From the Department of Pharmacology, University of
North Carolina, Chapel Hill, North Carolina 27599 and the
Department of Pharmacology, University of Texas Southwestern
Medical School, Dallas, Texas 75390
Received for publication, November 7, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Regulator of G-protein signaling (RGS) proteins
are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that
alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein Heterotrimeric guanine nucleotide-binding proteins (G-proteins)
act as molecular switches in multiple
GPCR1 signaling pathways via
regulation of specific effector molecules such as phospholipase C and
adenylyl cyclase. The biological activity of G-protein Although some effector proteins exhibit GAP activity (1-3), the
primary regulators of GTPase activity of G The R7 RGS family is a unique multidomain family, which consists of RGS
proteins containing a novel G- RGS proteins may modify GPCR signaling through selective increases in
GTP hydrolysis by a subset of G-proteins. For example, RZ family
members specifically accelerate GTPase activity of G In face of the uncertainty of the actions of R7 RGS proteins, we
purified the four members of the mammalian R7 family. The specificities
of action of G Protein Purification--
The generation of baculoviruses for
G Vesicle Reconstitution and
Characterization--
Detergent/phospholipid mixed micelles were
prepared by drying 110 µg of phosphatidylethanolamine, 70 µg of
phosphatidylserine, and 8 nmol of cholesteryl hemisuccinate under argon
and resuspending in detergent buffer (0.4% deoxycholate, 20 mM Hepes, 1 mM EDTA, 100 mM NaCl).
Fifty µl of this preparation was combined with 15 pmol of muscarinic
receptor, 50 pmol of G Steady State GTPase Assays--
One microliter (for
G The specificity of RGS proteins for G-protein substrates
determines in part their physiological effects on signaling. Previous in vitro studies with G G Vesicle Reconstitution--
G-protein Steady State GTPase Assays--
The GAP activity of
G
In the presence of a maximally effective concentration of RGS protein,
guanine nucleotide exchange is rate-limiting, and therefore stimulation
of GTPase activity by carbachol was observed with a concentration
dependence of agonist that approximated its occupancy curve for binding
to the M2 muscarinic receptor (data not shown). Similarly, in the
presence of a maximally effective concentration of carbachol, guanine
nucleotide exchange was no longer rate-limiting, and marked
concentration-dependent stimulation of GTPase was observed with
RGS4 (data not shown) and G G G G G The results of this study demonstrate G The drug selectivity of G-protein-coupled receptors has
been widely exploited therapeutically to manipulate specific cellular processes (30-34). Similarly, selectivities of G-proteins for effector activation and potential selectivities of RGS proteins for deactivation of G-proteins may provide equally rich targets for pharmacological modulation of G-protein-regulated signaling (35, 36). Whereas the role
of receptor activity and selectivity in regulating various classes of
G-proteins has been studied extensively in the past decades, the roles
of proteins exhibiting G Members of the G In this study, we examined the GAP activity of G Our results also differ from published reports that indirectly suggest
that G G G The differences observed in the maximal GAP activity of R7 family RGS
proteins may reflect differences in their interactions with G In summary, we have demonstrated that R7 family RGS proteins
selectively stimulate GTPase activity of Gi family
G-subunit selectivity of
purified Sf9 cell-derived R7 proteins, a subfamily of RGS
proteins (RGS6, -7, -9, and -11) containing a G
-like (GGL) domain
that mediates dimeric interaction with G
5.
G
5/R7 dimers stimulated steady state GTPase activity of
G
-subunits of the Gi family, but not of
G
q or G
11, when added to proteoliposomes
containing M2 or M1 muscarinic receptor-coupled G-protein
heterotrimers. Concentration effect curves of the G
5/R7
proteins revealed differences in potencies and efficacies toward
G
-subunits of the Gi family. Although all four
G
5/R7 proteins exhibited similar potencies toward
G
o, G
5/RGS9 and G
5/RGS11
were more potent GAPs of G
i1, G
i2, and
G
i3 than were G
5/RGS6 and
G
5/RGS7. The maximal GAP activity exhibited by
G
5/RGS11 was 2- to 4-fold higher than that of
G
5/RGS7 and G
5/RGS9, with
G
5/RGS6 exhibiting an intermediate maximal GAP activity.
