From the Department of Pharmacology, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27599-7365 and the
Department of Pharmacology, Emory University School of
Medicine, Atlanta, Georgia 30322-3090
Received for publication, August 23, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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RGS proteins (regulators of G protein signaling)
attenuate heterotrimeric G protein signaling by functioning as both
GTPase-activating proteins (GAPs) and inhibitors of G protein/effector
interaction. RGS2 has been shown to regulate
G A variety of hormone and neurotransmitter receptors transduce
signals through heterotrimeric G proteins. In their inactive state, G
proteins exist as heterotrimers consisting of A recently identified family of proteins termed RGS
(regulators of G protein signaling)
proteins interact directly with G Materials--
Hexahistidine-tagged human RGS2 was purified
after expression in Escherichia coli as described previously
(19). PS and 1,2-dioleoyl-sn-glycerol were obtained from
Avanti Polar Lipids (Alabaster, AL). 2MeSATP and isoproterenol were
purchased from RBI (Natick, MA). GTP, GTP [3H]Inositol Labeling of Turkey Erythrocytes and
Membrane Preparation--
Turkey erythrocytes were collected and
washed as described previously (12). Washed erythrocytes were
radiolabeled overnight in inositol-free DMEM supplemented with
myo-[3H]inositol (500 µCi/ml packed cells)
at 37 °C in a 95% O2, 5% CO2 atmosphere
with rapid stirring. Erythrocytes were lysed hypotonically in 40 ml of
ice-cold lysis buffer (5 mM
NaH2PO4, 5 mM MgCl2, 1 mM EGTA, pH 7.4), and membranes were isolated by
centrifugation at 13,500 × g for 10 min. The membranes
were washed with lysis buffer and 10 mM Hepes, pH 7.0, and
then resuspended (2 ml/ml packed erythrocytes) in 10 mM
Hepes, pH 7.0, for use in the PLC assay.
Assay of Phospholipase C- In Vitro Kinase Reactions--
For experiments with the mixture
of calcium and phospholipid-dependent PKC isozymes or with
the individual PKC isoforms ( [32P]Pi Labeling and Isolation of RGS2
from Mammalian Cells--
COS7 cells were transiently transfected
using FuGENE transfection reagent and His10-RGS2 plasmid
DNA (4 µg/100-mm plate), essentially as directed by the manufacturer.
Forty-eight h post-transfection, phosphate-free DMEM (4 ml/100-mm
plate) was applied for 1 h at 37 °C, and the medium was
supplemented with 500 µCi of [32P]Pi for an
additional 3 h. Drugs were added directly to the medium as
indicated, and incubation at 37 °C continued for 20 min. Following drug treatment, the medium was aspirated, and the cells were lysed isotonically in 1 ml of lysis buffer (20 mM Tris, pH 7.5, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 5 mM GTPase Assays--
Purified recombinant human P2Y1
receptor2 was reconstituted
with G Others reported previously that recombinant RGS2 blocks
GTPq-mediated inositol lipid signaling. Although
purified RGS2 blocks PLC-
activation by the nonhydrolyzable GTP
analog guanosine 5'-O-thiophosphate (GTP
S), its capacity to regulate
inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we
investigated regulation by RGS2 of both GTP and GTP
S-stimulated G
11 signaling. Different inhibitory potencies of RGS2
were observed under conditions assessing its activity as a GAP
versus as an effector antagonist; i.e. RGS2 was
a 10-20-fold more potent inhibitor of aluminum fluoride and
GTP-stimulated PLC-
t activity than of GTP
S-promoted PLC-
t
activity. We also examined whether RGS2 was regulated by downstream
components of the inositol lipid signaling pathway. RGS2 was
phosphorylated by PKC in vitro to a stoichiometry of
approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover,
RGS2 was phosphorylated in intact COS7 cells in response to PKC
activation by 4
-phorbol 12
-myristate 13
-acetate and, to a
lesser extent, by the P2Y2 receptor agonist UTP. In
vitro phosphorylation of RGS2 by PKC decreased its capacity to
attenuate both GTP and GTP
S-stimulated PLC-
t activation, with the
extent of attenuation correlating with the level of RGS2
phosphorylation. A phosphorylation-dependent inhibition of
RGS2 GAP activity was also observed in proteoliposomes reconstituted
with purified P2Y1 receptor and G
q
.
