From the Departments of Physiology and
¶ Pharmacology, University of Texas Southwestern Medical
Center, Dallas, Texas 75235,
Millenium Pharmaceuticals,
Inc., Cambridge, Massachusetts 02139, and the ** Institute for
Pharmacology, Klinikum Benjamin Franklin, Freie Universität
Berlin, 14159 Berlin, Germany
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ABSTRACT |
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Regulators of G protein signaling (RGS) proteins
accelerate GTP hydrolysis by G Heterotrimeric G proteins of the Gq class transduce
Ca2+ signaling by coupling heptahelical transmembrane
receptors to the An intriguing question in cell signaling is how different agonists can
stimulate the same Gq-coupled pathway to generate distinct spatial and/or temporal patterns of Ca2+ response within
the same cell. Although signaling in pancreatic acinar cells is
dependent on agonist binding to Gq-coupled receptors, several experiments suggested that Ca2+ release was also
regulated by a novel mechanism that controlled G protein signaling.
First, Ca2+ release evoked by carbachol, bombesin, or CCK
was equally inhibited by titration with proteins that sequestered
G G protein activity is regulated both by receptor-catalyzed GTP binding
to the RGS proteins were first identified by genetic techniques to be
inhibitors of G protein signaling (8, 16, 17). Recent work has
identified over 20 RGS proteins expressed in mammals (12, 16, 18-22).
In vitro studies suggest that several mammalian RGS
proteins, including RGS1, RGS4, and RGS16, have GAP activity toward
different Gi and/or Gq class In the present study, we examined the role of RGS4 and other RGS
proteins in regulating Ca2+ signaling in pancreatic acinar
cells. RGS4 inhibited Ca2+ signaling assayed either by
measuring agonist-dependent Ca2+ mobilization
in streptolysin O (SLO)-permeabilized cells or
Ca2+-activated Cl Expression and Purification of RGS Proteins--
Recombinant
RGS1, RGS2, RGS4, and RGS16 proteins were tagged at the N terminus with
the sequence MH6MG using the pET19b and a modified pQE60
expression vector, respectively (Qiagen), expressed in
Escherichia coli and purified as described (10, 14, 15). Briefly, a 10-ml overnight culture grown at 37 °C in T7 medium supplemented with 100 µg/ml ampicillin and 1% glucose was used to
inoculate 2 liter of T7/100 µg/ml ampicillin medium at 30 °C. Isopropyl-1-thio- Production of Knockout Mice--
Mice deficient in
G
Two strains of double homozygous null mice were unavailable for this
study. G Immunoblots--
Expression of G Preparation of Cells--
Pancreatic acini and single pancreatic
acinar cells from the rat and mouse pancreas were prepared by standard
collagenase and trypsin digestion procedures as described (3, 5, 7). The cells were suspended in solution A containing (in mM):
NaCl 140, KCl 5, Hepes 10 (pH 7.4), MgCl2 1, CaCl2 1, and glucose 10. The cells were kept on ice until use.
Measurement of Ca2+ Release and IP3
Production in Permeabilized Cells--
The procedures used to measure
agonist- and IP3-evoked Ca2+ release in
SLO-permeabilized pancreatic acini are essentially as described (3, 7).
In brief, the acini were washed and suspended in a KCl-based
permeabilization medium containing ATP regeneration system and SLO. The
cells were allowed to reduce medium [Ca2+] to 50-100
nM before stimulation. The same protocol was used to
measure IP3 production except that the volume of the
reaction medium was increased from 400 to 500 µl. At designated times
monitoring of [Ca2+] was interrupted to remove 25-50
µl of duplicate samples to 25-50 µl of an ice-cold 15% perchloric
acid. At the end of the incubation periods with all agonists and at
least 10 min after the last stimulation, precipitated proteins were
removed by centrifugation and IP3 content of the
supernatant were evaluated by a radioligand assay (3).
Measurement of Ca2+-activated Cl Inhibition of Ca2+ Signaling by RGS4 in Permeable
Cells--
Previous studies showed that RGS4 added to isolated
membranes (28, 29) or overexpressed in cell lines (24-27) inhibited Gq-dependent PLC Agonist Dependence of RGS4 Action--
An important finding shown
in Fig. 1 is that RGS4 inhibited Ca2+ release stimulated by
three different Gq-coupled receptors with markedly
different potencies. Fig. 1 (b, d, and
e) shows that, whereas 0.05 µM RGS4 inhibited
85% of Ca2+ release evoked by carbachol, 0.65 µM RGS4 only partially inhibited Ca2+ release
and 1.87 µM RGS4 inhibited 85% of Ca2+
release by CCK. The dependence of inhibition of Ca2+
release on RGS4 concentration is illustrated in Fig.
2. Half-maximal inhibition of
Ca2+ mobilization induced by carbachol, bombesin, and CCK
occurred at [RGS4] of approximately 35, 110, and 380 nM,
respectively. Hence, cholinergic receptors were 3- and 10-fold more
sensitive to RGS4 than were bombesin and CCK receptors. Addition of
higher concentrations of any agonist did not alter the IC50
of RGS4 (data not shown).