Moreover, the less efficacious G
5/RGS7 and
G
5/RGS9 inhibited G
5/RGS11-stimulated
GTPase activity of G
o. Therefore, R7 family RGS proteins
are Gi family-selective GAPs with potentially important
differences in activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits is
determined by the identity of the bound guanine nucleotide (GTP or
GDP), which in turn is governed by the relative rates of guanine
nucleotide exchange and hydrolysis of GTP by the intrinsic GTPase
activity of G
-subunits. These opposing reactions are stimulated by
agonist-occupied GPCR and GTPase-activating proteins (GAPs).
-subunits are a diverse
family of regulator of G-protein signaling (RGS) proteins that act as
GAPs for heterotrimeric G-protein
-subunits (4-7). This family is
defined by a conserved RGS domain, which markedly increases the rate of
GTP hydrolysis by G
-subunits and terminates effector activation by
both G
- and G
-subunits. More than 30 RGS proteins have been
identified and organized into subfamilies based on sequence similarity
and domain structure. These families vary in size and complexity, from
the R4 family whose structure is largely limited to the RGS domain to
the R12 and RhoGEF families whose members are large multifunctional
proteins containing several domains (for reviews see Refs. 8-10).
-like (GGL) domain homologous to the
G
-subunit of heterotrimeric G-proteins (11). This domain, found in
the mammalian proteins RGS6, -7, -9, and -11 and the
Caenorhabditis elegans proteins EAT16 and EGL10 (7, 12),
confers specific binding to G
5-subunits but not to
G
1-4 (11, 13). Heterodimeric association with
G
5 appears necessary for stability and biological
activity of R7 proteins (14-16). R7 proteins also contain a conserved
N-terminal DEP (dishevelled, EGL10, pleckstrin homology) domain of
unknown function (17).
z (18), and the recently characterized sorting nexin 13 (RGS-PX1) has
been reported to increase the rate of GTP hydrolysis by
G
s but not by G
i (19). The G-protein
selectivity of the R7 family of RGS proteins has not been clearly
defined. In single turnover GTPase assays, G
5/RGS6 and
G
5/RGS7 increased GTPase activity of G
o
(20) and G
5/RGS11 increased GTPase activity of
G
o and, to a much lesser degree, that of
G
i1, G
i2, and G
i3 (11). However, the R7 RGS proteins did not affect the single turnover GTPase
rates of other G
-subunits, including G
q (R183C),
G
s, and G
12. In contrast, when expressed
in cultured cell lines, RGS7 inhibited G
q-promoted
Ca2+ responses downstream of M3 receptors (14) and
5-HT2c receptors (21, 22) and inhibited
G
i-regulated K+ channel activity in a
G
5-dependent manner (23). Therefore, assays
of soluble G
-subunits suggest G
o selectivity, while
intact cell signaling studies implicate R7 proteins in regulation of Gq as well as Gi pathways.
5/R7 heterodimers were determined in
steady state GTPase assays of Gi and Gq family
G
-subunits reconstituted with GPCR in phospholipid vesicles.
G
5/RGS6, -7, -9, and -11 increased the GTPase activity
of G
o, G
i1, G
i2, and
G
i3 but not G
q or G
11.
Notable differences in maximal GAP activities were observed among
R7 family proteins, and the maximal activity of the most efficacious
RGS protein (G
5/RGS11) was inhibited by
G
5/RGS7 and G
5/RGS9.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5, RGS6, RGS7, and RGS11 have been described previously
(11, 20). A baculovirus for RGS9-1 in pFastbac Htb was prepared
similarly. Cultures of Sf9 insect cells (1.4 × 106 cells/ml) were co-infected with virus encoding the
RGS6, RGS7, or RGS9 gene (m.o.i. of 1) and a hexahistidine-tagged
G
5 (short isoform) (m.o.i. of 0.5). Forty-eight hours
postinfection, 4 liters of cells were collected by centrifugation. Due
to lower expression under these conditions, Sf9 cells were
infected with the RGS11:G
5 viruses at a 10:1 ratio
(m.o.i. of 0.5 and 0.05), and cell lysates were obtained 65 h
postinfection. Cells were resuspended and lysed in 600 ml of Buffer A
(20 mM KPO4, pH 8, 150 mM NaCl, 2 mM MgCl2, 5 mM
-mercaptoethanol,
protease inhibitors (500 nM aprotinin, 10 µM
leupeptin, 200 µM phenylmethylsulfonyl fluoride, 1 nM pepstatin, 10 µM
