From the Department of Pharmacology, Yale University
School of Medicine, New Haven, Connecticut 06520-8066 and
§ Department of Molecular and Cell Biology, University
of California, Berkeley, California 94720-3202
A ubiquitously employed mechanism for signal
transduction involves ligand binding to a cell surface receptor coupled
to a heterotrimeric guanine nucleotide-binding protein (G protein). Receptor activation stimulates nucleotide exchange and dissociation of
the G protein, releasing the G How are the strength and duration of signaling adjusted to achieve an
appropriate response? Attention in this regard has been devoted
primarily to receptors, where phosphorylation by protein kinases (9)
and receptor-binding proteins, like arrestins (10, 11), contribute to
signal desensitization. However, additional proteins participate in
signal attenuation at other levels, including phosducins (which act on
G The Cycle of G Protein Activation and Inactivation Activation of a G protein is initiated by agonist binding to a
receptor, eliciting conformational change that is transmitted to the G
protein, causing the G
Inactivation requires hydrolysis of the GTP bound to G Establishing a Paradigm: Identification of Yeast SST2 In yeast, signaling processes can be dissected genetically.
Because the cells can grow as haploids, recessive mutations can be
isolated and characterized. Haploid cells undergo mating in response to
a pheromone signal by transcribing new genes, by changing morphology,
and by transiently arresting in the G1 phase of the cell
cycle. Genes necessary for pheromone response were identified through
isolation of mutations that prevent mating, so-called sterile
(ste) mutations (20). If normal haploids fail to mate, they
become refractory to pheromone and resume cell division. Thus, yeast
cells display adaptation and recovery, as observed in mammalian
desensitization. In 1982, to identify gene products involved in this
down-regulation, Chan and Otte (21, 22) screened for mutant haploids
hypersensitive to pheromone-induced cell cycle arrest. These
supersensitive (sst) mutations fell into two classes. One
(sst1) was allelic to a gene (BAR1) encoding a
secreted protease responsible for inactivation of pheromone
( In addition to responding to doses of pheromone 2 orders of magnitude
lower than normal cells (23), sst2 mutants also fail to
emerge from pheromone-imposed cell cycle arrest (22). The SST2
gene was isolated (24) by selecting for genomic DNA clones that
allowed sst2 mutants to recover from pheromone-induced
growth arrest. At the time, however, knowledge about G
protein-dependent signaling was in its infancy, sequence data bases
were rudimentary, and relevance of signaling events in a unicellular
eukaryote to those in humans was not widely appreciated. By the late
1980s, however, it became clear that signaling pathways in yeast and mammalian cells bear considerable similarity (20). For example, the
yeast pheromone receptors have a topology resembling other G
protein-coupled receptors (2, 3), and the yeast G Because high-copy vectors were used to isolate
SST2, other genes that could rescue, when overexpressed, the
sst2 mutation were also obtained. One such dosage suppressor
was the KSS1 gene (for "kinase suppressor of
sst2") (27), the first MAPK cloned from any organism, thus
indicating a connection between G protein signaling and MAPKs. Another
dosage suppressor was the GPA1 gene (28, 29), which encodes
the The fact that overproduction of GPA1 (G Our recent biochemical and cytological studies (34) demonstrate that
SST2 and G RGS Proteins in Other Model Organisms The closest counterpart to SST2 in another organism is the
flbA gene product of the filamentous fungus, A. nidulans (36). Under conditions that should cause sporulation,
flbA mutants proliferate, remain undifferentiated, and
eventually lyse, a phenotype called "fluffy autolysis." Conversely,
overexpression of normal flbA permits sporulation under
conditions that would otherwise prevent it. A dominant fluffy autolysis
mutation is a substitution (G42R) in a G Another SST2-related gene was identified in the nematode,
C. elegans, during a genetic screen for mutants that alter
certain neuronal activities (39). When placed on a lawn of bacterial prey, C. elegans adjusts several of its behaviors, including
frequency of egg laying. Egg laying is controlled by serotonergic motor neurons that innervate the vulval and uterine muscle cells and is
suited to genetic analysis because the number of laid and unlaid eggs
(which are clearly visible inside the adult) can be readily compared. A
mutation (egl-10) that decreased the frequency of egg laying
was identified and the corresponding gene cloned (39). The C-terminal
portion of the 555-residue EGL-10 product bore similarity to
the C-terminal segment of the 698-residue SST2 protein (Fig. 2). While
egl-10 mutants rarely lay eggs, overexpression of normal
EGL-10 causes animals to lay eggs more frequently (39). These phenotypes suggest that EGL-10 is required for
serotonin-stimulated egg laying. In other animals, serotonin acts
through G protein-coupled receptors (2, 3). Indeed, the
goa-1 mutation, which resides in a gene homologous to
mammalian Go
Multiple homologs of SST2 and EGL-10 are present in higher
eukaryotes. Using the yeast two-hybrid system, a human protein, GAIP,
that interacts with human Gi3 BL34/1R20 cDNA was identified because the corresponding mRNA is
elevated in chronic lymphocytic leukemia (43). Expression is specific
to B lymphocytes and induced by mitogenic stimuli (44). The 196-residue
BL34/1R20 product has been renamed RGS1, in light of its presumed
function. Similarly, GOS8 cDNA, encoding a 211-residue protein
(RGS2), was isolated because expression of its corresponding mRNA
in human blood lymphocytes is induced by concanavalin A (a T-cell
mitogen) in combination with cycloheximide (45). However, GOS8 mRNA
is induced by cycloheximide alone (46). Yet another homolog, RGS3, was
identified by screening a B-cell cDNA library with an
oligonucleotide corresponding to sequences conserved between RGS1 and
RGS2 (47). RGS3 (519 residues) is much longer than RGS1 or RGS2.
