). The dried column was preequilibrated with
an assay buffer (25 mM HEPES, pH 7.4, 150 mM
NaCl, 0.5 mM 1,4-dithiothreitol, and 1.5 mM
MgCl2). The recovery was ~40-50%, as determined by
immunoblotting with the TGII and Gq/11
antibodies or
TGase activity measurement for TGII. The recovery of the
1BAR was ~50%, as determined by binding ability of
[3H]prazosin (29). In addition, it should be noted that a
nonhydrolyzable ATP analogue AppNHp (100 µM) was included
in the assay buffer throughout the study, since it has been reported
that TGII also binds ATP and hydrolyzes it (30).
1BAR (150 pM/tube), the
samples were preincubated in the presence of 1 × 10
5 M (
)-epinephrine or 1 × 10
4 phentolamine.
-32P]GTP·TGII was prepared by incubating
TGII (~50 nM) with 50 µM
[
-32P]GTP (100,000 cpm/nM) in 300 µl of
the assay buffer. After incubation at room temperature for 20 min,
unbound [
-32P]GTP and
[32P]Pi was removed by a dried Sephadex G-25
column which was preequilibrated with the assay buffer. The amounts of
[
-32P]GTP·TGII complex were determined by a
nitrocellulose membrane filter assay (29). Commercial
[3H]GDP was lyophilized to remove ethanol and then
reconstituted with water prior to use. The complex of
[3H]GDP·TGII was prepared by incubating TGII (50 nM) with [3H]GDP (100 µCi) in 300-500 µl
of the assay buffer at room temperature for 30 min. Unbound GDP was not
removed since TGII has a low affinity for GDP (28).
-32P]GTP·TGII complex (~1
nM/tube) preparation. The complex was mixed with and
without 4 nM PLC
1 or with 4 nM
heat-inactivated PLC
1 (boiled for 20 min) in the assay buffer. At
time 0, 100 µM cold GTP was added to prevent the
rebinding of radiolabeled guanine nucleotide. At the indicated time,
the samples were transferred to an ice-water bath, and the amount of
[
-32P]GTP·TGII remaining was determined by the
nitrocellulose filter method. A standard GTPase activity was also
performed by charcoal absorption method (29). Briefly, vesicles
containing proteins were mixed with 2 µM GTP plus 3 µCi
of [
-32P]GTP in the assay buffer in a 100-µl final
volume. The reaction was performed at room temperature for 20 min and
stopped by addition of ice-cold Norit A charcoal suspension (5%, w/v,
900 µl) in 50 mM sodium phosphate buffer (pH 7.4). The
reaction mixture was centrifuged for 20 min at 4 °C, and a 700-µl
aliquot of the supernatants was withdrawn and recentrifuged under the
same conditions. After a second centrifugation, the amount of
[32P]Pi released in a 500-µl aliquot was
determined by a
-counter. To determine turnover,
[35S]GTP
S binding was performed with the same samples
at room temperature for 20 min.
S
Binding--
Both experiments were carried out at 10 °C throughout
the study. [3H]GDP·TGII (~1 nM) was
incubated with 4 nM PLC
1 or Ca2+-bound
PLC-
1 or heat-inactivated PLC
1. Ca2+-bound PLC
1
was prepared by incubating of the enzyme with 30 µM
Ca2+ at room temperature for 20 min. At this concentration
of Ca2+, PLC
1 was fully activated (2). The final
concentration of Ca2+ was adjusted to 5 µM in
the reaction mixtures. At time 0, 2 µM GTP
S was added
to the reaction mixtures to prevent the rebinding of radiolabeled GDP.
For the GTP
S binding experiments, the reaction was started by adding
2 µM [35S]GTP
S (1500 cpm/nM,
final). Nonspecific binding was determined in the presence of 100 µM GTP or in the presence of 1 × 10
4 M phentolamine when the
1BAR was reconstituted with TGII or G
q
and PLC
1. The time-dependent experiments were performed
by transferring an aliquot (50 µl) to the ice-water bath at the
indicated time points. The amounts of GTP
S binding by TGII or
G
q and the remaining [3H]GDP·TGII
complex were determined by the nitrocellulose filter method (29).