L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone)) with 20 strokes of a Dounce homogenizer at 4 °C. The lysate was cleared by
low speed centrifugation, and the supernatant was centrifuged at
100,000 × g for 30 min. The soluble protein fraction
was loaded on a 3- to 5-ml column of Ni-NTA-agarose resin (Qiagen,
Germany) over 3 h. The column was washed with 15 ml of Buffer B
(20 mM KPO4, pH 8, 400 mM NaCl, 2 mM MgCl2, 5 mM
-mercaptoethanol,
and protease inhibitors) followed by 5 ml of Buffer C (20 mM KPO4, pH 8, 25 mM NaCl, 2 mM MgCl2, 5 mM
-mercaptoethanol,
and protease inhibitors). The G
5/R7 dimers eluted with
50-150 mM imidazole in Buffer C. This eluate was further
purified using 1 ml of HighTrap FPLC columns of either Q-Sepharose
(G
5/RGS6 and G
5/RGS7) or S-Sepharose
(G
5/RGS9 and G
5/RGS11) (Amersham
Biosciences). The Ni-NTA eluate was diluted 5:1 in starting buffer for
ion exchange chromatography (buffer for Q-Sepharose: 50 mM
Tris, pH 8, 2 mM dithiothreitol, protease inhibitors;
buffer for S-Sepharose: 50 mM Hepes, pH 8, 1 mM
EDTA, 2 mM dithiothreitol, 10% glycerol, protease
inhibitors), loaded onto the FPLC column, and eluted in the same buffer
with a 0-400 mM NaCl gradient over 30-column volumes. Each
dimer eluted at ~200 mM NaCl. Fractions were collected and concentrated using a Centricon centrifugal filter device
(Millipore, Bedford, CT). The concentration of purified
G
5/R7 dimers was determined by Coomassie staining
purified product and a standard curve of protein standards resolved by
SDS-PAGE. Yield was ~1 mg of G
5/R7 dimer per 4 liters
except for G
5/RGS11, whose purification yielded ~250
µg per 4 liters. G
- and G
-subunits (24) and muscarinic
receptors (25) were purified after expression from baculoviruses in
Sf9 insect cells as described.
, and 150 pmol of
G
1
2 in Buffer D (20 mM Hepes,
100 mM NaCl, 1 mM EDTA, 2 mM
MgCl2) and immediately loaded onto a G-50-Sepharose column
equilibrated with Buffer D. The eluate was collected in 200-µl
fractions. Fractions were assayed for the presence of muscarinic
receptor by incubation of 5 µl per fraction with 20 nM
[3H]quinuclidinyl benzilate (~200,000 cpm) in a volume
of 100 µl for 90 min at 30 °C and filtration over GF/F filters
(Whatman). Peak fractions also were assayed for G
incorporation by
incubation of 5 µl of the vesicle preparation with 1 µM
35S-labeled GTP
S (500,000 cpm) in the presence or
absence of 0.1% C12E10 detergent (total
volume = 100 µl) at 30 °C for 90 min. Samples labeled in the
absence of C12E10 were filtered over GF/F filters, which collect vesicles but not free protein, to quantitate incorporation of G
-subunits into vesicles, and
C12E10-containing samples were filtered over
nitrocellulose to quantitate total G
.
i/o-containing vesicles) or 5 µl (for
G
q/11-containing vesicles) of the vesicle preparations
was equilibrated on ice in Buffer D in the presence or absence of 100 µM carbachol and various concentrations of RGS protein.
[
-32P]GTP (2 µM; ~400,000 cpm) was
added to each 25 µl of reaction. The reaction was transferred to a
30 °C water bath for 15 min (for G
i/o-containing
vesicles) or 30 min (for G
q/11-containing vesicles) and
quenched on ice with 975 ml of cold 5% activated charcoal in 20 mM NaH2PO4. The charcoal was
pelleted by centrifugation, and a portion of the supernatant was added
to scintillant for 32Pi quantitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits in solution have
illustrated specificity of R7-RGS proteins for G
o,
whereas in vivo observations have suggested broader
activities. To more specifically address the selectivity of individual
G
5/R7 heterodimers for G
-subunits, we purified
G
5/RGS6, G
5/RGS7, G
5/RGS9,
and G
5/RGS11 to near homogeneity and directly measured
their selectivity in steady state GTPase assays with proteoliposomes
reconstituted with M1 or M2 muscarinic receptors and various
heterotrimeric G proteins of the Gq and Gi
families, respectively.
5/R7 Protein
Purification--
Full-length RGS6, -7, -9, and -11 were co-expressed
with hexahistidine-tagged G
5 in Sf9 insect cells
using the baculovirus expression system. Dimers were purified from the
soluble fraction using Ni-NTA-agarose and ion exchange chromatography.
