From the Department of Pharmacology, Boyer Center for Molecular
Medicine, Yale University School of Medicine, New Haven, Connecticut
06536 and the Department of Central Nervous
System Disorders and §§ Department of Structural
Biology, Wyeth-Ayerst Research, CN8000, Princeton, New
Jersey 08543
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heterotrimeric G proteins function as molecular
relays, shuttling between cell surface receptors and intracellular
effectors that propagate a signal. G protein signaling is governed by
the rates of GTP binding (catalyzed by the receptor) and GTP
hydrolysis. RGS proteins (regulators of G protein signaling) were
identified as potent negative regulators of G protein signaling
pathways in simple eukaryotes and are now known to act as
GTPase-activating proteins (GAPs) for G protein -subunits in
vitro. It is not known, however, if G
GAP activity is
responsible for the regulatory action of RGS proteins in
vivo. We describe here a G
mutant in yeast
(gpa1sst) that phenotypically mimics the loss of
its cognate RGS protein (SST2). The
gpa1sst mutant is resistant to an activated allele
of SST2 in vivo and is unresponsive to RGS GAP activity
in vitro. The analogous mutation in a mammalian
Gq
is also resistant to RGS action in transfected cells.
These mutants demonstrate that RGS proteins act through G
and that
RGS-GAP activity is responsible for their desensitizing activity in
cells. The G
sst mutant will be useful for uncoupling
RGS-mediated regulation from other modes of signal regulation in whole
cells and animals.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A wide variety of cellular signals (hormones, neurotransmitters,
light, odors) act through a three component system composed of cell
surface receptors, heterotrimeric G proteins, and effector proteins
(1). The mating pheromones in yeast Saccharomyces cerevisiae
act through receptors (STE2, STE3 gene products),
a G protein heterotrimer (GPA1, STE4,
STE18), and a mitogen-activated protein kinase signaling
cascade that promotes cell division arrest and fusion (2). If mating is
unsuccessful, however, the cells become refractory to pheromone
stimulation and will eventually resume normal growth.
RGS1 proteins have recently
been identified as a fourth component of the G protein signaling
pathway (2, 3). The founding member of the RGS family, called
SST2, was identified in a genetic screen for negative
regulators of the pheromone response pathway in yeast (4). Loss of
function sst2 mutants render cells supersensitive to a
pheromone stimulus and unable to recover from pheromone-induced growth
arrest. Dominant gain-of-function alleles of SST2 have the
opposite effect, rendering cells insensitive to pheromone stimulation
(5). Further genetic and biochemical experiments revealed that Sst2
interacts directly with the G protein -subunit, Gpa1 (6).
Behavioral genetic analyses in C. elegans uncovered a
homologue of Sst2, called EGL-10 (7). egl-10 was shown to
negatively regulate goa-1, which encodes the G that
mediates serotonin-dependent egg laying behavior. Two
mammalian homologues, GAIP and RGS10, were identified by their
interaction with G
-subunits in a two-hybrid screen (8, 9). An
additional 15 mammalian members of the family were found by expression
cloning, degenerate polymerase chain reaction, low stringency
hybridization, and as expressed sequence tags (7-11). All of the RGS
proteins share a conserved "RGS core domain" of ~120 amino acids,
with >20% sequence identity across all species. Several RGS proteins
have also been shown to attenuate G protein signaling in cultured cells
(12-15) and to partially substitute for the loss of SST2
expression in yeast (10, 12, 15).
RGS proteins were later shown to function as GTPase-activating proteins
(GAPs) for G-subunits in vitro (9, 11,
16-22).2 These findings
suggest that RGS proteins negatively regulate signaling via their
physical association with G
-subunits. By enhancing the rate of G
GTP hydrolysis, RGS proteins would shorten the lifetime of the active G
protein species and arrest signaling.
Does RGS GAP activity account for the negative regulatory properties of
these proteins in vivo? Proving this model would require that RGS knockout mutants, and G mutants that disrupt RGS
interaction, exhibit the same phenotype. RGS mutations have been
obtained in yeast and nematodes, but not in mammals. Indeed,
constructing knockout mutants in mammals will be complicated by the
fact that there are so many closely related (and possibly redundant)
RGS isoforms. An RGS-insensitive G
mutant has not been reported in any system.