The differential sensitivity of the Gq-coupled receptors to
RGS4 inhibition was also reflected in activation of PLC and
IP3 production in SLO-permeabilized cells. Fig.
3 shows that the three agonists
stimulated PLC activity to the same extent, indicating similar
activation of Gq class Selectivity of RGS4 Inhibition Depends on Receptor, Not
Gq Class
To evaluate the importance of the RGS4 Preferentially Inhibits the Response to Carbachol in Intact
Cells--
Measurements of Ca2+ activated Cl
Fig. 7 shows the inhibitory activity of
RGS4 at three concentrations with three different
Gq-coupled receptors. Cells were dialyzed with RGS4 for at
least 7 min, which is sufficient to allow equilibration of the protein
between pipette solution and the cytosol (39). In general, RGS4 was
between 100- and 1000-fold more effective in intact than in permeable
cells. This most likely reflects improved access of RGS4 to the inner
membrane surface in intact cells. In more than 20 experiments, 100 pM RGS4 either completely or almost completely inhibited
the effect of carbachol on Ca2+ signaling. This is the
highest potency for RGS4 in inhibiting G
To obtain unequivocal evidence for the differential sensitivity of the
Gq-coupled receptors to RGS4, we measured the effect of
RGS4 on carbachol and CCK stimulation in the same cells (Fig. 8a). Dialyzing cells with 10 pM RGS4 converted the large initial Cl
After addition of atropine to allow reloading of intracellular
Ca2+ stores, cells were challenged with CCK to assess the
effects of the same series of RGS4 concentrations on CCK stimulation
(Fig. 8). As shown, the CCK response was still substantial at 100 pM RGS4 and was clearly present at about 50% of maximal
amplitude at 1 nM RGS4. Dialysis with a relatively high
concentration of RGS4 (5 nM) finally inhibited
Ca2+ release both by carbachol and CCK (Fig.
8e). RGS4 did not interfere with activation of the
Cl RGS1 and RGS16 Preferentially Inhibit the Response of Pancreatic
Acinar Cells to Carbachol--
The results obtained with RGS4 raised
the question of whether other RGS proteins that interact with
Gq class RGS2 Equally Inhibits the Response of Pancreatic Acinar Cells to
Carbachol and CCK--
RGS2 was reported to specifically accelerate
GTPase activity of G
Estimations of the relative potency with which the different RGS
proteins inhibited the response to stimulation of the m3 and CCK
receptors are shown in Fig. 10e. The relative potency of RGS2 was estimated from all concentrations between 1 and 100 nM. The relative potencies of RGS4 and RGS1 were determined
from the concentrations needed for 89-95% inhibition because of the
large difference in potency in inhibiting carbachol and CCK responses. Since RGS16 only partially inhibits the response to CCK, the potency for inhibition of this response was estimated for the fraction inhibited by RGS16. Fig. 10e shows that the difference in
potency for the RGS proteins tested spans 3 log units. Hence, although the studies with RGS2, RGS1, and RGS16 are not as extensive as those
with RGS4, they corroborate the central finding that RGS proteins can
discriminate between receptor complexes to regulate Ca2+ signaling.
Like many cell types, pancreatic acini respond to a battery of
Ca2+ mobilizing agonists. Previous studies showed that at
least three agonists, carbachol, bombesin, and CCK, interact with
receptors coupled to Gq, stimulate PLC to the same extent
and mobilize the same Ca2+ pool (40, 41). The
Ca2+ signals evoked by all agonists are in the form of
[Ca2+]i waves that initiate in the
luminal pole and propagate through the cell periphery to the basal pole
(42-44). The [Ca2+]i waves
exhibit agonist specific initiation sites, speed, and propagation
patterns (3).
Coupling of receptors to G proteins may play a significant role in
conferring signaling specificity. Indeed, Ca2+ signaling
stimulated by several agonists displayed differential sensitivity to
guanine nucleotides (3). The present studies show that differences
exist in the interaction of receptor-Gq complexes with four
RGS proteins: RGS2, RGS4, RGS1, and RGS16. The sensitivity to RGS4 can
be best quantitated in permeabilized cells because an averaged response
from many cells is recorded. In permeabilized rat pancreatic acinar
cells, cholinergic receptors showed 3- and 10-fold higher apparent
affinity to RGS4 than bombesin and CCK receptors, respectively (Figs.
1-3). In mice, differences between the receptors were even greater
(Fig. 5). Species differences in CCK-dependent
Ca2+ signaling are well documented (41). Differential
sensitivity to RGS4 was corroborated in intact cells from the rat
pancreas (Figs. 7 and 8). The fact that RGS4 was a more potent
inhibitor of the response to carbachol relative to CCK within the same
cell excludes the possibilities of cell to cell variation and
restricted access of RGS4 to the inner leaflet of the plasma membrane.
Although it was difficult to accurately quantify inhibition in intact
cells, the CCK response was at least 10-fold less sensitive to RGS4
(see Figs. 7 and 8), consistent with experiments in permeable cells (Fig. 2). Therefore, the cumulative results from permeable and intact
cells indicated that pancreatic acini could be more readily stimulated
with CCK than with bombesin or acetylcholine in the presence of RGS4.