Twenty-five to seventy-five percent of the total cellular
immunoreactive R7 protein was recovered in the soluble fraction,
25-75% of the soluble protein was recovered following Ni-NTA
chromatography, and nearly 100% of the Ni-NTA eluate was recovered
following the final ion exchange purification. The results of a typical
purification (G
5/RGS7) are shown in Fig.
1A. Purified
G
5/RGS6, G
5/RGS9, and
G
5/RGS11 dimers are illustrated in Fig.
1B.
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Fig. 1.
G 5/R7
purification. A, the purification of
G
5/RGS7 is shown on a Coomassie Blue-stained SDS-PAGE.
The soluble fraction of Sf9 cells infected with RGS7 and
hexahistidine-tagged G
5 was loaded onto a Ni-NTA column.
The column was consecutively washed with 400 mM NaCl
(lane 1), 25 mM NaCl (lane 2), 50 mM imidazole (lane 3), 150 mM
imidazole (lane 4), and 300 mM imidazole
(lane 5). The 50 mM imidazole elution was
further purified on a HighTrap Q anionic exchange column and eluted
with a gradient of 0-400 mM NaCl. Lanes 6-13
show fractions eluting at ~175-225 mM NaCl. Fractions
shown in lanes 9-11 were pooled and concentrated.
B, the final purified products of all four
G
5/R7 dimers are shown: G
5/RGS6
(lane 1), G
5/RGS7 (lane 2),
G
5/RGS9 (lane 3), and G
5/RGS11
(lane 4).
-subunits
(G
o, G
i1, G
i2,
G
i3, G
q, G
11) were
reconstituted in phospholipid vesicles with
G
1
2 and either M1 (Gq family
G-proteins) or M2 (Gi family G-proteins) muscarinic receptors under conditions similar to those described by Ross and
coworkers (26, 27). Recovery of G
-subunits and M1 or M2 muscarinic
receptors in the various vesicle preparations was quantitated as
described under "Experimental Procedures." Essentially 100% of
added G
o, G
i1, G
i2, or
G
i3 was incorporated into vesicles, and receptor
recovery in the proteoliposomes was ~50%. We also prepared and
resolved G
o-containing vesicles using the higher exclusion limit Sephacryl S-300 gel filtration resin, which separates vesicles from free G
o, and observed nearly all of the
G
o immunoreactivity co-migrating with vesicles in the
void volume (data not shown). The four varieties of
M2·G
i/o vesicles contained similar G
protein levels
(~100 fmol/µl) and receptor: G
ratios (1:6) (data not shown).
Quantitation of G
q and G
11 is difficult
due to the low rates of guanine nucleotide turnover by these
G
-subunits. Therefore, calculations of GTPase activity reported
below were made assuming that incorporation of G
q/11
into vesicles was equal to that of Gi family
G
-subunits.
5/R7 proteins toward G
-subunits of the
Gi and Gq families was assessed in steady state
GTPase assays, which measure multiple rounds of GTP hydrolysis and, as
such, reflect both guanine nucleotide exchange and GTPase activity. RGS4, an effective GAP against Gq and Gi family
G
-subunits (28), was used as a reference RGS protein in all
experiments. Only minor increases were observed in the rate of GTP
hydrolysis in the presence of either agonist (100 µM
carbachol) or GAP (200 nM RGS4) alone in proteoliposomes
formed by reconstitution of M2 muscarinic receptor, G
o,
and G
1
2 (Fig.
2). In contrast, the combined presence of carbachol and RGS4 resulted in a markedly synergistic increase in
GTPase activity, and the rate of hydrolysis of GTP was linear for at
least 15 min.
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Fig. 2.
Steady state GTPase activity of reconstituted
G -subunits. The time course of GTP
hydrolysis by G
o reconstituted in phospholipid vesicles
with the M2 muscarinic receptor and G
1
2
under basal conditions (
), in the presence of 100 µM
carbachol (
), 200 nM RGS4 (
), or carbachol plus RGS4
(
) is shown.
5/R7 RGS proteins (see below).
5/R7 Proteins Stimulate Steady State
GTPase Activity of Gi Family G
-subunits--
To compare
the capacity of G
5/R7 proteins to accelerate GTPase
rates of Gi family G
-subunits, steady state GTPase
activities were determined in the presence and absence of 100 µM carbachol and in the presence and absence of 1 µM RGS protein (either RGS4 or each G
5/R7
dimer). RGS4 markedly increased GTPase activity for G
o,
G
i1, G
i2, and G
i3 in the
presence of 100 µM carbachol (Fig.