These results identify for the first time a phosphorylation-induced
change in the activity of an RGS protein and suggest a mechanism for
potentiation of inositol lipid signaling by PKC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits with GDP bound to G
. Upon agonist occupation, the receptor
promotes GDP/GTP exchange, and the active GTP-bound G
subunit and
G
dissociate to interact with target effector proteins. Signaling
is terminated by the hydrolysis of GTP to GDP and the subsequent
formation of the heterotrimer. Therefore, the magnitude and duration of
signaling is determined by the length of time G
remains in the
active GTP-bound conformation.
subunits to decrease the lifetime
of the active GTP-bound complex (1-4). RGS proteins attenuate
heterotrimeric G protein1
signaling by functioning as both GTPase-activating proteins (GAPs) (5,
6) and inhibitors of G protein/effector interaction (6, 7). In
vitro studies illustrate that RGS2 interacts with and functions as
a GAP for G
q (8), and in vivo studies demonstrate that RGS2 is a more potent inhibitor of G
q
signaling than is RGS4 in transfected cells (9). Members of the
Gq family of G proteins transmit signals from numerous cell
surface receptors, leading to activation of PLC-
isozymes and
subsequent cleavage of membrane phosphatidylinositol
4,5-bisphosphate to the second messengers inositol
1,4,5-trisphosphate and diacylglycerol (10). Inositol
1,4,5-trisphosphate initiates release of calcium from endoplasmic
reticulum stores, and diacylglycerol, in conjunction with calcium and
phospholipids, activates PKC (10). Purified RGS2 has been shown to
attenuate GTP
S-stimulated inositol lipid signaling in reconstitution
studies with both purified G
q and NG-108 cell membranes
(7). However, the capacity of RGS2 to modify inositol lipid signaling
under conditions where GTPase-promoted hydrolysis was operative was not
established. Utilizing the well characterized turkey erythrocyte model
of inositol lipid signaling (11-18), we have determined the effects of
RGS2 on both GTP and GTP
S-stimulated G
11 activation
of PLC-
t. Moreover, our results indicate that PKC promotes
phosphorylation of RGS2, both in intact mammalian cells in response to
PMA and in vitro with purified kinase. This modification
in vitro inhibits the capacity of RGS2 to attenuate PLC-
t
activation and significantly reduces RGS2-promoted GAP activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, FuGENE transfection
reagent, and PKC (calcium- and phospholipid-dependent enzyme) purified from bovine brain were purchased from Roche Molecular Biochemicals. Calyculin A, PKC (catalytic subunit) purified from rat
brain, and PKC isoforms (
,
1,
2, and
) were obtained from Calbiochem. myo-[3H]inositol was
purchased from American Radiolabeled Chemicals (St. Louis, MO).
Histidine-tagged (His10) RGS2 plasmid DNA
(pcDNA3.1(
); Invitrogen) was constructed from a bacterial RGS2
expression construct (provided by Dr. Scott Heximer) via standard
cloning techniques. Ni2+-NTA resin and the penta-His
monoclonal antibody were purchased from Qiagen (Valencia, CA).