1, the TGase activity was measured in the presence of 1%
N,N'-dimethylcasein, 1 µM putrescine (2.2 × 106 cpm/1 µM, final), and 300 µM Ca2+ at 25 °C for 20 min (28). The
nonspecific TGase activity was determined in the presence of 1 mM EGTA.
1BAR subtype only (32). TGII and its mutant
expressed cells were selected using 500 µg/ml G418 in Dulbecco's
modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal
bovine serum and 100 µg/ml penicillin, and 100 µg/ml streptomycin.
After completion of the selection, PLC
1 DNA (10 µg) inserted into
a hygromycin selection vector pCEP4 (Invitrogene) was transfected into
DDT1-MF2 cells expressing TGII and its mutants. The cells were selected
with 500 µg/ml hygromycin in a growth medium containing 300 µg/ml
G418. The established cells were maintained in the growth medium
containing 300 µg/ml G418 and 300 µg/ml hygromycin.
6 M
propranolol and 1 × 10
7 M
rawalscine. The
1BAR-mediated
[Ca2+]i was determined by addition
of 1 × 10
5 M
(
)-epinephrine (final). Five cells, which were within the beam light,
were selected to collect data. The reason for the selection of multiple
cells is to minimize the variability of outcome, since expression level
of proteins is expected to vary from a cell to a cell. The culture
dishes were placed in a temperature-regulated chamber (37 °C).
Fluorescence ratios were measured by an alternative wavelength time
scanning method (dual excitation at 340 and 380 nm, emission at 500 nm). Estimation of [Ca2+]i were
achieved by comparing the cellular ratio with fluorescence ratios
acquired using Fura 2 (free acid) in buffer containing known
concentrations of Ca2+.
[Ca2+]i was calculated as
described by Grynkiewicz et al. (34).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 on GTPase Function of TGII--
To assess
whether PLC
1 modulates GTPase activity of TGII, a single turnover
hydrolysis of [
-32P]GTP by TGII was determined using a
[
-32P]GTP·TGII preparation with and without PLC
1
(Fig. 1A). In the absence of
PLC
1, the [
-32P]GTP hydrolysis by TGII was observed
in a time-dependent manner and reached half-maximal GTP
hydrolysis within 8 min. In contrast, when PLC
1 was present, the
[
-32P]GTP hydrolysis was extremely slow (~21%
hydrolysis for 30-min incubation). The [
-32P]GTP
hydrolysis was not inhibited by heat-inactivated PLC
1, indicating
that the inhibition is caused by the interaction of PLC
1 with TGII.
The PLC
1-mediated inhibition of GTP hydrolysis by TGII was further
examined using the charcoal absorption method. Equimolar (4 nM) of TGII and PLC
1 or heat-inactivated PLC-
1 was
mixed and incubated at room temperature for 20 min. The vesicles containing TGII alone or the heat-inactivated PLC
1 produced
[32P]Pi with a turnover of 1.2-1.5
mol
1 min-1. On the other hand,
the vesicles containing TGII and PLC
1 produced less
[32P]Pi with a turnover of 0.24 mol
1 min
1. This
demonstrates that PLC
1 inhibits GTP hydrolysis by TGII, showing that
PLC
1 is not GAP for TGII.
View larger version (17K):
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Fig. 1.
Modulation of GTPase activity of TGII by
PLC 1. A, hydrolysis of
[
-32P]GTP by TGII in the presence and absence of
PLC
1. [
-32P]GTP·TGII complex (~1
nM/tube) was mixed with and without PLC
1 (4 nM/tube) as detailed under "Experimental Procedures."
Boiled PLC
1 was 4 nM. The reaction was performed at room
temperature and stopped by transferring the samples in an ice-water
bath. At time 0, amount of [
-32P]GTP·TGII was taken
as 100%. B, PLC
1 concentration-dependent GDP
release from TGII. [3H]GDP·TGII (~1 nM)
estimated by immunoblotting using TGII antibody was mixed with various
concentrations of PLC
1 (filled circle) or
heat-inactivated PLC
1 (open triangle). The GDP
release was determined at 10 °C for 10 min. At time 0, amount of
[3H]GDP·TGII without PLC
1 was taken as 100%.