This conclusion is reinforced by the findings with RGS2, RGS1, and
RGS16, which showed no or greater preferences for inhibition of
muscarinic stimulation (Fig. 10). Regulation by RGS proteins may
therefore provide the cell with a mechanism for intense (CCK),
intermediate (bombesin), and weak (acetylcholine) stimulation of
the same Gq-mediated signaling pathway. Intensity of
stimulation affects almost all parameters of the
[Ca2+]i signal (2, 38, 40).
Several findings of the present and previous work indicate that
differential sensitivity to RGS proteins was not the result of
different steady state levels of GTP-bound Gq class Receptor specificity of RGS protein action could reflect preferential
coupling of the receptors to different members of the Gq
class The finding that these RGS proteins inhibit Gq proteins
coupled to acetylcholine receptors in preference to other receptor types present in the same cell suggests selectivity in RGS protein regulation of G protein signaling. Studies with recombinant proteins in vitro showed that RGS4 similarly stimulated the GTPase
activity of several Gi class Another implication of the receptor-specific interaction of the RGS
proteins used in the present work is that RGS proteins may also
interact with the receptor-Gq complex, not only with activated Gq class In summary, the present study shows that regulating specificity of G
protein-coupled signaling by RGS proteins extends to single receptor
types. The identity of the Gq class subunits, thereby attenuating
signaling. RGS4 is a GTPase-activating protein for Gi
and Gq class
subunits. In the present study, we used
knockouts of Gq class genes in mice to evaluate the potency
and selectivity of RGS4 in modulating Ca2+ signaling
transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a
receptor-selective manner in both permeabilized cells and cells
dialyzed with RGS4 through a patch pipette.
Receptor-dependent inhibition of Ca2+ signaling
by RGS4 was observed in acini prepared from the rat and mouse pancreas.
The response of mouse pancreatic acini to carbachol was about 4- and
33-fold more sensitive to RGS4 than that of bombesin and
cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent
inhibitors of Gq-dependent Ca2+
signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than
CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially
inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of
receptor-selective inhibition by RGS4 was observed in acinar cells from
wild type and several single and double Gq class knockout
mice. Thus, these receptors appear to couple Gq class
subunit isotypes equally. Difference in receptor selectivity of RGS
proteins action indicates that regulatory specificity is conferred by
interaction of RGS proteins with receptor complexes.
INTRODUCTION
Top
Abstract
Introduction
References
isoforms of phospholipase C
(PLC)1 (1). Many cells
express multiple receptors that each activates the Gq
signaling pathway (2). For example, pancreatic acinar cells respond to
acetylcholine, bombesin and cholecystokinin (CCK) by intense activation
of PLC to generate IP3 and mobilize Ca2+ from
internal stores. In a recent study we showed that, although these three
agonists activate the same Gq-regulated signaling pathway
to mobilize the same Ca2+ pool, each agonist evokes a
distinct pattern of Ca2+ wave propagation (3).
Ca2+ signaling in pancreatic acini triggers the exocytotic
release of digestive enzymes from granules adjacent to the secretory
membrane (4).
and with antisera that specifically recognized Gq
class
subunits (3, 5). Second, these receptors promiscuously
coupled to members of the Gq class
subunits (6). Third,
maximal stimulation with each of these agonists generated roughly equal
levels of IP3. These results suggest that all three
agonists stimulate their receptors to activate the same amount of
G
q/11. Nevertheless, we found that GTP
S
differentially activated, and GDP
S differentially inhibited,
signaling by the various agonists acting in these cells (3, 7). These
effects of guanine nucleotides suggested that G protein activity was
differentially regulated by an unknown intracellular protein(s) that
acted in a receptor-dependent manner.
subunit and by the rate of GTP hydrolysis. Regulators of G
protein signaling (RGS) proteins are a recently identified family of
intracellular GTPase-activating proteins (GAPs) (8, 9) that accelerate
GTP hydrolysis by G
subunits (10-15), thus limiting the duration of
G protein activation. RGS proteins may regulate signaling by uncoupling
the cycle of GTP binding and hydrolysis from effector protein
activation by the G
subunit, even in the presence of persistent
agonist stimulation.
subunits but
not G
s or G
12 (10, 11, 15, 23-25). RGS4
inhibited Gq-dependent PLC
activation in
Xenopus oocytes and transfected COS and HEK293 cells (24-27). Furthermore, addition of recombinant RGS4 protein to NG-108 cell membranes inhibited Gq-dependent PLC
activity (28, 29). RGS2 was reported as a specific GAP for
G
q in an in vitro assay (30), although it
inhibited Gi-dependent signaling when expressed in transfected cells (31). Because RGS proteins with similar GAP
activities are co-expressed in cells within a single tissue (16, 18,
23, 29-31), the mechanisms that may provide more precise regulatory
specificity have been enigmatic. To date there is no information on the
potency or selectivity with which mammalian RGS proteins modulate the
same G protein
subunit during its response to different receptors.
This highlights the need to analyze RGS proteins in intact cells under
controlled conditions to assess their potency and specificity of action.
current in intact cells.