Activity in Turkey Erythrocyte
Membranes--
Turkey erythrocyte membranes (12.5 µl/assay) were
mixed with an equal volume of RGS2 diluted to the indicated
concentration in 10 mM Hepes, pH 7.0. Membrane/RGS samples
were incubated at 4 °C for 30 min and then added to 2× assay buffer
(10 mM Hepes, pH 7.0, 424 µM
CaCl2, 910 µM MgSO4, 2 mM EGTA, 115 mM KCl, 5 mM KH2PO4). The assay was initiated by the
addition of 25 µl of buffer, GTP
S, GTP
S with agonist, or GTP
with agonist and proceeded at 30 °C for 10 min. The reaction was
stopped by the addition of 500 µl of ice-cold CHCl3/MeOH
(1:2). 175 µl each of CHCl3 and H2O were
added, and the samples were centrifuged at 1800 × g
for 5 min. Inositol phosphates were isolated by anion exchange
chromatography by transferring 400 µl of the aqueous upper phase to
Bio-Rad AG1-X8 (200-400 mesh) columns containing 10 ml of
H2O. 10 ml of 200 mM ammonium formate, 100 mM formic acid were added, and the eluate was discarded.
Inositol phosphates were eluted with 5 ml of 1.2 M ammonium
formate, 100 mM formic acid, and [3H]inositol
phosphates were quantitated by liquid scintillation spectrometry.
,
1,
2, and
),
histidine-tagged RGS2 (4-18 pmol) was incubated with PKC at 30 °C
in a reaction containing 20 mM Tris, pH 7.5, 10 mM MgCl2, 500 µM
CaCl2, 100 µg/ml PS, 20 µg/ml
1,2-dioleoyl-sn-glycerol, 200 nM calyculin A,
and 200 µM [
-32P]ATP (~1500 cpm/pmol)
in a final volume of 20 µl. Concentrations of PKC and incubation
times are as listed in the figure legends. One unit of PKC activity is
defined as the amount of enzyme required to transfer 1 µmol of
phosphate from ATP to histone H1 per min at 30 °C. Reactions were
terminated by the addition of 20 µl of 2× Laemmli sample buffer.
Samples were separated by SDS-PAGE through 12.5% acrylamide according
to the method of Laemmli (20), and the protein bands were visualized by
silver or Coomassie stain. The gel was dried and exposed to
autoradiography film to detect radioactive bands. For experiments to
test the capacity of phosphorylated RGS2 to inhibit G
11
signaling, RGS2 was phosphorylated by the PKC catalytic subunit, which
does not require calcium and phospholipids for activation. Purified
RGS2 (~40 pmol) was incubated with the PKC catalytic subunit at
30 °C in a reaction containing 50 mM MES, pH 6.0, 12.5 mM MgCl2, 1.25 mM EGTA, 200 nM calyculin A, and 125 µM ATP in a final
volume of 16 µl. Concentrations of PKC and incubation times are as
indicated in the figure legends. Reaction mixtures were diluted in 10 mM Hepes, pH 7.0, 10 mM
-glycerophosphate, 200 nM calyculin A, and bovine serum albumin (2 mg/ml) and
mixed with turkey erythrocyte membranes at 4 °C for 30 min to obtain the indicated concentrations of RGS2. Membrane samples were assayed for
inositol phosphate production as described above.
-Glycerophosphate and calyculin A were included in PLC assays with phosphorylated RGS2 to
inhibit phosphatase activity.
-mercaptoethanol, 5 mM NaF, 10 mM
-glycerophosphate, 10 nM microcystin, 200 µM phenylmethylsulfonyl fluoride, 10 µM
tosylphenyl chloromethyl ketone, 1 µM pepstatin A, 2 µM leupeptin), and the lysates were centrifuged at
35,000 × g for 30 min. The resulting supernatant (1 ml) was incubated with 25-50 µl of Ni2+-NTA resin with
mixing for 1 h at 4 °C to isolate His10-tagged RGS2. The Ni2+-NTA resin was pelleted by centrifugation at
13,000 × g for 15 s, and the supernatant was
aspirated. The pellet was washed three times with 25 mM
imidazole, three times with 50 mM imidazole, and two times
with 75 mM imidazole. RGS2 was eluted from the resin twice
with 100 µl of 250 mM imidazole. Isolated proteins were resolved by SDS-PAGE (12.5% (w/v) gel) and subjected to protein staining or transferred electrophoretically to nitrocellulose. RGS2 was
detected by Coomassie staining or by Western blot with an
anti-penta-His monoclonal antibody. 32P incorporation into
RGS2 was assessed by autoradiography and PhosphorImager analysis
(Molecular Dynamics, Inc., Sunnyvale, CA).