Because of the evidence that yeast SST2 and nematode EGL-10 regulate G
protein signaling, it was presumed that mammalian homologs would act
analogously. Indeed, RGS4 was identified by screening for rat brain
cDNAs that, when expressed in a yeast sst2 RGS1, RGS2, RGS3, and RGS4 regulate G protein signaling in mammalian
cells. Elevation of RGS1 by transient transfection attenuated MAPK
activation in response to PAF and diminished the Ca2+ flux
provoked by either PAF or lysophosphatidic acid; similarly, expression
of each of the four RGS proteins attenuated MAPK activation by
interleukin 8 (47). Likewise, RGS4 expression attenuates MAPK
activation in response to agonist stimulation of M2 muscarinic acetylcholine receptors.2 In contrast, RGS3 had no effect
on MAPK stimulation by two post-G protein activators, phorbol ester and
activated Raf-1 kinase (47).
Searches of expressed sequence tag (EST) data bases and screening by
polymerase chain reaction amplification (39, 47, 48) have revealed more
RGS homologs in mammals and in the C. elegans genome,
including one with two tandem RGS domains (Fig. 2).
Evidence for Bifunctional RGS Proteins Some RGS proteins (including SST2, FlbA, and RGS3) possess long
N-terminal extensions. This number may increase as RGS clones identified only as EST fragments are fully characterized. In this regard, it is striking that the identity between RGS7 and EGL-10 (39)
is 75% across their N-termini versus 46% within their RGS cores, suggesting that the N-terminal segment is needed for function. Yeast SST2 provides a precedent for such a situation.
Whereas the C-terminal 230 residues of SST2 constitute its RGS domain,
the N-terminal 300 residues bear weak similarity to the catalytic
domain of mammalian Ras-GAP (34). Both the GAP-like and RGS segments
are required, since truncations at either the N- or C-terminal ends
completely eliminate biological activity (34). A dominant
gain-of-function allele, SST2(P20L), maps within the
GAP-like region, supporting its functional importance (33). The
GAP-like segment could stimulate the GTPase activity of G Another potentially multifunctional RGS is the mouse fused
gene product (49), which contains an N-terminal extension homologous to
proteins that bind to phosphoprotein phosphatase,
PP2A.3 Establishing the functions of other
domains is as important as sorting out specificity determinants in the
core RGS domain.
RGS Proteins Act as GAPs for G Effects of purified GAIP and RGS4 on purified Gi1 Selective binding to the transition-state conformation of G RGS Proteins and Ras-GAP: Similarities and Differences Crystal structures of Ras (55, 56) and G If G Why So Many RGS Family Members? The possibility that an RGS regulates one (or a small subset) of
G
subunit in its GTP-bound state from
the G
complex. The released subunits can stimulate a variety of
target (effector) enzymes (1), thereby eliciting biochemical responses
and changes in cellular physiology. Hundreds of G protein-coupled receptors have been identified (2, 3). These receptors share a common
architecture containing seven membrane-spanning segments (4, 5). G
proteins also comprise a superfamily that includes at least 17 distinct
G
(6), 5 G
, and 6 G
isoforms (1), allowing many combinatorial
possibilities. Three-dimensional structures of several G
subunits
and two different G
heterotrimers (7, 8) have been determined,
providing insights about how these molecular "switches"
operate.