Here, we report the identification and characterization of a yeast G
mutation that specifically disrupts Sst2 regulation in vivo
and in vitro. An analogous mutation in Gq
is
similarly insensitive to RGS action in cultured cells. An RGS-uncoupled G
mutation proves that G
is the primary target of RGS in cells. G
mutants of this type will be extremely useful for determining the
overall contribution of RGS proteins to signal regulation in
vivo.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains, Plasmids, and Antibodies--
Established methods were
used for the growth and genetic manipulation of yeast and bacteria
(23). Escherichia coli strain DH10B was used for the
maintenance of plasmids; strain BL21 (DE3) was used for expression of
recombinant Gpa1 and RGS. S. cerevisiae strains used in this
study were: YHD436 (MATa ura3-52
lys2-801am ade2-101oc
trp1-63 his3-
200 leu2-
1 bar1::hisG mf
1
mf
2::hisG FUS1-LacZ-URA3) for mutagenesis and
screening, YGS5 (24) or YRGS5 (YGS5, sst2-
2) (24) for
halo assays, and BJ2168 (25) for purification of Sst2-GST. Yeast
expression plasmids were pRS315, pRS316, pAD4M, pRS315-GPA1,
pAD4M-GPA1, pRS315-gpa1sst, pAD4M-gpa1sst (24),
pRS315-SST2, pRS315-SST2-1 (5), pAD4M-GST (24), 5HT2c, and RGS7 (38).
pcDNAamp-Gqsst was constucted by
oligonucleotide-directed mutagenesis in pcDNAamp-GqEE2 (26)
(Altered Sites, Promega). pAD4M-SST2-GST was provided by K. Blumer,
Washington University. Antibodies to Gpa1 (27), EE (Babco), and GST (J. Steitz, Yale University) are described elsewhere. Molecular modeling of
the Gi1
-GDP-AlF4
/RGS4
complex (23) (PDB accession number 1AGR) was performed using SYBYL
(Tripos, St. Louis, MO).
Mutagenesis Screening and Pheromone Assays--
YHD436 cells
were mutagenized with ethyl methanesulfonate (Sigma) as described (28)
and assayed for -factor supersensitivity using a reporter gene
(
-galactosidase) assay on nitrocellulose colony lifts (29). A
genomic DNA library in YCp50 was used for complementation cloning (ATCC
77162). The halo assay was performed as described (29).
Sst2-GST/Gpa1 Binding, GTPase Assays, and Mammalian Cell Culture
Assays--
Sst2-GST binding experiments were performed as described
(6) with the following modifications: 50 mM Hepes (pH 7.5)
and 0.1% Tween were used instead of 40 mM triethanolamine
and 1% Triton X-100. Washes were performed with binding buffer at
various salt concentrations instead of in phosphate-buffered saline.
Binding and wash buffers contained 5 mM MgCl2
and either 10 µM GDP or 10 µM GDP, 30 µM AlCl3, 10 mM NaF. Purification
of recombinant G and RGS proteins (>95% homogeneity), as well as
guanine nucleotide binding and hydrolysis assays, were performed as
described previously (19).2 Mammalian cell culture,
transfections, and second messenger assays are described elsewhere
(38). Data were analyzed using analysis of variance for a randomized
block design, with log transformation, followed by pairwise comparisons
employing the least significant difference method.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the course of a large scale genetic screen designed to identify
new desensitization components in yeast, we isolated a novel allele of
GPA1, designated gpa1sst (for
supersensitive allele of gpa1). Sequencing revealed a single missense mutation resulting in a Gly-to-Ser substitution at position 302. This glycine is conserved among G-subunit family members and is
located in the first of three switch regions known to undergo a
conformational change upon GTP hydrolysis (30-36).