C, time-dependent GDP release from TGII induced
by PLC
1. An equal amount of TGII (4 nM) and PLC
1
(filled circle) or Ca2+-PLC
1
(open square) was reconstituted.
Filled triangle indicates
[3H]GDP·TGII alone. The reactions were carried at
10 °C. Amount of [3H]GDP·TGII at time 0 was taken as
100%. The data present the means ± S.E. from one of the
representative experiments in triplicate.
1 influences exchange of GDP to GTP by TGII,
GDP release from TGII was determined (Fig. 1, B and C). To evaluate whether Ca2+-bound or unbound
PLC
1 exhibits this effect on TGII, PLC
1 preincubated with
Ca2+ (Ca2+-PLC
1) was also tested. A
[3H]GDP·TGII complex was reconstituted with various
concentrations of PLC
1. The results revealed that the GDP release
from TGII was accelerated as a function of PLC
1 concentration (Fig.
1B). The heat-inactivated PLC
1 was unable to catalyze the
[3H]GDP release, showing the specificity of PLC
1
action on TGII. Furthermore, the [3H]GDP release from
TGII induced by PLC
1 was time-dependent, reaching half-maximal [3H]GDP release within 4 min (Fig.
1C). The samples containing [3H]GDP·TGII
alone or [3H]GDP·TGII with Ca2+-PLC
1
showed a slow [3H]GDP release with a similar rate. The
result indicates that Ca2+-unbound PLC
1 acts as GEF for
TGII.
1 for TGII was further examined by determining
GTP
S binding to TGII (Fig. 2,
A and B). Consistent with the observations that
PLC
1 stimulated GDP release, the GTP
S binding of TGII was
increased as a function of PLC
1 concentration (Fig. 2A).
At a 1:2 ratio of TGII versus PLC
1, the GTP
S binding to TGII reached a plateau. PLC
1 alone showed no GTP
S binding activity, indicating that the increased GTP
S binding is caused by
the interaction of TGII with PLC
1. When the GTP
S binding by TGII
was determined as a function of incubation time with and without
PLC
1, the GTP
S binding in the presence of PLC
1 was higher
(~3-fold) than TGII alone and reached a maximum within 11 min (Fig.
2B). In the presence of Ca2+-PLC
1, the
GTP
S binding was similar to TGII alone, again demonstrating that
Ca2+-PLC
1 does not stimulate GTP
S binding to TGII. A
slight increase of basal GTP
S binding by TGII was also observed in
the presence of PLC
1, probably due to interaction of the enzyme with
TGII during preincubation.
View larger version (15K):
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Fig. 2.
Effects of PLC 1 on
GTP
S binding by TGII. A,
PLC
1 concentration-dependent GTP
S binding by TGII.
TGII (4 nM/tube) was mixed with various concentrations of
PLC
1. The reaction was carried out at 10 °C for 10 min. The
nonspecific GTP
S binding was determined in the presence of 100 µM GTP. B, time-dependent GTP
S
binding to TGII induced by PLC
1. TGII (4 nM) were mixed
with (filled circle) or without
(filled triangle) PLC
1 (8 nM) or
with Ca2+-PLC
1 (8 nM, open
square). The reaction mixtures were incubated at 10 °C.
C. Inhibition of TGase activity of TGII by PLC
1. TGII was
mixed with PLC
1 (filled circle) or
heat-inactivated PLC
1 (open triangle). The
TGase activity in the absence of PLC
1 was taken as 100%. The data
present the means ± S.E. from one of the representative
experiments in triplicate.
1
induces GTPase form of TGII, the TGase activity was determined in the presence of various concentrations of PLC
1 or heat-inactivated PLC
1 (Fig. 2C). The reaction was started with
Ca2+ to prevent the activation of PLC
1 and TGase of
TGII. The results showed that Ca2+-mediated TGase
activation was inhibited in a concentration-dependent manner by PLC
1. Heat-inactivated PLC
1 was unable to inhibit TGase
activity, demonstrating that the inhibition of TGase activity is due to
the interaction TGII with PLC
1. In addition, to discern whether the
cross-linking of proteins caused the decrease in the enzyme activity,
samples treated under the same conditions were subjected to
immunoblotting with TGII and PLC
1 antibodies. Cross-linking of
TGII-PLC
1 or TGII·TGII or PLC
1-PLC
1 was not observed (data not shown). Taken together, these data clearly show that PLC
1 is GEF
for TGII and that the interaction of TGII with PLC
1 induces a
conformational change in TGII to become GTPase.
