(Received for publication, December 23, 1996, and in revised form, February 21, 1997)
From the Department of Cellular and Molecular Pharmacology, Programs in Cell Biology and Biomedical Sciences, and the Cardiovascular Research Institute, University of California, San Francisco, California 94143-0450
Recombinant regulators of G protein-signaling
(RGS) proteins stimulate hydrolysis of GTP by subunits of the
Gi family but have not been reported to regulate
other G protein
subunits. Expression of recombinant RGS proteins in
cultured cells inhibits Gi-mediated hormonal signals
probably by acting as GTPase-activating proteins for G
i
subunits. To ask whether an RGS protein can also regulate cellular
responses mediated by G proteins in the Gq/11 family, we
compared activation of mitogen-activated protein kinase (MAPK) by a
Gq/11-coupled receptor, the bombesin receptor (BR), and a
Gi-coupled receptor, the D2 dopamine receptor,
transiently co-expressed with or without recombinant RGS4 in COS-7
cells. Pertussis toxin, which uncouples Gi from receptors,
blocked MAPK activation by the D2 dopamine receptor but not
by the BR. Co-expression of RGS4, however, inhibited activation of MAPK
by both receptors causing a rightward shift of the concentration-effect
curve for both receptor agonists. RGS4 also inhibited BR-stimulated
synthesis of inositol phosphates by an effector target of
Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol
phosphate synthesis activated by addition of
AlF4
to
cells overexpressing recombinant
q, probably by binding
to
q·GDP·AlF4
.
These results demonstrate that RGS4 can regulate
Gq/11-mediated cellular signals by competing for effector
binding as well as by acting as a GTPase-activating protein.
Heterotrimeric G proteins transduce extracellular signals detected
by transmembrane receptors into appropriate cellular responses (1, 2).
The intensity and duration of these responses depend on the relative
rates of biochemical reactions that turn G proteins on and off. The G
protein switch turns on when receptors promote replacement of GTP for
GDP bound by subunits of
trimers, leading to dissociation
of active G
·GTP from the
dimer and consequent regulation of
downstream effectors. A GTPase activity intrinsic to
subunits turns
off signals by converting
·GTP to inactive G
·GDP, which then
binds to and inactivates
. For pure G
subunits in
vitro the turnoff reaction is slow,
4 min
1 (2). In
contrast, many G protein-mediated physiological responses must turn off
much more rapidly, in fractions of a second.
Two classes of GTPase-activating protein
(GAP)1 have been reported to accelerate
deactivation of trimeric G proteins. One class includes G protein
effectors, such as phospholipase C (PLC) and the cGMP phosphodiesterase
subunit, which stimulate GTP hydrolysis by
q and
t, respectively (3, 4). Recent investigations have
discovered and characterized a second class of G
-GAPs, the RGS
(regulators of G protein signaling) proteins. Pure recombinant RGS
proteins display GAP activities for certain G protein
subunits (5-9). RGS proteins of mammals (8-12), yeast (13, 14), and Caenorhabditis elegans (11) share a conserved RGS domain and apparently share similar mechanisms of action. Indeed, a mammalian RGS
can partially complement yeast mutations that inactivate Sst2p, the RGS
of Saccharomyces cerevisiae (12).
Mammalian RGS proteins thus far examined appear to act selectively as
GAPs for G proteins in the
i family (5-8), including
i,
o,
z, and most recently
t (9). Transient expression of RGS4 in HEK293 cells
inhibits Gi-mediated activation of MAP kinase (MAPK) in
response to stimulation of the interleukin-8 receptor (12). In the
yeast two-hybrid system, in vitro binding, and
co-immunoprecipitation assays, RGS4 interacts with
i
family proteins but not with
s or
12
(5-8, 10).
Recent experiments indicate that RGS4 can interact with
q/11 proteins albeit less efficiently than with
i. A high concentration of
q·GDP bound
to AlF4
can inhibit the GAP activity
of RGS4 for
o·GTP, presumably because
q·GDP·AlF4
competes
against
o·GTP for binding the RGS protein (6).
Moreover, RGS4 can stimulate the GTPase activity of
q in
reconstituted vesicles (15). It is not known, however, whether RGS
proteins can serve in intact cells as
q-GAPs and
inhibitors of Gq-mediated cellular signals. Here we use
expression of recombinant RGS4 in COS-7 cells to show that RGS4 can
inhibit cellular signals mediated by Gq/11.