q and G
1
2 into
proteoliposomes by a modification of the method described by Brandt
et al. (21). Briefly, 15 pmol of P2Y1 receptor, 40 pmol of
G
q, and 150 pmol of G
1
2
were combined with a mixture of phosphatidylethanolamine,
phosphatidylserine, and cholesterol hemisuccinate in detergent
solution. Proteoliposomes were formed by Sephadex G-50 gel filtration.
RGS2 was phosphorylated by PKC essentially as described above and
diluted ~20-fold to the indicated final concentrations in the assay.
GTPase activity of the proteoliposomes was determined in the presence
of either phosphorylated or mock-phosphorylated RGS2, with or without
100 µM 2MeSADP. Assays were incubated for 30 min at
30 °C and contained 20 mM Hepes, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10 mM
-glycerophosphate, 10 nM microcystin, and 2 µM [
-32P]GTP (~4500 cpm/pmol). The
assays were terminated by the addition of 950 µl of a 4 °C
solution of 5% activated charcoal in 20 mM H3PO4. Following centrifugation, liberated
[32P]Pi in the supernatant was quantified in
a liquid scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S-stimulated G
q activation of PLC-
in NG-108
cell membranes and in reconstitution assays with purified
G
q and PLC-
1 (7). However, the effects of RGS2 on
G
q signaling activity in the presence of GTP
(i.e. under conditions where G
q GTPase
activity is operative) have not been fully characterized. We have
modified an established turkey erythrocyte membrane assay to
investigate and compare the capacities of
RGS23 to inhibit GTP and
GTP
S-stimulated PLC-
t activation. RGS2 was mixed with erythrocyte
membranes at various concentrations, and incubations were carried out
in the presence of aluminum fluoride, GTP plus 2MeSATP, GTP
S plus
2MeSATP, or GTP
S alone. RGS2 inhibited aluminum fluoride
(IC50 = 10 nM), GTP (IC50 = 14 nM, with 2MeSATP) and GTP
S-stimulated (IC50 = 192 and 223 nM, in the presence and absence of 2MeSATP,
respectively) PLC-
t activity in a concentration-dependent manner. However, the aluminum fluoride- and 2MeSATP plus GTP-promoted responses were inhibited by 10-20-fold lower concentrations of RGS2
than those necessary to inhibit GTP
S-stimulated PLC activity, both
in the presence and absence of the P2Y1 receptor agonist 2MeSATP (Fig. 1). Inositol lipid
signaling in the turkey erythrocyte is also stimulated by isoproterenol
through G
11 coupling to a
-adrenergic receptor
(22-24). RGS2 attenuated isoproterenol plus GTP
S-stimulated
inositol phosphate production half-maximally at an RGS concentration
~20-fold lower than that observed with 2MeSATP plus GTP
S
stimulation (Fig. 2). Although the
mechanism underlying this difference in potency has not been fully
investigated, this result suggests a receptor-selective component to
inhibition by RGS2.
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Fig. 1.
Inhibition of PLC-
activation in turkey erythrocyte membranes by RGS2. Turkey
erythrocyte membranes were radiolabeled overnight in inositol-free DMEM
with 0.5 mCi [3H]inositol, and membranes were prepared by
hypotonic lysis as described under "Experimental Procedures."
Membranes were incubated with the indicated concentrations of RGS2 for
30 min at 4 °C, and PLC activity was assayed as described. Reactions
were initiated by the addition of aluminum fluoride (A), 10 µM 2MeSATP plus 1 mM GTP (B), 10 µM 2MeSATP plus 10 µM GTP
S
(C), or 100 µM GTP
S (D). The
IC50 values for RGS2 obtained in A-D are
summarized in E. Basal levels of inositol phosphate
production with 10 mM Hepes, pH 7.0, were subtracted from
the values presented. Data are mean ± S.D. of triplicate
determinations and are representative of three experiments.