1 Is a Helper of
1BAR Function That Stabilizes
the GTPase Conformation of TGII--
G protein-coupled receptors are
GEFs for their cognate G proteins. Since the
1BAR
couples with both TGII and G
q (7, 8) but
G
q does not stimulate PLC
1 activity (27), we first
evaluated the specificity of GEF function of PLC
1 for TGII. The
1BAR was reconstituted with either TGII or
G
q in the presence and absence of PLC
1, and GTP
S
binding by TGII or G
q was determined (Fig. 3A). The results revealed
that, although the
1BAR was able to activate GTP
S
binding to both TGII and G
q, GEF activity of PLC
1 was
specific for TGII. Thus, in the presence of PLC
1, the
1BAR-mediated GTP
S binding to TGII was further
increased ~58%, whereas the level of the receptor-mediated GTP
S
binding to G
q did not change. Moreover, PLC
1 again
stimulated GTP
S binding by TGII, but not by G
q. This
observation is also consistent with the finding that PLC
1 is not
activated by G
q (27). Since GTP hydrolysis by TGII was
inhibited in the presence of PLC
1, we also determined whether the
inhibition of GTP hydrolysis occurs in the presence of the
1BAR (Fig. 3B). In the presence of PLC
1,
the rate of GTP hydrolysis was inhibited ~77% with the samples
containing TGII and PLC
1 and ~41% with the samples containing all
three components. These results again indicate that PLC
1 functions as a GTP hydrolysis-inhibiting factor (GHIF) and that there is a stable
association of TGII with PLC
1.
View larger version (20K):
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Fig. 3.
Effects of PLC 1 on
the
1BAR-mediated
GTP
S binding to TGII. A,
specificity of the GEF function of PLC
1 for TGII. The indicated G
proteins (4 nM/tube) were mixed with and without 4 nM PLC
1 or with and without 150 pM
1BAR. The reconstitution of proteins of interest was
achieved in phospholipid vesicles. The GTP
S binding was determined
in the presence of 2 µM [35S]GTP
S by
incubating of the reaction mixtures at 10 °C for 10 min.
B, effects of PLC
1 on the
1BAR-mediated
GTP hydrolysis by TGII. The reconstituted samples indicated in the
figure were incubated in the presence of 1 × 10
5 (
)-epinephrine or 20 µM
GTP
S at room temperature for 20 min. Pi release was
determined by charcoal absorption method as described under
"Experimental Procedures." The [35S]GTP
S binding
activity was also determined at room temperature for 20 min to
calculate turnover of the GTPase activity. The data shown are the
means ± S.E. from one of the representative experiments in
triplicate.
1BAR
versus PLC
1 for TGII, the
1BAR, TGII, and
PLC
1 were reconstituted, and the GTP
S binding activity of TGII
was assessed under various conditions (Fig.
4). The
1BAR-mediated
GTP
S binding to TGII was evident, reaching a plateau within 6 min
(Fig. 4A). When PLC
1 was present, the receptor-mediated
GTP
S binding was further increased (~47% at 2 min) and reached a
plateau within 4 min. PLC
1-mediated GTP
S binding to TGII was slow
compared with
1BAR-mediated GTP
S binding in both the
presence and absence of PLC
1. These data indicate that the
1BAR is the prime GEF for TGII and that PLC
1 functions secondarily. Although TGII alone showed no GTP
S binding at
time zero, when the receptor and/or PLC
1 were present, the basal
level of GTP
S binding by TGII was increased. The order of the basal
GTP
S binding was
1BAR + TGII + PLC
1 >
1BAR + TGII > TGII + PLC
1. To further
understand the role of PLC
1, the receptor and TGII were
reconstituted with various concentrations of PLC
1, and GTP
S
binding by TGII was determined at 2 and 4 min (Fig. 4B). At
the 2-min time point, GTP
S binding was increased as a function of
PLC
1 concentration. At the 4-min time point, GTP
S binding was
reached maximum at 1:1 ratio of TGII and PLC
1, and a further
increase in the concentration of PLC
1 did not increase the
1BAR-mediated GTP
S binding, probably due to the
limited amounts of TGII. When the TGII concentrations were varied at
fixed amounts of PLC
1, GTP
S binding by TGII was increased as a
function of TGII concentration (Fig. 4C). Although maximum
coupling efficacy was observed at 1:1 ratio of TGII and PLC
1, a
further increase of TGII concentration resulted in a decrease in the
coupling efficacy. These results indicate that a level of each
component governs the activation of GTP binding to TGII and that the
1BAR and PLC
1 induce GTPase conformation of TGII in a
concerted way. The sequence of conformational changes of TGII would be:
basal state of TGII, which can function as GTPase and TGase; the second
state, GTPase conformation that is induced by the receptor and can
reverse to the basal state; the third state, GTPase conformation
induced by the receptor and stabilized by interacting with PLC
1.