The potency of RGS4 was exceedingly high in intact cells, and GTP
S
reversed the inhibitory action of RGS4. This suggests that catalysis of
GTPase activity is the dominant mechanism by which RGS4 regulates
Ca2+ signaling. Furthermore, we provide the first evidence
for receptor selectivity in RGS4 inhibition of PLC
and
Ca2+ signaling. Even more pronounced receptor selectivity
was measured with RGS1 and RGS16. On the other hand, RGS2 showed
similar potency in inhibiting m3- and CCK-dependent
Ca2+ signaling. The potential role of the
subunits in
determining differential sensitivity to RGS4 was analyzed using
knockout mice. Deletion of individual Gq class
subunit
genes or combinations thereof had no effect on the receptor-specific
action of RGS4. Thus, specificity of RGS protein actions depends on
their interaction with the receptor complex rather than their
interaction with a specific Gq class
subunit.
EXPERIMENTAL PROCEDURES
-D-galactopyranoside (0.5 mM) induction was performed at OD600 of
0.6-0.8, and cell cultures were shaken 4 h prior to harvest.
Cells were pelleted and resuspended in TBP (50 mM Tris-HCl,
pH 8.0, 20 mM
-mercaptoethanol, and 0.1 mM
phenylmethylsulfonyl fluoride). Lysozyme (0.2 mg/ml) and DNase I (5 µg/ml) were added, and cells were inoculated on ice to complete lysis
and DNA digestion. The total lysate was centrifuged (12,000 × g for 30 min) at 4 °C, and the supernatant was loaded
onto a 2-ml nickel-NTA column pre-equilibrated with TBP buffer. The
column was washed with 20 ml of TBP and 0.2 M NaCl. The
final wash was performed using 10 ml of TBP with 5 mM
imidazole (pH 8.0). The protein was eluted twice with 2 ml elution
buffer (TBP containing 200 mM imidazole, pH 8.0), dialyzed
overnight against 1 liter of 50 mM HEPES, 2 mM
dithiothreitol buffer (pH 8.0) at 4 °C, and further concentrated with a Centricon 10 device (Amicon).
11 were produced as described (33). Briefly, the murine
G
11 gene was disrupted by homologous recombination in
mouse embryonic stem cells. Integration of the G
11
targeting vector replaced exons 3, 4, and a portion of 5 with several
translation termination codons present in the reverse orientation of
the PGK::Neo transgene. G
11
/
mice are
viable and fertile. G
14 knockout mice were produced by
disrupting the G
14 gene in ES cells by homologous
integration of a PGK::Neo expression cassette into the exon
that encodes amino acid residues Ser-121 to Lys-154. G
14
/
mice are viable and fertile with no apparent
behavioral or morphologic defects.
G
11
/
;G
14
/
double homozygous null mice were obtained from intercrossing the offspring of the single knockout mice. To produce G
15
/
knockout mice,
integration of the G
15 targeting vector replaced exons
3, 4, and a portion of 5 with PGK::Neo in the inverse
orientation. G
15
/
mice are viable and fertile with
no apparent behavioral or morphologic defects. Production of
G
q
/
mice was described (34).
G
q
/
;G
15
/
double homozygous null
mice were obtained by intercrossing the offspring of the single
knockout mice. All WT and mutant mice were of 129/SvEv × C57BL/6
genetic background.
q
/
;G
11
/
mice could not be
obtained as they die during embryogenesis (33), and obtaining
G
q
/
;G
14
/
mice from intercrossing
the single knockouts is impractical because these genes are tandemly
duplicated on mouse chromosome 19 (35) and are thus too close together
to expect recombination to place both null mutations on the same chromosome.
q,
G
11, G
14, and G
15 in WT
and knockout mice was assayed by Western blot as described (6, 36).
Analysis of membrane proteins from pancreatic acini showed that the
mutated
subunits were not expressed in the respective single and
double knockout mice and the remaining Gq class
subunits were expressed at similar levels in WT and mutant mice (see
also Refs. 6, 33, and 34).
Current--
The whole cell configuration of the patch clamp technique
(37) was used to measure the effect of agonists on Cl
current. The pipette solution and recording conditions were set to
optimize detection of the Ca2+-activated Cl
current as a reporter of changes in
[Ca2+]i (for details, see Ref. 5).
All experiments were performed at room temperature. The patch clamp
output was filtered at 20 Hz. Recording was performed with pClamp 6 and
a digi-Data 1200 interface (Axon Instruments). In all experiments, the
Cl
and cation equilibrium potentials were about 0 mV.
Cl
current was recorded at a holding potential of
40
mV. Current amplitude of stimulated cells was within 15% in a given
cell preparation but varied between 50 and 150 pA/pF between cell
preparations. Therefore, each experiment included at least one control
and the inhibition of signaling by RGS proteins was compared with a
control performed with a cell from the same preparation.
RESULTS
activation. Fig.
1 shows that addition of RGS4 to
permeabilized rat pancreatic acinar cells effectively blocked agonist-dependent, Gq-coupled Ca2+
release from internal stores. The permeabilized cells reduced [Ca2+] in the incubation medium to about 50 nM and responded to maximal stimulation with carbachol by
releasing about 75% of the Ca2+ that is accessible to
IP3 (Fig. 1a). Addition of 50 nM
RGS4 to the permeabilization medium inhibited about 85% of
Ca2+ release triggered by carbachol (Fig. 1b).