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Fig. 2.
Inhibition of
-adrenergic receptor-mediated
PLC-
activation in turkey erythrocyte
membranes by RGS2. A, turkey erythrocyte membranes were
radiolabeled overnight in inositol-free DMEM with 0.5 mCi of
[3H]inositol, and membranes were prepared by hypotonic
lysis as described under "Experimental Procedures." Membranes were
incubated with the indicated concentrations of RGS2 for 30 min at
4 °C, and PLC activity was assayed as described. Reactions were
initiated by the addition of 100 µM isoproterenol
(ISO) plus 10 µM GTP
S (
) or 10 µM 2MeSATP plus 10 µM GTP
S (
).
B, comparison of the IC50 values of RGS2 for
inhibition of purinergic and
-adrenergic receptor-mediated
activation of PLC-
t. Basal levels of inositol phosphate production
with 10 mM Hepes, pH 7.0, were subtracted from the values
presented. Data are mean ± S.D. of triplicate determinations and
are representative of three experiments.
Activation of PKC either indirectly through Ca2+-mobilizing
receptors or directly by phorbol esters results in desensitization of
receptor and G protein-promoted phosphoinositide hydrolysis (25).
Utilizing a turkey erythrocyte membrane reconstitution assay, we
previously reported that activation of PKC in intact cells inhibits the
capacity of G11 to activate purified PLC-
1. While the
effects of PKC were localized to the membrane, we were unable to
identify the membrane target for PKC (18). Therefore, since the
erythrocyte membrane assay provides a reliable assay of
G
11-interacting RGS proteins, we determined if RGS2 was
a substrate for PKC in vitro and utilized the erythrocyte
assay to identify phosphorylation-dependent changes in
RGS-promoted attenuation of inositol phosphate production. RGS2 was
phosphorylated to a stoichiometry near unity (0.77 ± 0.25 mol
phosphate/mol RGS2) by a bovine brain preparation consisting of a
mixture of PKC isoforms (Fig. 3).
Individual calcium and phospholipid-dependent PKC isoforms (
,
1,
2, and
) were also utilized and phosphorylated RGS2 to approximately the same level (Fig. 3). Minimal phosphorylation was
detected in the absence of PKC, indicating that little or no endogenous
kinase activity was present in the preparation of RGS2. The extent of
phosphorylation of RGS2 was dependent on PKC concentration (Fig.
4A) and the time of
incubation (Fig. 4B).
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Due to lack of an antibody for immunoprecipitation of RGS2 from turkey
erythrocytes, in vivo phosphorylation could not be assessed
in these cells. However, a histidine-tagged RGS2 construct for
expression in mammalian cells was engineered (see "Experimental Procedures") that permitted isolation of RGS2 from cell lysates with
Ni2+-NTA resin. COS7 cells were transiently transfected
with His10-RGS2 and radiolabeled with
[32P]Pi. Treatment of cells with PMA resulted
in a concentration-dependent increase in phosphorylation of
RGS2 (Fig. 5A), indicating
PKC-dependent phosphorylation of RGS2 in intact cells.
Pharmacological analysis indicates that COS7 cells possess an
endogenous P2Y2 receptor that couples to Gq to initiate
phosphoinositide hydrolysis and subsequently activate PKC. Incubation
of 32P-labeled COS7 cells expressing RGS2 with the P2Y2
receptor agonist UTP also resulted in an ~80% increase in RGS2
phosphorylation (Fig. 5B), indicating that physiological
activation of PKC through a G protein-coupled receptor promotes
in vivo phosphorylation of RGS2.
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To determine the effect of phosphorylation by PKC on the capacity of
RGS2 to attenuate activation of PLC-t, RGS2 was phosphorylated to a
stoichiometry near unity by PKC in vitro and reconstituted at the indicated concentrations with erythrocyte membranes as described
above. Nonphosphorylated RGS2, which was incubated in a standard kinase
reaction lacking PKC, inhibited GTP plus 2MeSATP-stimulated inositol
phosphate production in a concentration dependent manner (Fig.