View larger version (17K):
[in a new window]
Fig. 4.
GTP S binding to TGII
mediated by the
1BAR in the
presence and absence of PLC
1.
A, GTP
S binding to TGII in the presence and absence of
the
1BAR and/or PLC
1. Indicated proteins (150 pM
1BAR, 4 nM TGII, and
PLC-
1) in the figure were reconstituted in phospholipid vesicles. At
indicated time, an aliquot (50 µl) was transferred to a tube in an
ice-water bath. Nonspecific GTP
S binding was determined in the
presence 100 µM GTP or 1 × 10
4 M phentolamine. Standard
error was 7-10% of the specific GTP
S binding. B, rate
of the
1BAR-mediated GTP
S binding to TGII in the
presence of various concentrations of PLC
1. The samples were
incubated at 10 °C for 2 or 4 min. C, effects of TGII
level on the coupling efficacy involving
1BAR and
PLC
1. The samples were incubated at 10 °C for 4 min. All
experiments were performed three times in duplicate, and the specific
GTP
S binding is shown in the means ± S.E. from one of the
representative experiments.
1 Enhances [Ca2+] by
Activation of the
1BAR--
The role of PLC
1 in
facilitating coupling of the
1BAR with TGII was further
investigated using DDT1-MF2 cells stably expressing PLC
1 without and
with wild-type TGII (wtTG) and its mutants (Fig. 5). A TGII mutant (C-STG),
which lacks TGase activity by mutation of Cys277 to Ser at
TGase active site (37), was utilized to delineate GTPase
versus TGase activity of TGII. Moreover, if PLC
1 acts as
a stabilizer of GTPase conformation of TGII through its GEF/GHIF activity, wtTG would provide the same result as C-STG does.
To evaluate a specific interaction among
1BAR, TGII, and
PLC
1, two TGII mutants were utilized; m3TG in which an
1BAR interaction site on TGII was mutated (5), and
L656 (
1TG) in which a PLC
1 interaction site was deleted
(38). Proteins were highly expressed, and the expression levels were
comparable with each other (Fig. 5, A and B). It
should be noted that a fast mobility of the m3TG on SDS-PAGE was also
observed when the mutant was expressed in COS-1 cell (5). The reason is
not clearly understood. However, differences in an apparent molecular
weight were observed with TGIIs from different species, indicating that the mobility of TGII on SDS-PAGE gel is greatly affected by the primary
structure of the enzyme (39). The slow mobility of
1TG is
expected, because of the deletion of 30 amino acid residues from C
terminus (38). The coupling among
1BAR, TGII, and
PLC
1 was assessed by measuring
[Ca2+]i in a Ca2+-free
buffer (Fig. 5C). The control cells (vector) transfected with vectors (pcDNA3.1 and pCEP4) displayed an increase in the level of [Ca2+]i in response to
activation of the
1BAR with (
)-epinephrine. The
1-agonist-evoked peak increase in
[Ca2+]i was further increased by
~59% when PLC
1 (vector plus PLC
1) was expressed, demonstrating
that PLC
1 increases the coupling efficacy of this signaling system.