FLAG-tagged
human RGS4 cDNA was a generous gift from John H. Kehrl at the
Laboratory of Immunoregulation, NIAID, National Institutes of Health,
Bethesda, MD. cDNA constructs for the bombesin receptor (BR),
D2 dopamine receptor (D2R), and the
2-adrenoreceptor were as described (16). Chinese hamster
cDNA encoding an HA-tagged p44 MAPK was a gift from J. Pouysségur, Nice, France. pcDNAI and pCR3 were from
Invitrogen, San Diego.
COS-7 cells were maintained in Dulbecco's modified Eagle's H21 medium
with 10% calf serum. DNA was transfected with adenovirus and
DEAE-dextran as described (17). Transfection efficiencies were
determined by co-transfection of the plasmid pON249 encoding -galactosidase and assayed as described (17). Expression was consistently detected in over 90% of the cells. Expression of FLAG-tagged RGS4 and HA-tagged MAPK in total cell lysates was detected
by immunoblotting with monoclonal antibodies M2 (Eastman Kodak Co.) and 12CA5, respectively.
HA-MAPK activity was
assayed by a procedure modified from that described by Faure et
al. (18). COS-7 cells were transfected in 6-well plates at
0.8 × 106 cells/well and placed in serum-free medium
containing 0.1% bovine serum albumin after incubating for 24 h in
medium containing 10% calf serum. MAPK activity was measured 48 h
after transfection. After PTX pretreatment (100 ng/ml for 4 h),
where indicated, cells were stimulated for 10 min with appropriate
agonists. HA-MAPK immunoprecipitated from cell lysates was incubated
with bovine myelin basic protein (MBP) (Sigma) as a substrate in the
presence of [-32P]ATP (DuPont NEN).
32P-Phosphorylated MBP was quantitated with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after resolution on
a 14% polyacrylamide gel.
Total cellular inositol phosphates (IP) was measured according to Conklin et al. (19). 24 h after transfection cells were replated in a 24-well plate and labeled for 24 h with myo-[3H]inositol (4 µCi/ml, Amersham Corp.). After washing with medium containing 5 mM LiCl for 10 min, cells were incubated for 45 min at 37 °C with the appropriate agonist in the same medium containing LiCl. IP and total inositol fractions were resolved on a Dowex AG 1-X8 formate column (Bio-Rad) (12), and cellular IP content was expressed as the ratio of IP radioactivity to the sum of IP plus inositol radioactivity.
cAMP AssayIntracellular [3H]cAMP accumulation was estimated by determining the ratio of cAMP to the cellular pool of ATP plus ADP as described (16). 24 h after transfection each 60-mm dish of 1 × 106 cells was split into 9 wells in a 24-well plate and incubated in medium containing [3H]adenine (2 µCi/ml, Amersham). 18-24 h later, cells were washed once with 1 ml of assay medium and incubated in 1 ml of assay medium containing 1 mM 1-methyl-3-isobutylxanthine and agonist (isoproterenol) for 30 min as indicated.
To assess effects of an RGS protein on cellular
responses mediated by G proteins in the Gq/11 family, we
compared MAPK activation by a Gq/11-coupled receptor, BR,
and a Gi-coupled receptor, D2R, that
co-expressed with or without recombinant RGS4 in COS-7 cells (Figs.
1 and 2). Expression of RGS4 blocked MAPK
activation by the Gq/11-coupled receptor agonist, bombesin
(1 nM); PTX, which specifically blocks signaling by
receptors that activate Gi proteins, did not inhibit the
effect of bombesin (Fig. 1A). These results suggest that
RGS4 inhibited bombesin signaling to MAPK by inhibiting the action of a
G protein other than Gi, probably Gq/11. This inference was supported by additional experiments described below. Confirming a previous report (12) that recombinant RGS4 can inhibit
Gi-mediated signals in an intact cultured cell, RGS4
markedly inhibited Gi-dependent MAPK activation
by the D2R agonist, quinpirole (10 nM; Fig.
1B); PTX also blocked the D2R effect on MAPK
(Fig. 1B). RGS4 did not alter the expression of HA-MAPK in
these experiments (data not shown).
