From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Hundreds or thousands of chemical and physical
stimuli regulate the functions of eukaryotic cells by controlling the
activities of a surprisingly small number of core signaling units that
have been duplicated and adapted to achieve the necessary diversity. The most prevalent of these units, at least in animal cells, are three-protein modules consisting of signal recognition elements (receptors) and signal generators (effectors) whose activities are
linked and coordinated by heterotrimeric guanine nucleotide-binding proteins or G proteins.1
Collectively, mammalian cells contain hundreds of G protein-coupled receptors and dozens of effectors. It is difficult to count
functionally distinct G proteins because we do not understand the
significance of the heterogeneity offered by the possible combination
of 16 GDP-bound G protein The slow intrinsic rate of GTP hydrolysis by G RGS proteins were discovered functionally as negative regulators
of G protein signaling in Saccharomyces cerevisiae (Sst2p) (reviewed in Ref. 8) and Caenorhabditis elegans (EGL10) (9). This information converged quickly with demonstrations of interaction of a mammalian RGS protein with G
INTRODUCTION
Top
Introduction
References
, 5
, and at least 12
subunits (for reviews, see Refs.
1-5).
subunits have high affinity for a tight complex
of
and
subunits. This interaction of
with
occludes the sites of interaction of both of these signaling molecules with
downstream effectors, and the inactive state is maintained by an
extremely slow rate of dissociation of GDP from the oligomer (k ~ 0.01/min). An agonist-bound receptor (typically
a 35-60-kDa protein with seven plasma membrane-spanning helices)
activates an appropriate G protein by poorly understood interactions
that promote dissociation of GDP. High intracellular concentrations of
GTP ensure a transient existence of the nucleotide-free G protein, and
binding of GTP causes conformational changes in
that result in
dissociation of GTP-
from
. Both of these complexes can then
activate or inhibit signaling pathways by engaging in interactions with
effectors such as adenylyl cyclases, phospholipases, cyclic nucleotide
phosphodiesterases, and ion channels. Termination of signaling is
dependent on the GTPase activity of
. Typically slow
(kcat ~ 4/min) hydrolysis of GTP to GDP (which
remains protein bound) promotes dissociation of
from effectors and
reassociation with
.
proteins is regulated
by interactions with so-called GTPase-activating proteins or GAPs. GAPs
were first recognized as regulators of protein synthesis factors and
low molecular weight GTPases such as Ras. It is now appreciated that
certain effectors in G protein-regulated pathways act as GAPs on
cognate G
proteins (6, 7) and that there exists a large, newly
discovered family of GAPs for G
proteins known as
regulators of G protein signaling
or RGS proteins. Although one critical biochemical property of this
novel RGS protein family has been defined, knowledge of the requisite
regulation of these regulators is negligible. There are hints, however,
that these proteins may be poised at centers of signaling to intercept
activated G proteins, acting, from a G protein's point of view, as
"barbarians at the gate" of cellular signaling.
The RGS Protein Family
i3 in a
two-hybrid screen (10); induction of related messages by mitogenic
stimuli in human B (11, 12) and T (13) cells; and identification of
related sequences by data base searches, application of polymerase
chain reaction technology, rescue of the Sst2p-deficient phenotype, and
homology-based screening (9, 14-18). These developments have been
reviewed previously by others (8, 19-21). To date, 19 mammalian genes
are known to encode proteins that contain the diagnostic RGS core
domain (Fig. 1). Typically, this
120-amino acid core is flanked on both sides by highly variable arms to constitute a 25-kDa protein. However, the RGS core may be split (observed only in lower organisms), and larger family members have been
identified (e.g. RGS3, RGS7).
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Fig. 1.
A, primary and secondary structure of
RGS4. Only the residues observed in the crystal structure are shown in
the alignment, which includes other selected RGS proteins (see Ref.