3). Each of the G
5/R7
dimers also stimulated to varying degrees GTP hydrolysis by
G
o, G
i1, G
i2, and
G
i3 in the presence of agonist (Fig. 3 and Table
I). The rate observed with G
5/RGS11 was as high or
higher than the rate with RGS4 with all
four Gi family G
-subunits, while the GTPase rates in the
presence of 1 µM G
5/RGS6,
G
5/RGS7, and G
5/RGS9 were significantly
lower. Vesicles containing G
o achieved the highest
maximal GTPase rates irrespective of the RGS protein. However, the
basal rate of GTP hydrolysis by G
o in the absence of RGS
protein was also higher than that observed in
G
I-containing vesicles. Thus, the fold increase in
activity (GTPase rate in the presence of RGS and agonist divided by the
GTPase rate with agonist alone) of G
i3 was as high or
higher than that of G
o in response to
G
5/RGS6, -7, -9, and -11 stimulation. Further, the
effects of R7 proteins on GTPase activity of G
o-subunits
reconstituted with purified P2Y12 receptors was also determined (in the
presence of the agonist 2-methylthio ADP). Similar to the results
observed with M2 receptor-coupled G-proteins, each of the
G
5/RGS11 dimers stimulated steady state GTPase activity
of G
o, and G
5/RGS11 stimulated much
higher GTPase rates than the other R7 proteins (data not shown).
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Fig. 3.
G 5/R7
proteins stimulate GTPase activity of Gi family
G
-subunits. The effects of 100 µM carbachol and 1 µM of the indicated RGS
protein on the steady state GTPase activity of G
o,
G
i1, G
i2, and G
i3
reconstituted in phospholipid vesicles with the M2 muscarinic receptor
and G
1
2 were determined as described
under "Experimental Procedures." Open bars, no agonist.
Shaded bars, 100 µM carbachol. Results shown
are representative of at least three separate experiments using three
independent vesicle preparations.
GTPase rates of Gi family G-subunits in the presence of
RGS4 and G
5/R7 proteins
1) attained in the presence of 100 µM carbachol and 1 µM RGS protein are shown
with mean ± S.D.
5/R7 Proteins Do Not
Stimulate Steady State GTPase Activity of Gq Family
G
-subunits--
Regulation of GTPase activities of
G
q and G
11 was examined in vesicles
reconstituted with M1 muscarinic receptor and heterotrimeric G-proteins
(Fig. 4). RGS4 increased steady state
GTPase activity of G
q and G
11 in the
presence of agonist by nearly 5-fold to final GTPase rates of ~200
fmol of GTP/min/pmol of G
. Consistent with previous observations of
guanine nucleotide exchange/GTPase kinetics of Gq (29),
these rates are lower than those observed for Gi family
-subunits. In contrast to the activity of RGS4, none of the
G
5/R7 dimers significantly increased steady state GTPase
activity of G
q or G
11 in the presence of
carbachol (Fig. 4A). Likewise, G
5/R7 dimers
did not stimulate GTPase activity of G
q- or
G
11-subunits reconstituted with purified P2Y1 receptors (in the presence of the agonist 2-methylthio ADP) (data not shown). Further, 1 µM G
5/R7 dimers had no effect
on the GTPase activity of M1 receptor-coupled G
q and
G
11 stimulated by RGS4 and carbachol (Fig.
4B). The partial inhibition observed with
G
5/RGS11 was nonspecific as demonstrated by equivalent
inhibitory activity observed with boiled G
5/RGS11.
Therefore, under the conditions of these assays, R7 proteins neither
stimulate GTPase activity of G
q or G
11
nor affect GTPase activity stimulated by agonist and RGS4.
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Fig. 4.
G 5/R7
proteins do not stimulate GTPase activity of Gq family
G
-subunits. A, the effects of 100 µM carbachol and 1 µM of the indicated RGS
protein on the steady state GTPase activity of G
q and
G
11 reconstituted in phospholipid vesicles with the M1
muscarinic receptor and G
1
2 were
determined as described under "Experimental Procedures." Open
bars, no agonist. Shaded bars, 100 µM
carbachol. Results shown are representative of three separate
experiments using three independent vesicle preparations. B,
the effects of 1 µM G
5/RGS proteins on
agonist-stimulated steady state GTPase activity of M1 receptor-coupled
G
q and G
11 were determined in the
presence of 100 nM RGS4. The effects of heat inactivated
G
5/RGS11 were also determined. The results are presented
as a percentage of the agonist-stimulated activity.