Inhibition by RGS4 was fully reversible by addition of GTP
S (2.5 µM), a non-hydrolyzable GTP analog that activates G
subunits, suggesting that inhibition of signaling reflects the GAP
activity of RGS4 under these conditions. Consistent with this
interpretation, RGS4 at concentrations up to 1.87 µM had
no effect on Ca2+ release evoked by IP3 (Fig.
1e), which excludes the possibility that RGS4 inhibited the
IP3-activated Ca2+ channel.
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Fig. 1.
Inhibition of Ca2+ signaling by
exogenously added RGS4. Pancreatic acini were added to the
SLO-containing permeabilization medium and allowed to reduce
[Ca2+] of the incubation medium to about 50 nM. Where indicated, RGS4 was added from a stock solution
of 100-120 µM in 50 mM Hepes (pH 7.4) and 2 mM dithiothreitol. The cells were then stimulated with 2 mM carbachol (a and b) or 50 nM CCK (c-e). In experiments shown in
b-e, after stimulation with agonists, the cells were
exposed to 2.5 µM GTP S. To evaluate the extent of
Ca2+ release, at the end of each experiment, the cells were
treated with a maximal concentration of 2 µM
IP3.
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Fig. 2.
Agonist-dependent inhibition of
Ca2+ release by RGS4. The protocol of Fig. 1 was used
to evaluate the potency of RGS4 to inhibit Ca2+ release
evoked by each agonist. With all agonists, 2.5 µM GTP S
completely reversed the inhibition at all RGS4 concentrations. The
figure shows the mean ± S.E. of five experiments with carbachol
and three experiments with bombesin and CCK.
subunits by each agonist.
Consistent with the concentration dependence of Ca2+
signaling inhibition shown above, 50 nM and 0.2 µM RGS4 inhibited carbachol-stimulated IP3
production by 60 ± 4% and 91 ± 7%, respectively. By
contrast, 0.1 µM and 0.5 µM RGS4 was needed
to inhibit bombesin-stimulated IP3 production by 41 ± 6% and 94 ± 7%, respectively. Finally, 0.4 µM and
2 µM RGS4 inhibited the effect of CCK on IP3
production by 43 ± 7% and 77 ± 8%, respectively. Thus,
RGS4 inhibition of both PLC activity and Ca2+ signaling
were receptor-selective.
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Fig. 3.
Agonist-dependent inhibition of
PLC activity by RGS4. The protocol of Fig. 1 was used to
permeabilize cells and monitor [Ca2+]. RGS4 at the
indicated concentration was included in the permeabilization medium.
Samples were removed to determine the level of IP3
30 s before and after agonist stimulation and 30 s after
addition of GTP S. The figure shows the mean ± S.E. of three
experiments.
Subunit Identity--
The receptor-selective
action of RGS4 might reflect preferential coupling of the receptors to
different Gq class
subunits that respond differentially
to RGS4. Alternatively, selectivity might be determined by
receptor-specific interactions. We used mice genetically deficient in
one or more of the Gq class
subunits that are expressed
in pancreatic acinar cells (G
q, G
11, and G
14) (Fig. 4a;
Ref. 6) to distinguish these possibilities. When Ca2+
signaling was measured in intact acinar cells of homozygous knockout mice (G
q
/
, G
11
/
, and the double
knockout lines G
11
/
;G
14
/
and
G
q
/
;G
15
/
), maximal
Ca2+ responses were identical in wild type and all mutant
mice tested (6).
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Fig. 4.
Western blot analysis of Gq
class subunits and receptor-dependent
inhibition of Ca2+ signaling by RGS4 in pancreatic acini
from WT mice. Membrane proteins isolated from pancreatic acini of
WT mice express G
q, G
11, and
G
14, but not G
15 (a). The
protocol of Fig. 1 was used to test the effect of RGS4 on
agonist-evoked Ca2+ signaling. The response in the
absence of RGS4 (control) is shown only for carbachol
(b).
subunit identity in conferring
selectivity to RGS4 action, we compared the inhibition of
Ca2+ signaling by RGS4 in permeabilized pancreatic acini
from WT and Gq class knockout mice. Representative
experiments are illustrated in Figs. 4 and
5, and the results of all experiments are
summarized in Table I. Fig. 4
(b-e) shows the concentration of RGS4 needed to inhibit
50-60% of Ca2+ signaling evoked by each agonist in acini
from WT mice. Fig. 5 (a-d, e-h, and
i-l) show similar experiments for 50-60% inhibition of
Ca2+ signaling by comparable concentrations of RGS4 in
acini from the G
q
/
;G
15
/
,
G
11
/
, and
G
11
/
;G
14
/
mice, respectively. The relative inhibitory potency of RGS4 was unaltered for each of the
three agonists used here (Table I). The differential sensitivity to
RGS4 among the different receptors was somewhat higher in mouse than in
rat pancreas. In cells from WT mice, the response to carbachol was
about 4.5- and 33-fold more sensitive to RGS4 than that of bombesin and
CCK, respectively. Similar results were obtained in cells from
G
11
/
,
G
11
/
;G
14
/
, and
G
q
/
;G
15
/
mice. The findings in
Table I and Figs. 4 and 5 suggest that interactions with receptor,
rather than the identity of the G protein
subunit, dictate
differential sensitivity to RGS4.