Since the experiments were performed in Ca2+-free buffer,
the increase in [Ca2+]i is due to
the release of Ca2+ from an intracellular store that is
likely mediated by IP3 formed in response to PLC
1
activation. Expression of wtTG or C-STG resulted in an
increase in peak [Ca2+]i that was
~74% and ~63% greater than that observed in vector-transfected
cells, respectively. The cells coexpressing wtTG and C-STG
with PLC
1 exhibited ~23% increase in peak
[Ca2+]i compared with wtTG or
C-STG alone. The reason for this limited increase of
[Ca2+]i is probably due to a
limited number of the
1BAR, since the receptor is the
prime GEF in this signaling system (see Fig. 4). The
1-agonist-mediated Ca2+ release was due to
the coupling of TGII with the
1BAR and PLC
1, because
both m3TG- and
1TG-expressing cells showed an increase in peak
[Ca2+]i, which was ~53% less
than the cells expressing wtTG or C-STG. Moreover, the peak
[Ca2+]i was 20% less than the
control cell (vector plus PLC
1), and coexpression of PLC
1 with
these mutants did not significantly increase
[Ca2+]i. Although a residual
stimulation of Ca2+ release in activation of the
1BAR is most likely due to the incomplete blocking of
the interaction among these three proteins, it is also possible that
the increase in [Ca2+]i in these
cells is due to the coupling of the
1BAR with other G
proteins such as the Gq family of proteins. Preincubation of the cells with the
1-antagonist prazosin or
nonspecific PLC inhibitor U73122 completely abolished the
1-agonist-mediated increase in
[Ca2+]i (data not shown). To
assess endoplasmic reticulum Ca2+ content, we treated the
cells with thapsigargin (an inhibitor of endoplasmic reticulum
Ca2+ pump, which stimulates Ca2+ release) at
the end of the experiments (Fig. 5D). The
thapsigargin-induced release of
[Ca2+]i was the lowest in the
cells coexpressing of PLC
1 with wtTG or C-STG and
correlated with the amounts of
[Ca2+]i release induced by the
activation of the
1BAR with the cells expressing TGII
and its mutants without or with PLC
1. These data further demonstrate
that the increase in [Ca2+]i is
caused by release from the intracellular Ca2+ stores.
View larger version (40K):
[in a new window]
Fig. 5.
Expression of PLC 1
results in the increase in
[Ca2+]i in response to the
activation of the
1BAR.
A, expression level of TGIIs. Cell lysates (150 µg) were
subjected to immunoblotting using a TGII antibody, followed by SDS-PAGE
(10% gel). B, cell lysate (150 µg) was used to determine
PLC
1 by immunoblotting, followed by SDS-PAGE (10% gel).
C, effects of PLC
1 on the
1BAR-mediated
Ca2+ release in cells expressing PLC
1 with and without
wtTG and its mutants. D, thapsigargin-induced
Ca2+ release. Concentration of thapsigargin was 5 µM, and level of [Ca2+] with the vector
cells was taken as 100%. The data presented were obtained from the
experiments shown in panel C.
1 in regulation of
TGII activities reveal that PLC
1 exhibits GEF and GHIF activities
for GTPase function of TGII. Evidence for the GEF function of PLC
1
is that the enzyme facilitates GDP release from TGII and stimulation of
GTP
S binding (Figs. 1 and 2). The inhibition of TGase activity by
PLC
1 and the GHIF activity of the PLC
1 suggest that the
interaction of PLC
1 with TGII induces and stabilizes GTPase
conformation of TGII. The GEF/GHIF activity of PLC
1 displays
independently from the
1BAR. However, when the
1BAR is present, the receptor is the prime GEF (Fig. 4). This conclusion is based on the observations that (i) the
1BAR-mediated GTP
S binding is not additively enhanced
in the presence of PLC
1, (ii) PLC
1 increases the rate of GTP
S
binding mediated by the receptor, and (iii) PLC
1-mediated GTP
S
binding to TGII is slow as compared with that mediated by the
1BAR.
1 results in elevation of
the
1BAR-mediated Ca2+ release from the
intracellular Ca2+ stores is consistent with findings that
PLC
1 is the effector in TGII-mediated signaling pathway (2-5, 40).