6A), consistent with that
observed with untreated RGS2 (Fig. 1B). Phosphorylation by
PKC decreased the capacity of RGS2 to inhibit GTP-stimulated
phosphoinositide hydrolysis (Fig. 6A). Concentration of
PKC-dependent increases in phosphorylation of RGS2 (Fig.
4A) resulted in proportional decreases in RGS2-promoted attenuation of 2MeSATP plus GTP-stimulated inositol lipid signaling (Fig. 6B). Time-dependent increases in RGS2
phosphorylation (Fig. 4B) also produced corresponding
decreases in RGS2-mediated inhibition of GTP-promoted inositol
phosphate production (Fig. 6C).
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The effect of phosphorylation by PKC on the activity of RGS2 was also
determined under conditions where the RGS protein functions as an
inhibitor of G protein/effector interaction but not as a GAP
(i.e. in the presence of GTPS). The significantly higher concentrations of RGS2 required to inhibit GTP
S alone and GTP
S plus 2MeSATP-stimulated PLC-
t activation (Fig. 1, C-E)
limited our capacity to test the effect of phosphorylation on RGS2
activity with the nonhydrolyzable GTP analog under these conditions.
However, the observation that RGS2 is a significantly more potent
inhibitor of GTP
S-stimulated inositol phosphate production in the
presence of isoproterenol than with 2MeSATP (Fig. 2) provided
conditions for studying the capacity of RGS2 to inhibit PLC-
t
activation promoted by a nonhydrolyzable GTP analog. Nonphosphorylated
RGS2 attenuated isoproterenol plus GTP
S-stimulated inositol
phosphate production in a concentration-dependent manner,
and phosphorylation by PKC increased the concentration of RGS2 required
for half-maximal inhibition of PLC-
t activation (Fig.
7A). The extent of the
reversal of RGS2-mediated attenuation of inositol phosphate production correlated with the concentration of PKC (Fig. 7B) and time
of incubation with PKC (Fig. 7C), consistent with a
phosphorylation-dependent inhibition of RGS2 activity.
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The mechanism of phosphorylation-dependent inhibition of
RGS2 activity was further investigated via steady-state GTPase assays performed in proteoliposomes. Phosphorylated or mock-phosphorylated RGS2 was incubated with proteoliposomes containing purified
P2Y1 receptor and
Gq
1
2. The addition of
unphosphorylated RGS2 and the P2Y1 receptor agonist
2MeSADP stimulated GTP hydrolysis ~4-fold above the level
observed with RGS2 alone (Fig. 8).
Phosphorylation significantly reduced RGS2-promoted GTPase activity
both in the presence and absence of agonist, demonstrating a
phosphorylation-dependent inhibition of the GAP activity of
RGS2.
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DISCUSSION |
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RGS proteins inhibit heterotrimeric G protein signaling by
functioning as both GAPs and effector antagonists (1-3). Previous studies have demonstrated that RGS2 acts as a GAP for Gq
(8) and attenuates GTP
S-stimulated PLC-
activity (7). In the present study, we have examined the capacity of RGS2 to regulate inositol lipid signaling under conditions where GTPase
promoted hydrolysis was operative and have established that RGS2 is a
much more potent inhibitor of P2Y receptor-stimulated PLC-
activity in the presence of GTP than in the presence of the hydrolysis-resistant GTP analog GTP
S. Similar concentrations of RGS2 were required for
half-maximal inhibition of GTP-stimulated inositol lipid signaling and
that occurring in the presence of aluminum fluoride, which mimics the
structure of the
subunit at the transition state of the GTPase
reaction (26). Thus, RGS2 is a more potent inhibitor of inositol lipid
signaling under conditions in which the G protein
subunit exists in
the GTPase transition state, either transiently (GTP-promoted
signaling) or stably (aluminum fluoride-stimulated signaling). Our
results are consistent with the observation that RGS proteins interact
with higher affinity to the GDP-AlF4
complex than to the GTP
S-bound form of G
(5, 27, 28).