To further assess the effectiveness of RGS4 in blocking cellular
responses mediated by Gq/11 and Gi, we measured
MAPK activation by a range of concentrations of both agonists (Fig. 2).
In both cases, a higher concentration of each agonist was required to produce equivalent MAPK activation in cells overexpressing RGS4, that
is expression of RGS4 shifted the agonist concentration curve to the
right. In RGS4-expressing cells, the apparent EC50 of
bombesin was increased ~5-fold (Fig. 2A). The quinpirole
concentration-effect curve was shifted to the right ~10-fold (Fig.
2B) indicating a relatively greater effectiveness of RGS4
for inhibiting the Gi-mediated effect in comparison with
that mediated by Gq/11. The apparent difference in potency
could reflect different mechanisms by which RGS4 inhibits the two
effects, but it is also consistent with a simpler interpretation that
the RGS protein catalyzes GTP hydrolysis less efficiently with
q/11 than with
i proteins.
The RGS-induced rightward shift of signaling concentration-effect
curves (Fig. 2) is predictable from the GAP mechanism of RGS action.
When a GAP increases the rate of GTP hydrolysis, equivalent steady-state concentrations of G·GTP can only be achieved by an
increased rate of receptor-catalyzed GTP-for-GDP exchange and thus by
higher concentrations of agonist-occupied receptor. The extent of an
RGS-induced rightward shift would be limited by the kd of the agonist ligand for the relevant receptor.
Although a GAP-induced shift in concentration-response curves has not
been previously documented, it would be an attractive way to fine tune responsiveness of cells to extracellular stimuli. In phospholipid vesicles reconstituted with a M1-muscarinic acetylcholine
receptor and Gq, the EC50 of carbachol for
stimulating GTP hydrolysis was increased by addition of purified
PLC
1, an
q-GAP; in this case, addition of the GAP
also markedly increased the maximal rate of GTP hydrolysis (3, 20).
To determine whether similar concentrations of cellular RGS4 are
required to inhibit Gi- and
Gq-dependent hormonal signals, we transfected
cells with graded amounts of RGS4 plasmid (Fig. 3),
which produced graded cellular amounts of RGS4 protein (Fig. 3C). RGS4 inhibited Gi- and
Gq-mediated elevation of MAPK activity with similar
dose-effect curves over a 16-fold range of transfected DNA (Fig. 3,
A and B). Although the endogenous amounts of
cellular RGS proteins are unknown, this result argues that similar
amounts of RGS4 protein are required to produce both inhibitory
effects. It is unlikely that all RGS proteins exhibit quantitatively
similar abilities to inhibit Gi- and
Gq-dependent hormonal signals. Indeed, another
member of the RGS protein family, GAIP, inhibited
Gi-mediated activation of MAPK much more effectively than
that mediated by Gq/11.2
RGS4 Reduces Accumulation of Inositol Phosphates Stimulated by Bombesin
If RGS4 inhibits BR stimulation of MAPK activity by
inactivating Gq/11, the RGS protein should also reduce
BR-stimulated synthesis of IP by PLC, the principal effector of
Gq/11. Indeed, expression of RGS4 reduced bombesin-induced
IP accumulation by about 50% (Fig. 4A). The
inhibitory effect of RGS4 was probably exerted on Gq/11
rather than on G
i because PTX failed to inhibit
BR-induced IP accumulation (not shown). The BR is likely to stimulate
IP accumulation via the
subunit of Gq/11 rather than
via its
subunit because PLC
1 and PLC
3, the G
protein-responsive PLC isozymes of COS cells, are sensitive to
q/11 stimulation but relatively insensitive to
stimulation by G
; COS cells lack PLC
2, the PLC isozyme that is
most sensitive to
(21).