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Fig. 5.
Receptor-dependent inhibition of
Ca2+ signaling by RGS4 in pancreatic acini from
G q
/
;G
15
/
,
G
11
/
, and
G
11
/
;G
14
/
mice. The protocol of Fig. 1 was used to test the effect of RGS4
on agonist-evoked Ca2+ signaling. Acini from the pancreas
of G
q
/
;G
15
/
(a-d),
G
11
/
(e-h), and
G
11
/
;G
14
/
(i-l) mice
were used to measure Ca2+ signaling. Examples of the
responses in the absence of RGS4 are shown in trace
a for carbachol, in trace f for
bombesin, and in trace k for CCK.
Receptor selectivity of RGS4 is unaltered in mice deficient in
Gq class genes
current using the whole cell configuration of the patch clamp technique
were used to independently assess the potency and receptor-selective
action of RGS4. This Cl
current faithfully reflects
changes in [Ca2+]i in pancreatic
acinar cells (5, 38). Fig. 6 establishes a dose-response relationship that identified the minimal concentration of carbachol (100 µM) necessary to evoke a maximal
Ca2+ response in acinar cells. After dialysis with the
pipette solution (7 min), stimulation by 100 µM carbachol
generated a typical biphasic Ca2+ response that consists of
an initial spike followed by a plateau of Ca2+-activated
Cl
current. Maximal Ca2+ response was
demonstrated by the fact that subsequent addition of high
concentrations of carbachol (1 mM) or CCK (10 nM) did not evoke additional activation of the
Cl
current because internal Ca2+ stores were
depleted upon the first stimulation. In contrast, stimulation with 10 µM carbachol also evoked a significant but submaximal
Ca2+ response because subsequent addition of 1 mM carbachol further activated the Cl
current
(Fig. 6b). The absence of a response to later addition of
CCK indicated that 1 mM carbachol maintained the
Ca2+ stores in a depleted state. Ca2+ signaling
evoked by 30 µM carbachol was also submaximal (data not
shown). Reducing the carbachol concentration to 2.5 µM
initiated rapid oscillations in the Cl
current (Fig.
6c), whereas stimulation with an even lower concentration of
carbachol initiated lower frequency oscillations (Fig. 6d). Many cell types, including pancreatic acini, typically respond to
submaximal agonist stimulation with oscillations in
[Ca2+]i (2, 5, 38). Based on the
agonist dose-response relationship, we next tested RGS4 inhibition of
Ca2+ signaling.
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Fig. 6.
Effect of stimulus intensity on the pattern
of Ca2+ signaling in patch-clamped cells. The whole
cell configuration of the patch-clamp technique was used to measure the
effect of different concentrations of carbachol on Cl
current in pancreatic acinar cells. Single cells were dialyzed with the
pipette solution for at least 7 min prior to stimulation with 100 µM carbachol, followed by 1 mM carbachol and
then 10 nM CCK, as indicated by the bars
(a). b-d, same protocol as above, except that
the initial stimulation was with 10, 2.5, or 0.5 µM
carbachol, respectively.
-dependent
activity reported to date. The agonist-dependent effect of
RGS4 seen in permeable cells could be reproduced in intact cells. Thus,
bombesin-stimulated Ca2+ signaling was only partially
inhibited by 100 pM RGS4 (Fig. 7), whereas 500 pM RGS4 was needed for complete inhibition (Fig. 7). RGS4
at 500 pM only partially inhibited the effect of CCK (Fig. 7i), and 5-10 nM were needed for maximal
inhibition of CCK-dependent Ca2+ signaling
(Fig. 7j). Control experiments showed that boiling RGS4
prevents the inhibition of agonist-evoked Ca2+
signaling by this protein (Fig. 7, b and
h).
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Fig. 7.
RGS4 is a potent inhibitor of
Ca2+ signaling in intact cells. Single pancreatic
acinar cells were dialyzed with the pipette solution for at least 7 min
before the first stimulation. As indicated in the figure, the pipette
solution also contained 100 pM (b, c,
and e), 500 pM (f, h, and
i), or 10 nM (j) RGS4. In control
experiments like those in b and h, the pipette
solution was incubated in a boiling water bath for 15-20 min before
use. The experiment in j shows that elevating
[Ca2+]i with A23187 activated the
current, indicating that RGS4 does not affect responses downstream of
the [Ca2+]i increase. Similar
sensitivity to RGS4 was observed in at least 20 experiments in which
the cells were dialyzed with 10, 100, or 1000 pM RGS4 and
stimulated with carbachol, 4 experiments in which the cells were
dialyzed with 0.1, 0.5, or 2 nM RGS4 and stimulated with
bombesin, and 7 experiments in which the cells were dialyzed with 0.1, 1, or 10 nM RGS4 and stimulated with CCK.
current to an oscillatory response (Fig. 8b). 100 pM RGS4 markedly decreased the amplitude and frequency of
oscillation, and 1 nM RGS4 totally blocked the response to
carbachol. Increasing the concentration of carbachol had no effect on
this pattern of inhibition (data not shown). Comparison of the
oscillatory responses obtained at 100 µM carbachol and
various concentrations of RGS4 with the responses elicited by
suboptimal carbachol concentrations indicated that 100 pM
RGS4 inhibited the maximal response to carbachol to a level usually
attained at a 100-fold lower carbachol concentration.
