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Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia
Received June 10, 2003; accepted June 19, 2003
G protein signaling pathways are essential for all aspects of cell and
organ physiology, and the involved proteins have long served as primary drug
targets. At the most basic level, these proteins include a signal-receiving G
protein-coupled receptor (GPCR), a transducing heterotrimeric G protein
(G
subunits), and a signal-generating downstream target
effector. These proteins work together to transmit signals across the plasma
membrane. A neurotransmitter or hormone activated GPCR stimulates the exchange
of GDP for GTP on G to initiate heterotrimer dissociation and
activation of effector proteins that, in turn, initiate a cascade of cellular
signaling events. The regulators of G protein signaling (RGS proteins)
participate in this process by binding directly to activated G-GTP to
serve as GTPase-activating proteins (GAPs), which limit the lifetime of
G-GTP and terminate signaling event(s).
RGS proteins are relatively new actors on this stage. All family members
contain a signature RGS domain responsible for GAP activity. So far, more than
30 mammalian family members have been identified and classified into seven
subfamilies based on sequence identity and functional similarities
(De Vries et al., 2000
;
Ross and Wilkie, 2000;
Hollinger and Hepler, 2002).
Although many RGS proteins are relatively simple, others are more complex and
contain multiple domains for binding various signaling proteins, and
accumulated evidence now suggests that RGS proteins act as tightly regulated
modulators and integrators of G protein signaling. Much has been learned in
recent years about the biochemical mechanisms whereby RGS proteins stimulate
the GTPase activity of G (Ross and
Wilkie, 2000). However, much less is known about how RGS
function(s) are regulated in living cells
(Hollinger and Hepler, 2002).
After RGS proteins were first shown to act as G GAPs, questions
immediately emerged about which RGS proteins talked to which G subunits
and how selectivity for these interactions is determined in a cellular
context. Given that there are more than 20 G subunits and more than 30
RGS proteins, early speculation predicted that G and RGS proteins form
discrete functional pairs. Surprisingly, this has not turned out to be true in
most cases. Although certain RGS protein subfamilies do selectively bind and
regulate the activity of a specific class of G (for example, p115RhoGEF
and G12/13), this is an exception. Most RGS proteins are perplexingly
promiscuous regarding which G they can bind. In reconstitution assays
using purified proteins, most can regulate the activity of many members of the
Gi subfamily or Gq (De Vries et al.,
2000). So the question remains: exactly how do RGS proteins and
G subunits decide to pair up in living cells?
In this issue of Molecular Pharmacology, Roy et al.
(2003) provide evidence that
G may receive critical help from their linked receptors to recruit a
preferred RGS protein. The authors show that two simple RGS proteins, RGS2 and
RGS4, are recruited to the plasma membrane by expressing either G
subunits (Gi, Gq, or Gs) or linked GPCRs (M2-muscarinic
cholinergic, AT1a-angiotensin, or 2-adrenergic, respectively). Not
surprisingly, expression of G proteins initiates RGS protein membrane
recruitment, whereas expression of RGS-insensitive G-protein mutants does not.
However, among the remarkable observations the authors report is that RGS
protein recruitment to membranes also occurs with receptors alone, is specific
for receptors functionally linked to the target G protein, and is independent
of the activation state of either receptor or G protein. Furthermore, RGS
protein membrane recruitment mirrors RGS regulation of G protein function.
Together, these findings suggest that GPCRs, either alone or in coordinated
effort with their linked G proteins, can selectively recruit certain RGS
proteins to the plasma membrane to determine their signaling functions.
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RGS-Receptor Interactions |
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). A more recent study
(Wang et al., 2002) shows that
selective "knock-down" of mRNA and protein levels for RGS3 and
RGS5 in target cells selectively regulates signaling through M3-muscarinic
cholinergic and AT1a-angiotensin receptors, respectively. These findings are
consistent with the conclusions of Roy et al.
(2003) and suggest selective
RGS protein regulation of receptor signaling. An important difference between
these reports is in the approaches used. Whereas the previous studies examined
the effects of RGS proteins on receptor and G protein signaling, the study in
this issue focuses on the influence of G protein-linked receptors on the
subcellular localization of the RGS protein.
One unexpected finding is that the activation state of either the receptor
or the linked G protein does not seem to matter to the RGS protein; the mere
expression of receptor or linked G protein is sufficient to provide membrane
binding sites for RGS protein. Consistent with this observation, receptor- or
G protein-independent activation of the relevant signaling pathways did not
cause translocation of RGS proteins to the plasma membrane. These findings
suggest that initial RGS protein association with the plasma membrane may be
constitutive. In support of this idea, a previous report shows that the N
terminus of RGS4 contains lipid modifications and positively charged patches
that dictate membrane association
(Srinivasa et al., 1998). In
addition, RGS4 can spontaneously associate with anionic phospholipid vesicles,
and this association is stabilized by addition of functionally paired GPCR and
G protein (M1-muscarinic and Gq or M2-muscarinic and Gi)
(Tu et al., 2001). Still
unresolved is whether RGS proteins associate directly with receptors or a
receptor/G protein complex or somehow indirectly influence RGS protein
membrane localization by intermediary proteins and/or lipids.
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; Brady and Limbird,
2002; Hall and Lefkowitz,
2002). Many of these proteins are regulatory in their functions,
but others have established signaling roles of their own. As such, newly
emerging models of GPCR signaling suggest that receptors can serve as
platforms for scaffolding proteins that assemble multiprotein complexes. The
clear advantage of such complexes is that they provide a cellular mechanism
for dictating local organization of functionally related signaling proteins.