Zeng et al. (29) and Xu et al. (30) recently
reported receptor-selective inhibition by RGS4 of calcium release and
PLC activity in pancreatic acinar cells dialyzed with RGS4.
G11-mediated inositol lipid signaling in the turkey
erythrocyte membrane model is stimulated by both P2Y1 and
-adrenergic receptors (22-24), and thereby provides conditions for
more directly investigating potential receptor-selective activity of
RGS2. RGS2 inhibited isoproterenol plus GTP
S-stimulated inositol
phosphate production half-maximally at RGS concentrations 20-fold lower
than those observed with 2MeSATP and GTP
S. While our results do not
reveal the mechanism underlying these differences in RGS2 potency, they suggest the occurrence of receptor-selective activity of RGS2. The
N-terminal domain of RGS4 was proposed to impart high affinity and
receptor-selective inhibition of Gq signaling (29). Little similarity exists between the N-terminal domain of RGS4 and RGS2, and
it will be important to establish the basis of the apparent receptor
selectivity found in our study with RGS2.
PKC is activated as a downstream consequence of PLC activation and has
been implicated in regulation of inositol lipid signaling (25). We
previously demonstrated that membranes isolated from turkey
erythrocytes pretreated with PMA exhibit a decreased capacity for
G11-mediated activation of purified, reconstituted
PLC-
1 (18). Additionally, we illustrated that PLC-
t is
phosphorylated in intact erythrocytes in response to PMA treatment, and
in vitro phosphorylation of PLC-
t by PKC reduces its
basal catalytic activity (17). We demonstrate here that RGS2 is
phosphorylated stoichiometrically by PKC in vitro and in
intact mammalian cells stimulated with PMA or the P2Y2
receptor agonist UTP. Phosphorylation decreases the capacity of
reconstituted RGS2 to attenuate PLC-
t activity in turkey erythrocyte
membranes and significantly reduces RGS2 GAP activity in
P2Y1 receptor/G
q
vesicles, supporting
a role for phosphorylation in regulation of RGS protein activity. This result may be surprising in light of the general conception that RGS
proteins have a "desensitizing" activity on G protein-mediated signaling and the observations in our laboratory and numerous others
illustrating that PKC activation generally promotes desensitization of
inositol lipid signaling (31-34). Phosphorylation of RGS2 by PKC would
potentiate receptor-stimulated inositol lipid hydrolysis, and
therefore, our results suggest that inositol lipid signaling in
vivo is regulated temporally by a balance of PKC-promoted
inhibitory and stimulatory signals. Such a view is not inconsistent
with reports suggesting that receptor-promoted phospholipid signaling is not simply a result of straightforward negative feedback regulation by PKC. Rapid desensitization of phosphoinositide hydrolysis is often
only partial and accumulation of inositol 1,4,5-trisphosphate is
biphasic (35-37). Moreover, levels of intracellular calcium have been
shown to oscillate in the presence of hormone (38-41), and PKC has
been strongly implicated in these calcium oscillations (42-45). Thus,
studies of both receptor-promoted inositol 1,4,5-trisphosphate production and intracellular calcium levels are consistent with the
concept that the role of PKC in regulating phosphoinositide hydrolysis
may be more complex than mere signal inactivation.
PKC is activated as a consequence of signaling events other than Gq-promoted phosphoinositide hydrolysis, and phospholipid signaling does not occur in isolation in the intact cell. Thus, in vivo phosphorylation of RGS2 may be involved in cross-talk regulation between signaling pathways. More detailed studies will be needed to determine whether involvement of different PKC isoforms with different temporal patterns of activation and substrate selectivities also underlies our observations with PKC-promoted phosphorylation of RGS2.