RGS4 reduced maximal stimulation of IP accumulation by BR stimulation but did not alter the EC50 for bombesin (Fig. 4A). In contrast, RGS4 expression did not affect maximal activation of MAPK by bombesin but did cause a rightward shift of the bombesin concentration-effect curve (Fig. 2A). How could the relations between agonist concentration and response be different, if as seems likely, both BR responses are mediated by Gq/11 and stimulation of PLC? Although we do not know the reason for this discrepancy, the two assays were performed under different conditions and reflect activation of Gq/11 and PLC in different ways. BR-mediated elevation of MAPK activity measured 10 min after addition of agonist probably results from some (undefined) combination of signals triggered by diacylglycerol activating protein kinase C isozymes and by inositol trisphosphate (InsP3) elevating cytoplasmic Ca2+. The IP measurements, in contrast, assessed accumulation at 45 min of total radioactive inositol phosphates in cells labeled with radioactive inositol and exposed to LiCl, which inhibits IP degradation. InsP3 constitutes only a fraction of the total IP pool, and LiCl may not alter concentrations of InsP3 and total inositol phosphates in the same way. Consequently, the extent and time course of bombesin-induced changes in InsP3 under conditions used in the MAPK experiments need not parallel bombesin-induced changes in total IP accumulation.
As expected from the reported (5-8) inability of RGS4 to stimulate GTP
hydrolysis by the subunit of Gs, the RGS protein had no
effect on cAMP production stimulated by the
2-adrenoreceptor agonist, isoproterenol (Fig.
4B) or on the cAMP-mediated activation of MAPK by
isoproterenol (not shown).
To support the idea that RGS4 inhibits PLC
stimulation by an effect on q/11, we took advantage of a
recently discovered property of RGS proteins in vitro, their
ability to bind G
proteins whose nucleotide binding pockets contain
GDP complexed with AlF4
(6, 8, 9).
This property is thought to reflect enhanced affinity of RGS proteins
for a G
conformation that mimics the transition state of GTP
hydrolysis; it was useful for our purposes because the conformation of
G
·GDP·AlF4
also allows it to
regulate activity of the appropriate effector. Although RGS4 reportedly
(6) binds
G
q·GDP·AlF4
less
tightly than
G
i·GDP·AlF4
, we
imagined that an RGS4-
q interaction would inhibit
stimulation of IP accumulation in cells transfected with recombinant
G
q and exposed to
AlF4
.
This turned out to be the case (Fig. 5).
AlF4 elevated cellular IP accumulation
in cells expressing recombinant G
q but had no effect in
untransfected cells; by itself, recombinant G
q produced a smaller but reproducible elevation of cellular IP content. These results suggest that increased abundance of G
q caused a
modest elevation in the cellular concentration of its GTP-bound form, and that addition of AlF4
activated
PLC still further by binding to the GDP-bound form of transfected
G
q, rendering it capable of activating the effector enzyme. Co-expression of RGS4 with G
q substantially
inhibited IP accumulation in response to
AlF4
(Fig. 5). RGS4 presumably
inhibited effector stimulation in this case not by accelerating GTP
hydrolysis but by binding to and sequestering
G
q·GDP·AlF4
. RGS4
also decreased the elevation of MAPK activity seen in untreated cells
transfected with G
q (not shown), an effect that probably reflects acceleration of GTP hydrolysis by G
q.
In summary, we present two new sets of observations. While RGS proteins
are known to inhibit signals mediated by Gi, we show for
the first time that an RGS protein can interact with
Gq/11 and inhibit signals transduced by
G
q/11 in intact cells. RGS4 probably inhibits bombesin
responses by acting as a GAP, that is, by stimulating the intrinsic
GTPase activity of G
q/11. Our experiments also raise the
possibility that RGS4 inhibits bombesin responses by sequestering the
GTP-bound active conformation of G
q/11 (that is, by the
mechanism that probably inhibits the
AlF4
response), in addition to
stimulating GTP hydrolysis.
Second, we found that RGS4 induces rightward shifts in
concentration-effect curves for agonists acting on receptors coupled to
either Gi or Gq/11. It is likely that other RGS
proteins modulate hormonal signals mediated by Gi and
Gq/11 in much the same way. For each response, the extent
of the rightward shift will depend on the local concentration of RGS
protein and its relative affinity for the G protein involved. Thus
different complements of RGS proteins could allow two cells to mount
quantitatively different responses to the same concentration of a
physiological agonist even when both cells use the same receptors and G
proteins. If the relevant receptor couples to two distinct G proteins
(for instance, to Gq and Gs or to
Gi and Gq), differing cellular complements of
RGS proteins with distinct G selectivities could even produce qualitatively different responses of two cells to the same agonist.
We thank Tom Baranski, Simon Fishburn, Pablo Garcia, Paul Herzmark, Taroh Iiri, Janine Morales, Enid Neptune, and Soren Sheikh for valuable discussions.