5/R7 Proteins Exhibit
Differences in Maximal Activity and Potency toward Gi
Family G
-subunits--
To more fully elucidate any selectivity of
G
5/R7 proteins as GAPs for Gi family
subunits, full concentration effect curves of each G
5/R7
protein were generated in the presence of a maximally effective
concentration of carbachol. Consistent with the data in Fig. 3,
maximally effective concentrations of G
5/RGS11 produced larger effects than G
5/RGS7, G
5/RGS6, and
G
5/RGS9 on the GTPase activity of each G
-subunit
(Fig. 5 and data not shown), and the highest maximal rate observed with each RGS protein was observed with
G
o as substrate (not shown). Each of the
G
5/RGS dimers produced a near maximal effect at a
concentration of 1 µM, and therefore each activation
curve was normalized to 100% of maximal activity for comparison of
EC50 values (Fig. 6). All
four G
5/R7 dimers exhibited similar potency for
G
o (EC50 = 16-47 nM), while G
5/RGS9 and G
5/RGS11 were more potent
(EC50 = 25-80 nM) than G
5/RGS6
and G
5/RGS7
(EC50 = 150-350 nM) for G
i1,
G
i2, and G
i3 (Table II).
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Fig. 5.
G 5/R7
proteins stimulate different maximal GTPase activity. Steady state
GTPase rates were determined for
M2·G
o·G
1
2 vesicles in
the presence of 100 µM carbachol and various
concentrations of R7 proteins to generate concentration effect curves.
Values observed at each RGS concentration in the absence of vesicles
have been subtracted from the data. Data are plotted as GTPase rates
(min
1). Symbols are G
5/RGS11 (
),
G
5/RGS9 (
), G
5/RGS7 (
), and
G
5/RGS6 (
). Results are representative of at least
three independent determinations using three separate vesicle
preparations.
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Fig. 6.
G 5/R7
proteins exhibit differences in potency toward Gi family
G
-subunits. Steady state GTPase rates
were determined for
M2·G
i/o·G
1
2 vesicles
in the presence of 100 µM carbachol and various
concentrations of R7 proteins to generate concentration effect curves.
Data are normalized to 100% activity for comparison of
EC50 values. Symbols are G
5/RGS11 (
),
G
5/RGS9 (
), G
5/RGS7 (
), and
G
5/RGS6 (
). Results are representative of at least
three independent determinations using three separate vesicle
preparations.
EC50 values G5/R7 proteins for stimulation
of GTPase activity of Gi family G
-subunits
-subunits using three different vesicle preparations.
5/RGS7 and
G
5/RGS9 Inhibit
G
5/RGS11-stimulated G
o GTPase
Activity--
Marked differences in the maximal GTPase rate of
G
-subunits were observed across the G
5/R7 protein
family. For example, G
5/RGS11-stimulated GTPase activity
of G
o was twice that achieved in the presence of
G
5/RGS9 or G
5/RGS7. These results suggest that G
5/RGS7 and G
5/RGS9 interaction with
G-proteins results in a less active G
conformation with respect to
GTPase activity than that promoted by G
5/RGS11
interaction. To test this hypothesis, steady state GTPase activity of
M2·G
o·
1
2 vesicles was
measured in the presence of 100 nM G
5/RGS11
or 1 µM G
5/RGS7 alone or with 100 nM G
5/RGS11 plus 1 µM
G
5/RGS7. As illustrated in Fig. 7A, GTPase activity in the
presence of G
5/RGS11 was nearly twice that observed with
a 10-fold higher concentration of G
5/RGS7. However, the
combined presence of 1 µM G
5/RGS7 and 100 nM G
5/RGS11 resulted in activity only
slightly greater than that of G
5/RGS7 alone, suggesting
that G
5/RGS7 competitively antagonizes the action of the
more efficacious G
5/RGS11. Heat-inactivated
G
5/RGS7 neither stimulated GTPase activity nor inhibited
the stimulatory effect of G
5/RGS11 on GTPase activity
(Fig. 7A), demonstrating that both effects of
G
5/RGS7 are dependent on protein activity. To further
characterize the interaction of RGS proteins and G
o, the
concentration dependence of the inhibitory effect of
G
5/RGS7 and G
5/RGS9 was determined by
varying the concentrations of these proteins in the presence of
carbachol and 100 nM G
5/RGS11. Both G
5/RGS7 and G
5/RGS9 significantly
inhibited G
5/RGS11-stimulated GTPase activity of
M2·G
o vesicles with IC50 values of
100-200 nM (Fig. 7B). These data indicate that
while G
5/RGS7 and G
5/RGS9 are less
efficacious activators of GTPase activity, they interact with a similar
region of G
o as does G
5/RGS11.