) (12) and recoverins (13, 14). Here we focus on discovery of
another superfamily of evolutionarily conserved proteins, dubbed RGS
proteins, for "regulators of G protein signaling." RGS proteins act
as negative regulators of G protein-dependent signaling, at least in
part, because they stimulate hydrolysis of the GTP bound to activated
G
subunits.
subunit to release GDP and to bind GTP (Fig.
1). GTP binding alters the conformation of three "switch" regions
in G
that are its primary contact sites with G
, promoting
subunit dissociation (7, 8). Guanine nucleotide exchange can occur
spontaneously, but is accelerated by agonist-activated receptor and
retarded by G
binding. Thus, the receptor acts as a
GDS,1 whereas G
acts as a GDI
(15).
Fig. 1.
The cycle of G protein activation and
inactivation. Top panel, when GDP-bound, G is inactive
and associated with G
. Agonist binding to a receptor promotes
guanine nucleotide exchange; G
releases GDP, binds GTP, and
dissociates from G
. Dissociated subunits activate target proteins
(effectors). When GTP is hydrolyzed, subunits reassociate. G
antagonizes receptor action by inhibiting guanine nucleotide exchange.
RGS proteins bind to G
, stimulate GTP hydrolysis, and thereby
reverse G protein activation. Bottom panel, the roles of a
receptor, G
, and an RGS are completely analogous to the GDSs,
GDIs, and GAPs that regulate small monomeric G proteins like Ras.
[View Larger Version of this Image (17K GIF file)]
, shifting the
equilibrium in favor of subunit reassociation, preventing further
signaling. Purified G
subunits display a measurable intrinsic rate
of GTP hydrolysis, but the turnover number in vitro cannot account, in some systems, for the rate at which signaling is terminated in vivo (16, 17). Other regulatory processes (such as those mediated by arrestin or phosducin) could be rate-limiting. Nonetheless, the importance of GTP hydrolysis provoked a search for factors that
accelerate the GTPase activity of G
subunits, termed GAPs (for
"GTPase-activating proteins") (Fig. 1). Effector enzymes can serve
this role: phospholipase C
stimulates the GTPase activity of
Gq
in vitro (18); and
subunit of cGMP
phosphodiesterase stimulates GTP hydrolysis by Gt
(19).
On the other hand, recent findings have converged on the conclusion
that RGS proteins are GAPs for Gi
and Go
.
Identification of the RGS proteins provides an instructive example of
how model organisms, like the yeast Saccharomyces cerevisiae
and the nematode Caenorhabditis elegans, can reveal new
mechanisms of signal regulation applicable to more complex organisms,
including humans.
-factor). The sst2 mutations defined a novel gene and
the first RGS family member.
subunit shares
45% amino acid identity with mammalian Gi
(6). This evolutionary conservation even allows some mammalian receptors and G
proteins to function in yeast (25, 26).
subunit of the pheromone receptor-coupled G protein. Cells
lacking GPA1 display constitutive activation of the pheromone response
pathway (28, 29), suggesting that G
transmits the signal and
providing the first in vivo evidence that a G
complex
has a positive and direct role in regulating downstream effectors.
) overcame the need for
functional SST2 implied, in a formal genetic sense, that G
operates
in the same pathway, but downstream of, SST2. G
overexpression helps
attenuate signaling through sequestration of G
. Other genetic
observations suggested a direct interaction between SST2 and GPA1.
First, certain mutations in the G
gene could bypass the requirement
for a fully active SST2 gene (30, 31). Second, the
recovery-promoting effect of G
overexpression was more efficient if
residual SST2 function was present (32, 33). Third, dominant gain-of-function mutations in SST2 confer resistance to
receptor-mediated activation of the pathway but not to activation by
mutations that permit constitutive release of G
(33). Fourth,
stimulating the pheromone response pathway induces SST2 production;
yet, resumption of growth does not occur in cells that lack G
,
indicating that SST2 cannot stimulate adaptation when G
is absent
(34). Indeed, it was proposed in 1992 that Sst2 serves as a GAP for
G
(20). An alternative model, that Sst2 promotes G
degradation
(35), is inconsistent with the genetic findings and with a subsequent study showing that GPA1 half-life is the same in sst2
and
SST2 cells (33).
colocalize in the cell. Both proteins are associated with
the plasma membrane and, to a lesser extent, with the Golgi body (34).