Precedent exists for sensitivity of RGS protein-G protein interactions
to protein phosphorylation state. Gz is phosphorylated by PKC in vitro, thereby reducing the capacity of RGSZ1 to
accelerate G
z GTPase activity (46, 47). The effect of
G
z phosphorylation on its interaction with RGSZ1 also
suggests that stimulation of PKC potentiates G
z
signaling by lengthening the time that the G protein remains in the
active GTP-bound conformation. Therefore, while the target of
PKC-promoted phosphorylation in our study is the RGS protein rather
than the G protein, the observation that PKC-mediated phosphorylation
of RGS2 decreases its capacity to attenuate G
11
signaling is consistent with the sensitivity of RGS-G protein
interactions to phosphorylation demonstrated with G
z and
RGSZ1. Phosphorylation of RGS2 decreased its ability to inhibit
GTP-promoted PLC-
t activation and its capacity to promote GTP
hydrolysis in vesicles containing the P2Y1 receptor and
G
q
. In addition to inhibition of RGS2 activities
under conditions where GTPase activity was operative, phosphorylation also attenuated RGS2 inhibition of GTP
S-stimulated signaling. While
the details of the association are still unclear, these observations
suggest that PKC-promoted phosphorylation of RGS2 affects interactions
with G
q/11 in such a way that RGS2 cannot promote GTPase
activity or block G
q/11 interactions with its effector
PLC-
as effectively as in the nonphosphorylated state.
Phosphorylation may provide a common method of regulation of RGS
proteins, impacting on both their subcellular localization and their
GAP activity. Pedram et al. (48) observed phosphorylation of
RGS3 and RGS4 by cGMP-dependent protein kinase and
implicated phosphorylation in translocation of these RGS proteins from
a cytosolic to a membrane localization. Farquhar and colleagues reported that membrane-associated GAIP, but not soluble GAIP, exists as
a phosphoprotein and that GAIP can be phosphorylated in
vitro by purified casein kinase 2 and by isolated clathrin-coated vesicles (49). Benzing et al. (50) recently reported
PKC-promoted phosphorylation of RGS7 and
phosphorylation-dependent association of RGS7 and 14-3-3 proteins. Moreover, binding of 14-3-3 to phosphorylated RGS7 inhibited
the capacity of RGS7 to promote GTP hydrolysis by Gi.
Interestingly, several of the residues in the putative 14-3-3 binding
motif of RGS7, including the serine residue phosphorylated by PKC, are
conserved in RGS2 and other RGS proteins, suggesting another possible
level of RGS2 regulation by PKC in the intact cell. Although the
precise mechanisms have not been fully elucidated, phosphorylation by
PKC probably plays an important role in regulating cellular RGS2 activity.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Josè Boyer and Dr. Scott Heximer for helpful discussions and advice.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grants GM29536 and GM38213 and a Howard Hughes Medical Institute Predoctoral Fellowship (to M. L. C.).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: CB# 7365, Mary Ellen Jones Bldg., University of North Carolina School of Medicine, Chapel Hill, NC 27599. Tel.: 919-966-5356; Fax: 919-966-5640; E-mail: tkh@med.unc.edu.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M007699200
2 G. L. Waldo and T. K. Harden, manuscript in preparation.
3 Human RGS2 was used in this study; however, we have cloned turkey RGS2, which is ~69% identical and 80% homologous to the human homologue (G. L. Waldo and T. K. Harden, unpublished observations).
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ABBREVIATIONS |
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The abbreviations used are:
G protein, guanine nucleotide-binding protein;
PKC, protein kinase C;
PLC, phospholipase C;
GTPS, guanosine 5'-O-thiophosphate;
DMEM, Dulbecco's modified Eagle's medium;
PAGE, polyacrylamide gel
electrophoresis;
2MeSADP, 2-methylthioadenosine diphosphate;
2MeSATP, 2-methylthioadenosine triphosphate;
PMA, 4
-phorbol 12
-myristate
13
-acetate;
GAP, GTPase-activating protein;
NTA, nitrilotriacetic
acid;
MES, 4-morpholineethanesulfonic acid.
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