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Fig. 8.
Agonist dependence of RGS4 action in the same
cell. The protocol of Fig. 7 was used to evaluate the action of
RGS4. In all experiments (a-e) at the times indicated by
the bars, the cells were first stimulated with 100 µM carbachol, inhibited by 10 µM atropine,
and, after reloading Ca2+ stores, restimulated with 10 nM CCK. Note that CCK stimulation followed carbachol
stimulation allowing for longer dialysis time with RGS4 in the pipette
solution to ensure that the differential sensitivity was not due to
different concentrations of RGS4 in the cytosol during the stimulation
with CCK. Similar differential sensitivity between carbachol and CCK
was observed in more than 20 experiments.
current by Ca2+ because the ionophore
A23187 stimulated normal activation of the current in cells containing
5 nM RGS4 (Fig. 8e). Carbachol-evoked signaling
showed the same 10-fold greater sensitivity to RGS4 inhibition in over
20 cells exposed only to carbachol or CCK (data not shown).
subunits show similar receptor-selective
inhibition of Ca2+ signaling. RGS1 and RGS16 dialyzed into
acinar cells inhibited Ca2+ signaling evoked by carbachol
and CCK (Fig. 9). Furthermore, both RGS
proteins showed similar potency in inhibiting the response to
carbachol. At a concentration of 0.1 nM, RGS1 and RGS16
inhibited the response to carbachol by 23 ± 5%
(n = 3) and an average of 21% (n = 2),
respectively. Increasing the concentration of RGS1 and RGS16 to 1 nM resulted in 81 ± 4% (n = 11) and
72 ± 7% (n = 5) inhibition of the response to
carbachol, respectively (see Fig. 9, b and e). By
contrast, these RGS proteins were relatively poor inhibitors of the
response to CCK stimulation. Increasing RGS1 concentration to between 1 and 3 µM was needed to inhibit the response to CCK by
85 ± 8% (n = 4), whereas RGS16 only partially inhibited the response to CCK8. RGS16 (100 nM) inhibited
CCK evoked Ca2+ release by 57 ± 4%
(n = 3), and increasing the concentration to 1 µM (n = 2) or 5 µM
(n = 2) did not cause further inhibition (Fig. 9,
compare g and h).
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Fig. 9.
Agonist-dependent action of RGS1
and RGS16 in inhibiting Ca2+ signaling. The protocol
of Fig. 8 was used to evaluate the effect of different concentrations
of RGS1 (panels b-d) or RGS16 (panels
e-h) on Ca2+ signaling. In all experiments,
during the indicated time, the cells were stimulated with 1 mM carbachol, inhibited by 10 µM atropine,
and restimulated with 10 nM CCK8. The number of similar
experiments under each condition is given in the text.
q (30), although when expressed in
COS cells, it affected signaling mediated by Gq and
Gi (31). Fig. 10 shows the
effect of RGS2 on the response of mouse pancreatic acinar cells to
carbachol and CCK stimulation. Equivalent results were obtained in rat
pancreatic acini. RGS2 inhibited carbachol-evoked Ca2+
signaling in the same concentration range measured with other RGS
proteins. Thus, 1 nM RGS2 inhibited the carbachol-induced response by 57 ± 6% (n = 3) and 100 nM RGS2 inhibited the response by 94 ± 5%
(n = 4). Unlike the findings with other RGS proteins, RGS2 inhibited the response to carbachol and CCK stimulation with equal
potency. For example, in the same cells, 10 nM RGS2
inhibited the response to carbachol by 83 ± 6% and the response
to CCK by 81 ± 7% (n = 6).
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Fig. 10.
Agonist-independent action of RGS2 in
inhibiting Ca2+ signaling. The protocol of Fig. 8 was
used to evaluate the effect of different concentrations of RGS2
(panels a-d) on carbachol and
CCK-dependent Ca2+ signaling in mouse
pancreatic acini. Very similar results were obtained in two full
dose-response studies with RGS2 in rat pancreatic acini.
Panel e shows the estimated relative potency of
RGS proteins to inhibit the carbachol and CCK responses. The results
are plotted as the mean ± S.E. of 5-14 determinations. Note the
log scale of the y axis.
DISCUSSION
subunits generated by the three agonists. First, the three RGS proteins showed similar potency toward carbachol stimulation but highly variable
potency toward CCK stimulation. Second, acinar cells express similar
number of cholinergic and CCK receptors (45). Third, titrating the
level of all activated Gq class
subunits with antibody
raised against a C-terminal sequence common to G
q, G
11, and G
14 showed similar activation of
Gq by all agonists (3). Fourth, each agonist activated
PLC
to the same extent (Fig. 3).
subunits, which, in turn, are differentially regulated by RGS
proteins. Alternatively, interaction between RGS proteins and receptors
may determine receptor specificity. To distinguish between these
possibilities, we studied the role of the Gq class
subunits in determining receptor specificity and inhibition by RGS4.