34). Secondary structure is depicted as either a helix for -helical
residues or a thick line for coil. The RGS box consists of four
segments, each defined by the color of its secondary structure:
red (segment 1), gold (segment 2),
green (segment 3), and blue (segment 4). The same
scheme identifies these segments in the tertiary structure of RGS4
(B). Residues highlighted in yellow are conserved
and form the hydrophobic core of the RGS box. Residues highlighted in
gray are conserved and make direct contacts with
G
i1. The numbers beneath the alignment
indicate RGS4 residues that contact switch regions of
G
i1 and the specific switch with which they interact. B, the RGS4-G
i1
complex. RGS4 is drawn with the colored segments defined in
A. The Ras-like domain of G
i1 is
drawn in dark gray, whereas the
-helical domain is in
light gray. The three switch regions of
G
i1 are shown in red.
GDP-Mg2+, bound in the active site of
G
i1, is shown as a ball-and-stick model.
AlF4
is omitted from the figure for
clarity. Modified from Ref. 34, with permission.
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RGS Proteins Are G![]() |
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Given the genetic evidence that RGS proteins are negative
regulators of G protein signaling that act at the level of the
dissociated G subunit or above (but not on
or below), the
most obvious hypotheses were that RGS proteins might act as inhibitors
of GDP dissociation, blocking G protein activation, or stimulators of GTPase activity, facilitating deactivation. There is no evidence for
the former mechanism. However, of the eight mammalian RGS proteins that
have been examined biochemically, all act as GAPs (15, 17, 22-24).
In the absence of a receptor, the rate-limiting step in
steady-state hydrolysis of GTP by a G protein is release
of product (GDP dissociation). To examine the effect of an RGS protein
on actual hydrolysis of GTP, it is necessary to bypass the
rate-limiting step or accelerate it substantially. The first approach
requires preparation of substrate, GTP-G
, by incubation of the G
protein with GTP in the absence of Mg2+. Catalysis is then
initiated by addition of Mg2+ in the presence or absence of
an RGS protein, and a single round of GTP hydrolysis is monitored over
a typical time course of seconds to minutes. Alternatively,
reconstitution of a heterotrimeric G protein with an appropriate
receptor in phospholipid vesicles permits addition of a receptor
agonist to speed product dissociation; the accelerating effect of a GAP
on steady-state GTP hydrolysis can then be measured.
RGS4 is readily expressed in bacteria and purified, and its GAP
activity has been studied most extensively. Whatever the actual mechanism for acceleration of GTP hydrolysis, GAPs such as RGS4 can be
conceptualized to act as enzymes, binding substrate (e.g. G-GTP) and facilitating its conversion to product (G
-GDP)
(25-27). Parameters that characterize the reaction include affinity of the GAP for a pseudosubstrate complex (e.g. G
-GTP
S),
Km for "substrate" and
Vmax (estimated by measurement of initial rates
of GTP hydrolysis at varying concentrations of G
-GTP), and the
pre-steady state rate of GTP hydrolysis (kcat)
at saturating concentrations of the GAP. This intrinsic rate of
hydrolysis may exceed the Vmax for steady-state
turnover of G
-GTP by the GAP if, for example, dissociation of the
GAP from G
-GDP is slow. The interactions of RGS4 with
G
o and G
t are catalytic; a single molecule of RGS4 can accelerate the GTPase activity of multiple molecules of G
. The maximal rate enhancement is estimated to be
roughly 100-fold with G
t (to 0.5/s at 4 °C).
Vmax and Km (at 22 °C) are
roughly 3/s and 2 µM, respectively; the affinity of RGS4
for GTP
S-bound G
t or G
o is
approximately equal to the Km.
The capacities of G proteins to serve as substrates for RGS protein
GAPs are influenced by palmitoylation of G
, at least in
vitro. Thus, palmitoylation of G
z and
G
i decreased their affinities for certain RGS proteins
by at least 90%, as well as the maximal rate of GTP hydrolysis (28).
These observations are particularly intriguing because palmitoylation
of G
is reversible and, at least in some cases, is regulated in
response to activation of cognate receptors.