Furthermore, overexpression of wtTG and C-STG substantially
enhances the
1BAR-mediated Ca2+ release, and
the TGII mutants m3TG and
1TG greatly reduce the level of the
1BAR-evoked Ca2+ release with or without
overexpression of PLC
1. Interestingly, the increase in
[Ca2+]i was somewhat small when
PLC
1 was coexpressed with wtTG or C-STG (Fig.
5C). Although the reason for the limited Ca2+
release is probably due the limited number of cognate receptors, other
mechanisms may be involved. Thus, the PLC
1 activity is positively
and negatively regulated by TGII depending on the Ca2+
level, expression level of TGII, and binding of guanine nucleotides (3,
11, 12). PLC
1 can also be inhibited by its metabolite IP3 (13-15). Since coexpression of TGII with PLC
1
increases basal IP3 formation (5), all of these factors
would reduce the interaction capability of PLC
1 with TGII. There
were no significant differences in peak
[Ca2+]i between wtTG- and
C-STG-expressing cells, indicating that at the initiation
of coupling of these three molecules, the increased Ca2+
level in cell does not affect the GTP binding by TGII. These results
also support the findings that Ca2+-unbound PLC
1 is GEF
for TGII (Figs. 1C and 2B). Our results also
indicate that PLC
1 is catalytically activated by GTP·TGII, since
the level of the endogenous PLC
1 is sufficient to increase [Ca2+]i maximally when wtTG as
well as C-STG was highly expressed (Fig.
5C).
1 and cGMP phosphodiesterase as well as RGS
proteins terminate GTPase function to prevent further activation of the
effector themselves (19-26). In contrast, PLC
1 facilitates the
signaling involving the
1BAR and TGII through its
GEF/GHIF activity for TGII when the cognate receptors are present. This novel role of PLC
1 is probably necessary to promote the production of second messengers and to overcome the nature of its regulation by
multiple factors. The coupling efficacy of the
1BAR with
TGII in respect to the activation of the cognate PLC is poor as
compared with that with G
q (7, 8, 12). In addition, two
mechanisms for the regulation of PLC
1 activity have been studied
intensely: (i) a supporting role of other Ca2+-mobilizing
receptor systems and (ii) an effector role in the receptor·TGII
coupling system. Our results clearly support the latter mechanism;
PLC
1 is activated at the basal Ca2+ level in an intact
cell by the
1BAR through TGII. Operation of these two
mechanisms probably depends on whether the cells express the cognate
receptors and TGII. It has been reported that PLC
1 is activated by
the capacitative Ca2+ entry, following bradykinin
stimulation in rat pheochromocytoma (PC-12) cell, which does not
express TGII (17). In these regards, further study is required to
establish physiological relevance.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Elliott Ross for kindly
providing us with virus for Gq expression in Sf9
cells and valuable suggestions for the experiments. We also thank Dr.
Jian-Fang Feng for purification of recombinant PLC
1.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant RO1-GM45985 from the National Institutes of Health.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.
§ Permanent address: Dept. of Biochemistry, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
To whom correspondence should be addressed: Dept. of Molecular
Cardiology (NB50), Lerner Research Inst., Cleveland Clinic Foundation,
9500 Euclid Ave., Cleveland, OH 44195. Tel.: 444-216-8860; Fax:
444-216-9263; E-mail: imm@ccf.org.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008252200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PLC, phospholipase
C;
G protein, guanine nucleotide-binding protein;
GAP, GTPase-activating protein;
GEF, guanine nucleotide exchanging factor;
RGS, regulator of G protein signaling;
GHIF, GTP hydrolysis-inhibiting
factor (the terminology was used as to counterpart of GEF);
GTPS, guanosine 5-O-(3-thiotriphosphate);
IP3, inositol 1,4,5-triphosphate;
PIP2, phosphatidylinositol
4,5-bisphosphate;
SM, sucrose monolaurate;
PAGE, polyacrylamide gel
electrophoresis;
TGase, transglutaminase;
TGII, tissue
transglutaminase;
AR, adrenoreceptor;
AppNHp, adenyl-5'-yl
imidodiphosphate;
wtTG, wild-type tissue transglutaminase;
C-STG, tissue transglutaminase mutant (Cys277
Ser);
DMEM, Dulbecco's modified Eagle's medium;
KR, Krebs
Ringer.
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