Our previous finding, that all three receptor types in pancreatic acini
showed similar values of Kapp toward their
respective agonist in WT and the various Gq class knockout
mice (6), provides strong evidence that the identity of these
subunits does not play a role in conferring agonist specificity. More
importantly, RGS4 was an equally effective inhibitor of
Ca2+ signaling in pancreatic acinar cells isolated from WT
and mutant mice. This indicates that, regardless of whether the
acetylcholine, bombesin, and CCK receptors normally couple to
G
q, G
11, or G
14, it is the
receptors, rather than the identity of the G protein
subunit, that
dictate differential sensitivity to RGS4. This unexpected finding
further indicates that signaling specificity is regulated not only by
receptor-catalyzed GTP loading to activate G proteins but
also by GTP hydrolysis to limit the duration of signaling. Thus,
RGS proteins play an important role in conferring signaling specificity.
subunits, as did RGS1,
GAIP, and RGS10 (11, 13-16). RGS1 (15, 16) and RGS16 (24) were shown
to interact with G
i1 and G
o subunits.
Overexpression in cells by transient or stable transfection of RGS4 and
RGS16 equally inhibited signaling evoked by receptors coupled to
Gi and Gq (25, 26, 46). Similarly, in
vitro RGS2 bound to and activated G
q but not to
G
i1 (30, 31), but when expressed in COS cells it
inhibited G
q- and
G
i1-dependent signaling (31). Our ability to
control the concentration of RGS proteins in the cytosol allowed us to
demonstrate selectivity for interaction of the RGS proteins with
several Gq-coupled receptors present in the same cell. As
discussed above, results in cells from the knockout mice indicate that
the receptors must play a central role in conferring selectivity to
RGS4 action. Thus, selectivity of RGS proteins appears to extend beyond
classes of G
subunits to the level of specific receptor types. With
increasing knowledge of the distinct properties of numerous RGS
proteins, it is clear that experimental systems similar to those used
in the present studies will be instrumental in evaluating regulatory
specificity of G protein signaling by RGS proteins.
subunits. This may be suggested from
the finding that the RGS proteins blocked the initiation of signaling. If RGS proteins interacted only with the activated G
, we would expect them to preferentially inhibit Ca2+ signaling after
the initial activation of PLC and Ca2+ release. This type
of inhibition was observed with recombinant protein containing only the
RGS domain of RGS4, which has Gq-GAP activity but is not
receptor-selective (47). The fact that full-length RGS4 and other RGS
proteins inhibit initial activation of PLC and Ca2+ release
(Figs. 1, 4, and 5 in permeable cells and Figs. 7-10 in intact cells)
suggests that RGS proteins may interact with the receptor-Gq complex. RGS4 interaction with a receptor-G
protein-effector complex was also inferred by the mode of activation
and inhibition of GIRK channels via Gi-coupled receptors in
Xenopus oocytes overexpressing RGS4 (25). The structure of
the RGS4-G
i1 complex showed that only the RGS core
domain was visible in the crystal (48). It is possible that amino acids
in RGS4 which flank the core domain directly interact with receptors
and/or G proteins complexed with receptors. In agreement with this
suggestion, we demonstrated that the N-terminal domain of RGS4 confers
receptor selectivity (47).
subunit is not
essential for receptor specificity. Rather, interaction of RGS proteins
with the receptor complex appears to confer specificity of action. Our
study also provides the first demonstration that RGS1, RGS2, RGS4, and
RGS16 are potent regulators of Ca2+ signaling in intact
cells, which points to the importance of the catalytic action of RGS
proteins in regulating G
-mediated signaling.
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ACKNOWLEDGEMENTS |
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We thank K. Blumer and S. Heximer (Washington University) for RGS1; D. Forsdyke (Queens University) for RGS2; M. Gosselin (Millennium) for RGS16; C. Gowan, D. Smith (University of Texas Southwestern), and S. Pease (Caltech) for technical assistance; P. Sternweis and J. Hepler for antisera; and E. Ross, S. Mumby, and our colleagues for comments on the manuscript.
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FOOTNOTES |
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* This work was funded by NIH Grants DK38938, DK46591, and DE12309 (to S. M.) and DK47890 (to T. M. W.) and by additional support from the Welch Foundation (Grant I-1382), the Leukemia Association of North Central Texas, and the American Heart Association (to T. M. W.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-2593; Fax: 214-648-5274; E-mail: smuall{at}mednet.swmed.edu.
The abbreviations used are:
PLC, phospholipase
C; RGS proteins, regulators of G protein signaling; GAP, GTPase-activating proteins; WT, wild type; CCK, cholecystokinin; SLO, streptolysin O; GTP
S, guanosine
5'-3-O-(thio)triphosphate; GDP
S, guanyl-5'-yl
thiophosphate; IP3, inositol 1,4,5-trisphosphate.
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REFERENCES |
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