Berman et al. (26) compared affinities of GTPS-, GDP-,
and GDP-AlF4
-bound forms of
G
o for RGS4 by testing their capacity to compete with
GTP-G
o for the GAP. The
GDP-AlF4
-bound forms of G
proteins
approximate transition-state complexes (29, 30), and RGS4 has markedly
higher affinity for this complex than for the substrate or product
complexes. Others have made similar observations with different RGS
proteins (15, 23, 31, 32). However, preferential affinity for the
transition-state complex is not always manifest. A protein designated
Gz GAP, now known to be a member of the RGS family (28),
has equally high affinities for both GTP
S-G
z and
GDP-AlF4
-G
z (33).
The high affinity interaction between RGS4 and
Gi1-GDP-AlF4
permitted crystallization and solution of the structure of the complex
at 2.8-Å resolution (34) (Fig. 1). Only the core domain of RGS4 was
visible in the crystal. Importantly, it has been demonstrated that this
domain contains all of the crucial elements for GAP activity (10, 17,
35, 36). The core of RGS4 contains a classic right-handed, antiparallel
four-helix bundle that interacts (via loops along its base) with
switches I, II, and III of G
i1. The switches
of G
proteins are those regions whose conformations are sensitive to
the identity of the bound nucleotide (GTP or GDP), and the residues of
switches I and II are intimately involved with binding and hydrolysis
of GTP. RGS4 does not contribute any residues that interact directly
with either GDP or AlF4
. However, a
conserved Asn residue in RGS4 (Asn128) may interact in the
ground state with the hydrolytic water molecule or with the side chain
of G
i1 residue Gln204, a
critical residue that orients and polarizes the catalytic water in the
transition state. It is thus suggested that RGS4 acts as a GAP by
stabilizing the flexible switch regions of G
proteins in
conformations resembling those found in the transition state, thus
lowering the activation energy barrier; Asn128 may further
contribute to the chemistry of hydrolysis by interactions with water or
Gln204.
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Comparison with RasGAP |
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Comparisons of the activities and structures of low molecular
weight GTPases with those of heterotrimeric G proteins continue to be
useful. The basal rate of GTP hydrolysis catalyzed by Ras is 2 orders
of magnitude below that catalyzed by a typical G protein; the rates
of GAP-stimulated GTP hydrolysis by both proteins are similar. Much of
the difference in the basal activities and, thus, the correspondingly
greater efficiency of RasGAP is ascribable to a single Arg residue at
the active site of G protein
subunits. Arg178 in
G
i1 participates directly in catalysis by
stabilization of the negative charge on
-phosphoryl oxygen atoms in
the transition state. Ras lacks such a residue and is essentially
inactive as a GTPase when compared with G
i1.
RasGAP participates directly in GTP hydrolysis by insertion of an
"arginine finger" into the active site (37). RasGAP also binds to
the mobile switches of Ras, orienting Gln61 appropriately
(Gln61 corresponds to Gln204 in
G
i1). The active sites of the
transition-state structures of Ras and G
i1
(associated with their respective GAPs) are amazingly similar. In
particular, the critical Arg and Gln residues are in nearly identical
positions, even though the Arg residues point into the active site from
different directions, and one (G
i1) is
contributed in cis, the other in trans (Fig.
2).
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Specificity of RGS-G![]() |
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Early recognition of the large number of RGS proteins, a number
roughly comparable with that of G subunits, prompted speculation of
high specificity in their interactions; this has not been realized to
the extent anticipated. G protein
subunits are usually categorized as members of one of four subfamilies, designated Gs (two
genes), Gi (eight genes), Gq (four genes), and
G12 (two genes). Most RGS proteins tested to date act as
GAPs toward members of the Gi subfamily and appear to
discriminate minimally among them; Gi proteins inhibit adenylyl cyclases, activate K+ channels, inhibit
Ca2+ channels, and activate cyclic nucleotide
phosphodiesterases. Some of these RGS proteins also act as GAPs toward
members of the Gq subfamily (phospholipase C
activators)
(38). Because GAP assays with G
q are difficult
technically, this interaction has been tested in only a few cases. GAPs
for G
s and G
12 subfamily members have not
been detected, although inhibitory effects of RGS proteins on
Gs-mediated signaling pathways have been observed (see
below). Although we suspect that greater specificity of RGS-G
protein interactions than is currently evident will be uncovered, it
seems clear that an explanation for the large number of RGS proteins
does not lie with any scenario resembling their 1:1 correspondence with
G protein
subunits.