Since the gpa1sst mutant mimics the loss of
SST2, we investigated its ability to be regulated by
SST2. First we compared the pheromone response in cells
expressing GPA1 or gpa1sst, using the
growth inhibition ("halo") assay (29). In
SST2+ cells, the pheromone response through
gpa1sst was potentiated compared with
GPA1. In the absence of SST2, however, the wild
type and mutant forms of Gpa1 responded equally (Fig. 1A). In cells expressing
SST2-1 (a dominant mutant that promotes pheromone
desensitization), there was a striking difference between wild type and
mutant GPA1 (Fig. 1B). Cells expressing
GPA1 and SST2-1 exhibited a greatly attenuated
response to pheromone, resulting in a "filled in" halo. However,
cells containing SST2-1 and gpa1sst
responded no differently than cells containing
gpa1sst alone. Clearly, the Gly-to-Ser mutation
blocks the negative regulatory effect of Sst2 in vivo. Since
there is no functional difference between Gpa1 and Gpa1sst
in the absence of Sst2 expression, we conclude that Gpa1sst
is fully competent to transmit a pheromone signal and interacts normally with G and the receptor.
|
Sst2 is thought to act by binding to Gpa1 and stimulating its GTPase
activity. Therefore, genetic uncoupling of SST2 and
gpa1sst should accompany a physical and/or
functional uncoupling of the two proteins. To test this, we purified
each of the proteins and compared the ability of Gpa1 and
Gpa1sst to bind and hydrolyze GTP. We first measured the
rate of [35S]GTPS binding and steady state
[
-32P]GTP hydrolysis and found no difference between
the wild type and mutant forms of Gpa1 (Fig.
2, A and B). We
then compared the ability of each protein to catalyze the rate-limiting
hydrolytic step of the reaction, using a single turnover assay in the
absence or presence of a purified RGS protein (GAIP, Fig.
2C). GAIP is functionally equivalent to Sst2, but is more
stabile and can be purified in much larger quantities.2 In
the absence of RGS, the initial kcat of
hydrolysis was ~0.006 min
1 for both wild type and
mutant. With the addition of RGS, however, the rate of hydrolysis was
greatly accelerated (at least 20-fold) for Gpa1 but not at all for
Gpa1sst. A more accurate determination of the
RGS-stimulated GTPase rate could not be made, as the reaction was
essentially complete at the first time point. Thus Gpa1sst
can bind and hydrolyze GTP normally, but is completely unresponsive to
RGS GAP activity. These results are consistent with the in vivo experiments where, in the absence of SST2
expression, Gpa1sst can signal as well as the wild
type.
|
Despite the complete loss of GAP activity in vitro, Gpa1sst does not equal the loss of SST2 in vivo. There are at least two possible explanations for this difference. First, Sst2 could regulate proteins other than Gpa1. Such interactions could involve the N-terminal 300 amino acid region of Sst2, a domain that is not found in any other RGS protein. A less likely alternative is that Gpa1sst is weakly activated by RGS in vivo but not in vitro.
The x-ray crystal structure has recently been solved for RGS4 complexed
with Gi1 and GDP-AlF4
,
a transition state mimic (37). The structure suggests a mechanism in
which RGS promotes hydrolysis by stabilizing the transition state
conformation of G
. A key prediction of this model is that loss of
GAP activity should accompany a loss of RGS binding. Accordingly, we
compared the ability of mutant and wild type Gpa1 to bind Sst2 in
vitro. Yeast lysates containing an Sst2-GST fusion protein were
adsorbed onto glutathione-Sepharose and mixed with similarly prepared
lysates containing Gpa1 or Gpa1sst. The resin was washed at
various NaCl concentrations, in the presence of either GDP or
GDP-AlF4
(Fig.
3). At 50 mM NaCl, both the
wild type and mutant were retained by Sst2-GST, but binding was not
AlF4
-dependent and is
therefore likely to be nonspecific or nonfunctional. At higher salt
concentrations (150-250 mM) Gpa1 was selectively retained
when GDP-AlF4
was present, but
Gpa1sst did not bind at all. At very high concentrations
(350 mM), neither protein was retained. Thus the inability
of the RGS protein to act as a GAP for Gpa1sst appears to
be due to a weakened protein-protein interaction.