View larger version (20K):
[in a new window]
Fig. 7.
G 5/RGS7
and G
5/RGS9 inhibit
G
5/RGS11-stimulated GTPase
activity of G
o. A,
steady state GTPase activity of
M2·G
o·G
1
2 vesicles was
determined in the presence of 100 µM carbachol alone or
in the presence of G
5/R7 proteins. The effects of 100 nM G
5/RGS11, 1 µM
G
5/RGS7, and heat-inactivated G
5/RGS7 (1 µM) were assayed separately and in combination.
Results are representative of two independent experiments with two
RGS11 preparations. B, G
5/R11-stimulated
steady state GTPase activity of G
o was determined at
various concentrations of added G
5/RGS7 and
G
5/RGS9.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i/o
specificity of G
5/R7 proteins and found no evidence of
regulation of G
q GTPase activity by these proteins.
Further, we demonstrated that differences exist in the potencies and
relative efficacies of the G
5/R7 proteins for their
G
i/o substrates. Finally, we illustrated that
G
5/RGS7 and G
5/RGS9, which are less
effective promoters of maximal GTPase activity than is
G
5/RGS11, inhibit G
5/RGS11-stimulated
GTPase activity of G
o.
GAP activity and selectivities within this
class of proteins remain undefined.
5/R7 family previously were reported to
be specific for G
o in single turnover assays of soluble
G
-subunits (11, 20). These assays may not accurately represent
physiological interactions between G-proteins and RGS proteins for
several reasons including the lack of a lipid bilayer with which
G-proteins and RGS proteins may associate, the lack of a GPCR, which
may form a complex with RGS proteins (37-39) and facilitate
interaction with G
-subunits, and the necessity of using
GTPase-deficient mutants of Gq family G-proteins given
their low rates of exchange. These limitations may explain
discrepancies between selectivities for G-protein
-subunits observed
in single turnover versus cell-based assays. Indeed, RGS2
behaves as a G
q-specific GAP in single turnover assays,
but exhibits GAP activity toward G
i as well as
G
q in steady state GTPase assays of proteoliposomes
reconstituted with GPCR and heterotrimeric G-proteins (28).
5/R7
proteins using steady state GTPase assays of receptor-coupled
G-proteins reconstituted in phospholipid bilayers. Because these assays
measure multiple rounds of hydrolysis in the presence of
receptor-stimulated guanine nucleotide exchange, wild type
G
q or G
11 may be used, and the
contributions made by agonist, receptor, G
-subunits, and the
phospholipid bilayer to GTPase activity are likely more representative
of a cellular environment. Our results differ from those from single
turnover assays (11, 20) with respect to the selectivity within the
G
i/o family since all four G
-subunits of this family
are substrates for G
5/R7 proteins.
5/R7 proteins stimulate GTPase activity of
G
q or G
11 (14, 21, 22) in that we did not
observe stimulation of G
q or G
11 GTPase
activity in response to R7 proteins. A trivial explanation for our
observation is that R7 proteins inhibit agonist promoted exchange and
thereby mask GAP activity in steady state GTPase assays. However, the
lack of an effect of R7 proteins on the steady state GTPase activity
achieved in the presence of carbachol and RGS4 demonstrates that
G
5/R7 heterodimers do not significantly affect
agonist-promoted exchange of guanine nucleotides under the conditions
of these assays (Fig. 4B). We also observed minimal to no
effects of G
5/R7 proteins on agonist-stimulated guanine nucleotide exchange measured directly in GTP
S binding assays (not
shown). Therefore, the inability of G
5/R7 proteins to
stimulate steady state GTPase activity of G
q and
G
11 indicates that they do not function as
Gq family GAPs under the conditions of our assay. In the
absence of GAP activity, the reported effects of RGS7 on Gq
family G-protein signaling could reflect direct inhibition of
phospholipase enzymes, as observed by Posner et al. (20). Although we have observed some inhibition of receptor-stimulated inositol phosphate accumulation in cells cotransfected with R7 RGS
proteins and G
5, this inhibition is less pronounced and
requires expression to much higher levels than does the marked
inhibition of phospholipase C response observed in cells overexpressing
RGS2 or RGS4 (data not shown). Thus, the reported effects of R7
proteins on cellular Gq pathways may reflect either loss of
GAP selectivity due to protein overexpression or a more complex
interaction of G
5/R7 dimers with the G-protein signaling cycle.