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RGS Proteins May Be Effector Antagonists |
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As noted above, RGS4 binds to the switch regions of
Gi1. Logically, these switches are also
involved in effector binding, because effectors interact preferentially
with GTP-bound
subunits. This proximity or overlap of binding
surfaces raises the possibility that RGS proteins can compete for
effector binding to G
. Such competition has been observed between
RGS4 and phospholipase C
, which compete for binding to
GTP
S-G
q (38). In addition, both RGS4 and GAIP prevent
pertussis toxin-insensitive activation of phospholipase C
by
GTP
S. Because RGS4 does not stimulate hydrolysis of GTP
S by G
proteins, a GAP mechanism cannot be operative. Transient expression of
RGS4 in COS-7 cells blocked activation of inositol phosphate synthesis
by AlF4
, also consistent with an
effector-antagonist mechanism (39). Similar observations have been made
with RGS proteins, G
t, and its effector, the
subunit
of a retinal cyclic GMP phosphodiesterase (27, 32).
Competition for effector binding would not be an effective mechanism
for negative regulation of G protein-mediated signaling by a protein
like RGS4, which is an active GAP. The interaction of RGS4 with G
proteins is transient and itself sufficient to interrupt signaling via
the GAP mechanism. The affinity of RGS4 for GDP-bound G
proteins is
low and presumably insufficient to prevent recycling of G
. However,
it is possible that other members of the RGS protein family might have
preferential affinity for GTP- or GDP-bound forms of
, rather than
the transition-state complex. These proteins could then, in theory, act
as effector antagonists or sequestrants of
such that it could not
interact with effectors or
. Either of these mechanisms would
have consequences substantially different from those of a GAP
mechanism. Simple stimulation of the GTPase activity of G
deactivates the protein and facilitates association with
,
blocking downstream interactions by both
and
. The other two
mechanisms would block signaling by
but would leave
-mediated
signaling intact.
Chatterjee et al. (40) demonstrated that expression of a
truncated form of RGS3 (RGS3T) in baby hamster kidney cells impaired stimulation of cyclic AMP accumulation by the calcitonin gene-related peptide or pituitary adenylyl cyclase-activating peptide. These peptides are presumably activating Gs-coupled receptors,
and the question of mechanism is thus of considerable interest. We have tested RGS3T in vitro and detected GAP activity toward
Gi and G
o but not
G
s.2 Direct
interactions between RGS3T and G
s have not yet been
examined to evaluate the possibility that this RGS protein might act as an antagonist, blocking the binding of G
s to adenylyl
cyclase. The first crystal structure of a G
protein associated with
an effector has now been solved, that of G
s with the
catalytic domain of adenylyl
cyclase.3 Superposition of
the structure of G
i1 associated with RGS4 on that of G
s associated with adenylyl cyclase suggests
that the interaction of an RGS protein with a G protein
subunit
would not block binding of a G
protein to adenylyl cyclase. Although both RGS4 and adenylyl cyclase bind to switch II of G
, the
structures suggest that the two interactions could be accommodated
simultaneously. Speculatively, the GAP activity of certain RGS proteins
might be manifest only in the presence of ancillary molecules, such as
an effector.
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Regulation of RGS Proteins |
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RGS proteins have voracious catalytic appetites. Their
overexpression in various cultured cells demonstrates that they are capable barbarians, in general obliterating the activities of pathways
in manners predictable from their specificities in vitro as
GAPs. For example, RGS4 and GAIP block Gi-mediated
inhibition of adenylyl cyclase (41), whereas RGS3T, RGS4, and to a
lesser extent RGS1 and RGS2 attenuate Gq- or
Gi-regulated activation of MAP kinase (14, 39, 40).
Similarly, RGS3, RGS4, and GAIP suppress Gq-mediated
synthesis of inositol trisphosphate (39-42). To date, the
unanticipated result from such studies is attenuation of
receptor-mediated activation of adenylyl cyclase by RGS3T, discussed
above.