|
Since we have shown that Gpa1sst can specifically block RGS
action both in vivo and in vitro, we examined if
a similar mutation in a mammalian G would also block RGS action in
cultured cells. We created a Gly188
Ser mutation in
Gq
(Gq
sst) and examined its
sensitivity to RGS7 in cells co-transfected with the 5HT2c
receptor. This particular combination of signaling proteins was chosen
because they have overlapping expression patterns in the brain and are
known to interact in cells (38). In cells expressing wild type
Gq
, 5-HT stimulation resulted in a typical calcium
response (using Fura-2), which was attenuated ~40% by RGS7
expression (Fig. 4A). In cells
expressing Gq
sst, however, the response was
completely refractory to RGS7 (Fig. 4B). To confirm the
results obtained by calcium release, measurements of agonist-induced
[3H]inositol trisphosphate (IP3) production
were performed on the same cells. Co-expression of RGS7 with wild type
Gq
reduced maximal IP3 generation by
~30%, while co-expression with Gq
sst had
no effect (Fig. 4C). Thus, like Gpa1sst,
Gq
sst effectively couples to receptor and
G
, yet is resistant to the effects of RGS regulation. Since
signaling in this case is mediated by G
, rather than G
(as it
is in yeast), we can also conclude that effector coupling is unaltered
by the Gly188
Ser mutation.
|
To determine how the Gly to Ser substitution can disrupt G-RGS
interactions in such a selective manner, we employed molecular modeling
using the coordinates of the RGS4-Gi1
crystal structure (37). The conserved Gly in Gi1
(Gly183) is
located directly opposite Glu83 in RGS4 at the binding
interface (Fig. 5). Buried surface area in this region accounts for 120 Å2, or 22%, of the G
binding site. Substituting Gly with Ser would introduce a hydroxyl
group less than 1 Å from the backbone carbonyl of Glu83,
an energetically unfavorable position both electrostatically and
sterically (compare Fig. 5, B versus C). When mapped onto the crystal structure of Gi1
-
or
Gt
-
, however, the same substitution shows no
crowding at the
binding interface, no interference with guanine
nucleotide binding, and no effect on the conformational changes that
occur during GTP hydrolysis. In a direct test of this model, the
corresponding mutation in Gi1
was shown to cause a
>100-fold reduction in RGS4 binding (by flow cytometry measurements)
and >1000-fold reduction in GAP
activity.3
|
RGS proteins were first identified through genetic studies carried out
in yeast (4, 5). G proteins were identified as potential targets of RGS
regulation, through enzymological studies carried out in mammals (9,
19, 21). With this first description of a mutation that selectively
blocks G interaction with RGS, their cellular target and mechanism
of action are now firmly established. The next major challenge will be
to disrupt G
-RGS interactions in animals. Knockout mutations of the
>20 RGS isoforms may be impractical, so G
sst mutants
could be used instead to determine which signaling pathways are subject
to RGS regulation and how desensitization of G proteins compares with
desensitization of receptors. Thus we believe that G
sst
mutants will prove useful for determining how RGS proteins regulate signaling, not just in cultured cells but also in whole animals.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Takatoshi Karasawa for plasmid
construction, Deborah Nusskern for technical assistance, Charlie Boone
for discussion, and Steven Sprang for RGS4-Gi1
coordinates.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant GM 55316 from the National Institutes of Health and by a grant from the American Cyanamid Company (to H. G. D.).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.
§ Supported by Grant 95009550 from the American Heart Association.
¶ National Institutes of Health Predoctoral Trainee T 32CA 09085.
American Heart Association Postdoctoral Fellow
CT-96-FW-32.
** National Science Foundation Predoctoral Fellow 45037.
¶¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06536-0812. Tel.: 203-737-2203; Fax: 203-737-2290; E-mail: henrik.dohlman{at}yale.edu.
1
The abbreviations used are: RGS, regulator of G
protein signaling; GAP, GTPase-activating protein; GAIP, G protein
-interacting protein; GST, glutathione S-transferase;
5-HT, serotonin; IP3 inositol trisphosphate; GTP
S,
guanosine 5'-3-O-(thio)triphosphate.
2 Apanovitch, D. M., Slep, K. C., Sigler, P. B., and Dohlman, H. G. (1998) Biochemistry, in press.
3 K.-L. Lan, N. A. Sarvazyan, R. Taussig, R. G. Mackenzie, P. R. DiBello, H. G. Dohlman, and R. R. Neubig, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|