Membrane localization of SST2 is due, at least in part, to direct
binding to G
because SST2 can be isolated from cell lysates in a
complex with a GPA1-glutathione S-transferase fusion, even
after detergent and salt extraction of membranes (34), but binding
seems unaffected by the nature of the nucleotide added (GDP or
GTP
S). Membrane association of Sst2 is striking because its deduced
sequence is hydrophilic, lacks any significant stretches of hydrophobic
residues, and is devoid of myristoylation or isoprenylation sites.
Overexpression of a GTPase-deficient allele of G
(gpa1R297H) in wild-type cells enhanced their
pheromone sensitivity, resembling the loss of SST2 (34).
This G
mutant presumably remains in the GTP-bound state, cannot bind
G
, but still forms a complex with SST2. By sequestering SST2,
less is available to act on the normal G
, thereby explaining the
observed enhancement in pheromone sensitivity.
gene, fadA (37).
Alteration of the analogous Gly in mammalian Gs
slows
its rate of GTP hydrolysis and impairs its ability to release G
(38). On the other hand, certain other fadA (G
)
mutations, including a substitution predicted to block G
dissociation, actually suppress a flbA mutation (37). One interpretation of these findings is that free G
somehow blocks sporulation and that flbA is required for sporulation
because it promotes the ability of G
to remain associated with
G
.
(40, 41), results in elevated egg laying,
and conversely, overexpression of normal GOA-1 causes
reduced egg laying (40, 41). The fact that egl-10 and
goa-1 mutations have related (but opposite) phenotypes and the fact that an egl-10 mutation has no effect if a
goa-1 mutation is present suggest that the normal role of
EGL-10 is to down-modulate the activity of GOA-1.
Fig. 2.
Sequence alignment of known and presumed RGS
proteins. Conserved regions (originally termed GAIP or GOS8
homology ("GH") domains) of the indicated 14 RGS proteins were
aligned essentially as described by Siderovski et al. (48).
For accession numbers, see references cited in the text. Identities and
conventional conservative substitutions (D/E; K/R; E/Q; D/N; N/Q;
I/L/M/V; S/T; A/C/S; P/G; and F/Y/W) shared between five or more
members are given as white-on-black letters.
Dashes indicate single-residue gaps; larger gaps or
C-terminal extensions are given as numbers in
parentheses. Numbers to the left and
right of each line indicate the position of the
residue at the beginning and end of each sequence segment shown; an
apostrophe indicates tentative numbering derived from
cDNAs known to be incomplete. Sc, S. cerevisiae; An, A. nidulans; Ce,
C. elegans; and Hs, Homo
sapiens.
[View Larger Version of this Image (115K GIF file)]
was identified (42).
Gi3
(prepared by in vitro translation) binds
to a GAIP-glutathione S-transferase fusion protein. GAIP is
expressed in many tissues and is similar over most of its 217-residue
length to the C-terminal portions of SST2, FlbA, and EGL-10. GAIP is
more related to products of two other short mammalian cDNAs, GOS8 and
BL34/1R20 (42). In these proteins, similarity to EGL-10 extends over
~130 contiguous amino acids, whereas in SST2 and FlbA, similarity is
divided into three discontinuous blocks (Fig. 2). This ~130-residue
core domain defines the RGS superfamily and, in GAIP, is both necessary
and sufficient for interaction with Gi3
(42). GAIP also
interacts more weakly with Gi2
, but not with
Gq
.
mutant, could stimulate recovery from pheromone-induced growth arrest and
partially block pheromone-induced gene transcription (47). In an
independent study, RGS2 was also able to confer pheromone resistance to
yeast sst2 cells (48). The 205-residue RGS4 product is
expressed exclusively in the brain
(47).2
(in the
manner that GAP acts on Ras) after SST2 associates (via its RGS domain)
with G
. However, as discussed above, RGS proteins lacking a GAP-like
domain can partially substitute for loss of SST2 in yeast and, as
discussed below, are presumably able to stimulate the GTPase activity
of G
subunits in situ. Hence, the GAP-like domain of SST2
may contribute to adaptation through another mechanism.