5/R7 proteins exhibited differences in the potency and
efficacy of their GAP activity against the G
i/o family.
G
5/RGS6 and G
5/RGS7 each exhibited
10-fold lower potency for G
i
-subunits than for
G
o. In contrast, G
5/RGS9 and
G
5/RGS11 exhibited similar potency for all four
Gi family G
-subunits. This pattern mirrors the grouping
of R7 proteins by sequence similarity; that is, RGS6 and RGS7 have
higher sequence identity to each other than to RGS9 and RGS11 and
vice versa (13). The R7 proteins group differently with
respect to their apparent efficacies for stimulation of GTPase activity. G
5/RGS11 exhibited the highest maximal effect, while the
maximal effects of G
5/RGS7 and G
5/RGS9 were much less and G
5/RGS6 exhibited an intermediate maximal effect. These
differences in activity inversely correlate with the expression of R7
transcripts in rat brain where RGS11 is expressed at much lower levels
than RGS7 and -9, and again RGS6 is intermediate (42). We speculate that expression of the robustly active RGS11 may be regulated differently than the less active proteins.
i1, G
i2, and G
i3 share
high sequence homology, and these signaling proteins are essentially
interchangeable in many signaling processes. However, although these
proteins often are expressed in the same cell, they may not be entirely
functionally redundant. For example, selectivity of coupling of certain
G protein-coupled receptors among these three G
i
proteins has been illustrated (43-46), and several reports suggest
selective coupling of receptors to ion channels through specific
G
i-subunits (for review see Ref. 47). Our observation of
selectivity of action of
5/RGS proteins among
G
i1, G
i2, G
i3, and
G
i/o likely has physiologically important ramifications.
For example, this family of RGS proteins is highly expressed in the
central nervous system as are Gi family G
-subunits.
Given the broadly different patterns of expression of RGS6, RGS7, RGS9,
and RGS11 in the brain (42), we hypothesize that otherwise functionally
redundant G
i-subunits may exhibit cell-specific
differences in signaling activities as a consequence of the presence of
different RGS proteins.
.
Unlike GAP proteins for the Ras superfamily GTPases, which contribute a
catalytic arginine required for GTPase activity, RGS proteins are
considered to enhance GTPase activity solely by stabilizing G
switch
regions (48). Presumably, R7 RGS proteins bind G
-subunits through
interactions similar to those observed in the RGS4/G
i
(48) and the RGS9/G
t/i1 crystal structures (49), in
which the base of a 4-helix bundle (
4,
5,
6,
7) of the RGS domain directly contacts
portions of the three switch regions of G
. Martemyanov and Arshavsky
recently reported that mutation of RGS9 residues in the base of
G
-interacting helices
5 and
6
(L353E/R360P) resulted in markedly higher maximal GAP activity toward
G
transducin than observed in wild type RGS9, consistent with
a role for this region in determining maximal GAP activity (50). It
will be equally important to determine if regions outside the RGS box
play important roles in defining selectivities of
5-RGS
proteins among G
-subunits as suggested in studies of G
selectivity of C. elegans R7 family members (40, 41).
-subunits. We have shown differences in potency and efficacy of
G
5/R7 dimers as GAPs among the Gi family
G
-subunits. Further, lower efficacy GAPs were shown to inhibit
GTPase activity achieved in the presence of a more efficacious GAP,
indicating that RGS proteins apparently interacting with the same
activating surface of a G
-subunit promote different maximal rates of
catalysis by the G
GTPase.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors would like to thank Dr. Alfred Gilman for input and support and Drs. David Siderovski and John Sondek for thoughtful suggestions during preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grants GM29536, GM65533, and GM34497.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.
§ Supported by postdoctoral National Research Service Award GM66561 from the United States Public Health Service. To whom correspondence should be addressed: Department of Pharmacology, Campus Box 7365, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5356; Fax: 919-966-5640; E-mail: shelleyb@med.unc.edu.
¶ Supported by a postdoctoral fellowship from the Pharma Foundation.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M211382200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G-protein
coupled receptor;
RGS, regulator of G-protein signaling;
GAP, GTPase
activating protein;
GGL, G- like;
m.o.i., multiplicity of
infection;
Ni-NTA, nickel-nitrilotriacetic acid;
C12E10, polyoxyethylene 10-lauryl ether.
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