The existence of such activities dictates their scrupulous regulation
in cells. Although there are hints, we do not yet know how RGS proteins
are regulated in vivo or to what purpose. The most obvious
paradigm involves feedback desensitization of activated, G
protein-regulated pathways. Such desensitization is, of course, well
known in these signaling systems, and regulation is exerted at many
levels. The first RGS protein to be characterized, Sst2p in S. cerevisiae, fills this role and is itself regulated by control of
gene transcription (36, 43). It is likely that this will be at least a
part of the RGS protein story in mammalian cells, but broad adoption of
this paradigm is premature. There has been only one example of enhanced
transcription of a mammalian RGS protein gene in response to activation
of a G protein-coupled receptor (14), whereas regulation of
transcription of other RGS proteins has been noted in response to
expression of p53 (44) or polyclonal activation of T cells and B cells
(11-13). The apparently broad specificity of the interactions of many
RGS proteins with G proteins also dictates caution in interpretation
of the physiological consequences of RGS protein action. Will RGS
proteins act predominantly as feedback inhibitors of activated
pathways, transducers of cross-talk between signaling systems, or
both?
Even less well understood are potential roles of subcellular
localization and posttranslational modification in regulation of RGS
protein function. There are indications that these will be fruitful
areas for experimentation. Many RGS proteins have no obvious motifs to
specify localization at sites of action, but some do and there are
suggestions of others. Ret-RGS1 contains a hydrophobic domain near the
amino terminus suggestive of transmembrane insertion (17). GAIP and
Ret-RGS1 contain a cysteine string (17, 45), and GAIP is palmitoylated
and membrane-bound (45). An RGS protein was discovered as a partner
that interacts with the carboxyl-terminal cytoplasmic tail of
polycystin, a large integral membrane protein of unknown function
implicated in the genesis of polycystic renal
disease.4 RGS4 and other
members of the family appear to interact directly with adenylyl
cyclases.5 Perhaps RGS
proteins bind to effectors for G protein action, poised to intercept
and deactivate GTP-bound subunits after their dissociation
from
. Muallem and
Wilkie6 have demonstrated
that RGS protein boxes, which retain full potency and efficacy when
tested in vitro, are markedly less effective than their
full-length, "armed" counterparts when tested in
vivo.
Although the observations mentioned briefly in the previous paragraph
are in some ways disjointed and several of them are quite preliminary,
we believe they speak to an exciting future for research in this area.
Only simplistic models of RGS protein action can be drawn now (Fig.
3), and we believe they will be proven
entirely inadequate. The near future will almost certainly yield a
great deal of new information about regulation of RGS protein
concentration, activity, and localization. The specificities of their
interactions with G protein subunits will be defined, as will
interactions between RGS proteins and other cellular constituents. We
suspect that the various members of the family will play broad biological roles, not only in feedback regulation of G protein function
but also in coordination of the activities of G protein-regulated signaling systems with other related pathways.
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FOOTNOTES |
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* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This is the second article of three in the "Signaling by Heterotrimeric G Proteins Minireview Series." Title adapted from Barbarians at the Gate. The Fall of RJR Nabisco (Burrough, B., and Helyar, J. (1991) Harper & Row, New York).
To whom correspondence should be addressed. Tel.: 214-648-2370;
Fax: 214-648-8812.
1
The abbreviations used are: G protein,
heterotrimeric guanine nucleotide-binding regulatory protein; GAP,
GTPase-activating protein; RGS, regulator of G protein signaling;
GTPS, guanosine 5
-3-O-(thio)triphosphate; GAIP,
G
-interacting protein, an RGS protein.
2 D. M. Berman and A. G. Gilman, unpublished observation.
3 J. J. G. Tesmer, R. K. Sunahara, A. G. Gilman, and S. R. Sprang, manuscript in preparation.
4 G. Walz, personal communication.
5 R. K. Sunahara and A. G. Gilman, unpublished observations.
6 S. Muallem and T. Wilkie, personal communication.
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REFERENCES |
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