One well-studied example of this is the InaD protein found in Drosophila
melanogaster. In the fly, Gq and inositol
lipid/Ca2+ signaling mediate visual signal transduction.
InaD is a scaffolding protein containing multiple PDZ domains that
simultaneously bind the carboxyl-terminal tail of Gq-linked rhodopsin along
with the functionally related signaling proteins phospholipase C, protein
kinase C, and the transient receptor potential Ca2+
channel (Xu et al., 1998). The
net effect of this complex is the assembly and tight regulation of all
proteins necessary for a shared signaling task. Since the discovery of InaD, a
number of novel binding partners and parallel signaling complexes have been
identified for mammalian GPCR systems
(Brady and Limbird, 2002;
Hall and Lefkowitz, 2002). One
complex RGS protein (RGS12) contains a PDZ domain which may bind a GPCR
(Snow et al., 1998). However,
no other RGS proteins contain PDZ domains, and the findings described here and
elsewhere indicate that simple RGS proteins also participate in scaffolding
complexes with GPCRs and proteins involved in a shared signaling task. As
mentioned above, RGS4 association with artificial liposomes is enhanced by
Gq-linked muscarinic receptors (Tu et al.,
2001). Add to this the fact that RGS4 also can form a stable
ternary complex with phospholipase C, Gq, and G
(Dowal et al., 2001), and one
could easily envision a scenario in which RGS4 is part of a larger
multiprotein scaffolding complex of shared signaling function, as has been
proposed (Sierra et al.,
2000). Indeed, this may be the best explanation for the unexpected
observation in this study that RGS2 is selectively recruited to the plasma
membrane by expressing Gs-linked 2-adrenergic receptors or Gs.
This was unexpected because RGS2 is not a GAP for Gs but has been shown
to bind certain isoforms of adenylyl cyclase to inhibit catalytic activity
(Sinnarajah et al., 2001).
Therefore, the emerging picture is that RGS proteins are recruited to the
plasma membrane in cells by a GPCR-centered multiprotein complex to modulate
the activity of the linked G and/or effector
(Fig. 1). If RGS protein
functions are dictated by the receptor and not G, then this would
explain why RGS proteins are so promiscuous with regard to their G
interactions in vitro. In a cellular environment, RGS proteins would not be
free to find any available G. Receptors would selectively sort RGS
proteins at the plasma membrane to orient and optimize their GAP activity
toward the linked G. Where and how RGS proteins and receptors interact
at the plasma membrane is still unclear. Recent evidence suggests that certain
GPCR and linked proteins with shared signaling function assemble within
specialized microdomains at the plasma membrane known as lipid rafts and in
cavolin-enriched rafts termed caveolae
(Steinberg and Brunton, 2001;
Ostrom, 2002). It is
conceivable (although not yet demonstrated) that some RGS proteins and
receptors may colocalize within lipid rafts to modulate signaling events. Many
RGS proteins contain multiple binding domains for various protein binding
partners. Once bound to their target receptor, RGS proteins also could serve
as secondary scaffolds that recruit other signaling proteins in much the same
way that -arrestins do with 2-adrenergic receptors
(Hall and Lefkowitz, 2002). Add
to this our new appreciation that GPCRs can oligomerize and couple to multiple
G proteins (Angers et al.,
2002), and the possibilities for future study (despite the
bewildering complexity) become very exciting.
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Implication for RGS Proteins as Potential Drug Targets |
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; Neubig and Siderovski,
2002). GPCRs and their linked signaling pathways are the direct
targets for a large number of currently used drug classes. What makes many RGS
proteins such attractive new drug targets is their unique capacity to modulate
G protein signaling combined with their highly regionalized localization, most
notably within the central nervous system
(Gold et al., 1997). Small
molecules that inhibit RGS protein/G interactions have been proposed as
novel drugs to potentiate the actions of endogenous neurotransmitters in
various disease states such as Alzheimer's and others. Alternatively, such
therapeutic agents could be used to boost the effects of existing
GPCR-directed drugs by decreasing the therapeutic dose needed while increasing
the agonist's regional specificity, thereby reducing unwanted side effects
(Neubig and Siderovski, 2002).
If RGS proteins are recruited to specific receptor signaling complexes, then
identifying which receptor(s) are paired with which RGS proteins warrants
further investigation. The design of small molecules that block or mimic RGS
protein/receptor interactions could become a highly specific therapeutic tool
that is effective only in those cell types in which both the RGS protein and
the receptor are localized. Identifying binding partners and specific sites of
RGS/receptor interaction will be important both for a better understanding of
G protein signaling and for future drug development. |
Acknowledgements |
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Footnotes |
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ABBREVIATIONS: GPCR, G protein-coupled receptor; RGS, regulator of G protein signaling; GAP, GTPase-activating protein; PDZ, PSD-95, Disc-large, ZO-1.
Address correspondence to: John R. Hepler, Department of Pharmacology, 1510 Clifton Road, 5001 Rollins Research Center, Emory University School of Medicine, Atlanta, GA 30322-3090. E-mail: jhepler{at}emory.edu
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References |
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TopRGS-Receptor InteractionsGPCR Scaffolding Complexes: Have...Implication for RGS Proteins...References |
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