Subunits
,
Gi2
, Gi3
, Go
, and
Gs
have been examined in vitro (50). Neither
affected the steady-state rate of GTP hydrolysis by these G
subtypes. Under such conditions, however, GTP hydrolysis is limited by
GDP dissociation. Hence, the steady-state assay actually measures the
rate of guanine nucleotide exchange and suggests that RGS proteins do
not function as either GDIs or GDSs. When a single round of GTP
hydrolysis was measured, either RGS4 or GAIP stimulated the rate of
hydrolysis more than 40-fold for all of the G
subtypes, except
Gs
. Thus, RGS proteins act as GAPs (at least for the
Gi
subfamily). RGS4 partially restored GTPase activity
to Gi1
(R178C) and to Gi1
(S47N) but not to
Gi1
(Q204L) (50). Arg-178 stabilizes the developing
negative charge on the
-phosphate leaving group in the transition
state during hydrolysis; Ser-47 contributes to Mg2+
binding, which is required for nucleotide hydrolysis and subunit dissociation; and Gln-204 is essential for orienting the attacking water molecule and for transition-state stabilization (51, 52). Restoration of function by RGS4 suggests that RGS proteins may accelerate GTP hydrolysis by stabilizing G
proteins in their active
conformation. Indeed, RGS1 (53) (also
RGS44) interact weakly with
Go
in the presence of GDP or GTP
S but strongly in the
presence of both GDP and AlF4
, a
combination that mimics the transition-state of the nucleotide during
hydrolysis.
may not
be a general feature of RGS proteins, however. A human 173-residue
RGS10, isolated via its interaction with a GTPase-deficient G
mutant, Gi3
(Q204L) (54), can co-immunoprecipitate with
either Gi3
(Q204L) or Gz
(Q204L), both
presumably GTP-bound, but not with wild-type (presumably GDP-bound)
Gi3
or Gz
or with either wild-type or
mutationally activated Gs
. Like RGS4 (50), GAIP (50),
and RGS1 (53), RGS10 (54) stimulates the GTPase activity of several
members of the Gi
subfamily but is ineffective against Gs
.
(7, 8, 57, 58) in
their GTP- and GDP-bound forms have been solved. Ras and G
likely
hydrolyze GTP by similar catalytic mechanisms. Nonetheless, by itself,
Ras hydrolyzes GTP at a rate about 100-fold slower than
Gs
(15, 59). In the presence of Ras-GAP, however, Ras
hydrolyzes GTP at least 100-fold faster than
Gs
(60, 61). One model to explain this difference (62)
is that Ras-GAP resembles the so-called "helical domain" that is
present in Gs
, but absent in Ras, and that Ras-GAP and
the helical domain both introduce into the catalytic cleft an Arg
residue that helps to stabilize the transition state. Indeed, Ras-GAP
permits Ras to bind GDP and AlF4
(63)
supporting a common mechanism for catalysis.
subunits have a "tethered GAP", how does an RGS stimulate
GTPase activity? Ras-GAP interaction with Ras may provide a clue.
Ras-GAP contacts the GTP-binding pocket and the "effector domain"
of Ras, a loop that undergoes significant conformational change upon
GTP hydrolysis. In G
, the helical domain interacts with the
GTP-binding pocket, but not with the "switch" regions that undergo
conformational change upon GTP hydrolysis. Hence, an RGS protein could
accelerate GTP hydrolysis by binding to one or more of the switch
elements, so that the conformation of the RGS-G
-GTP complex
approximates that of Ras-GAP bound to Ras-GTP. Alternatively, an RGS
could introduce an Arg into the catalytic center. In any event,
>40-fold stimulation of G
GTPase by RGS proteins brings the rate of
GTP hydrolysis in line with that of Ras stimulated by Ras-GAP.
classes is unlikely since divergent RGS proteins (RGS1, RGS4,
RGS10, and GAIP) all stimulate GTPase activity of the Gi subfamily (59, 63). Other RGS members may act on other G
classes.
Differences among the RGS members may not be apparent under available
assay conditions. Association with additional regulatory factors or
post-translational modifications may impose specificity on RGS
function. RGS action may be restricted by expression pattern or agonist
induction. Indeed, SST2 is induced by pheromone as a
feedback mechanism to limit the extent of G protein-mediated signaling
(34). Similarly, RGS1 expression is induced by PAF, and elevated RGS1
expression blocks PAF-induced MAPK activation (47). Combinatorial,
spatial, temporal, and developmental regulation of RGS function clearly
could modulate the intensity, duration, localization, and cell-type
specificity of G protein signaling.
We are grateful to T. Adams, H. Bourne, K. Blumer, P. Casey, A. Gilman, M. Koelle, and E. Neer for helpful comments and communication of unpublished results.