From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, November 1, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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RGS proteins negatively regulate heterotrimeric G
proteins at the plasma membrane. RGS2-GFP localizes to the nucleus,
plasma membrane, and cytoplasm of HEK293 cells. Expression of activated Gq increased RGS2 association with the plasma
membrane and decreased accumulation in the nucleus, suggesting that
signal-induced redistribution may regulate RGS2 function. Thus, we
identified and characterized a conserved N-terminal domain in RGS2 that
is necessary and sufficient for plasma membrane localization.
Mutational and biophysical analyses indicated that this domain is an
amphipathic Many hormones, neurotransmitters, and sensory stimuli elicit
specific physiological responses in target tissues by activating receptors that are coupled to heterotrimeric G
proteins1 (1, 2). Activated
receptors promote exchange of GTP for GDP on G The regulators of G protein signaling (RGS) proteins are a large family
that regulate G protein signaling in part by acting as
GTPase-activating proteins (GAPs) for several classes of G protein Genetic studies in budding yeast (13) and Caenorhabditis
elegans (14) indicate that eukaryotes express several types of RGS
proteins to provide selective regulation of distinct G protein signaling pathways, and a means of differentially regulating a single
pathway in response to various physiologic conditions. Similarly,
certain mammalian RGS proteins have been shown to regulate selectively
different G protein signaling pathways. For example, deletion of RGS2
in mice results in defects in T-cell activation, antiviral immunity,
and hippocampal neuron function (15), whereas an RGS9L knockout shows a
slowed recovery of rod photoresponse (7). Such differences in function
are reflective of both differences in cell-specific RGS expression
patterns as well as selectivity of RGS proteins for different signaling
pathways. Many RGS proteins are co-expressed in a wide number of cell
types and tissues. In such cases, specificity may be mediated in part
through selectivity of RGS proteins for particular G protein substrates
(16, 17). More generally, however, RGS proteins are relatively
nonselective toward G protein substrates in vitro. This
suggests that the regulatory selectivity observed in cells (18) is
achieved by interaction with other signaling molecules. For example,
the N terminus of RGS4 confers receptor-selective regulation of
Gq-coupled responses (19, 20); the PDZ domain of RGS12
binds peptides from the C termini of the interleukin-8-RB receptor
(21); the GGL domains of RGS6, RGS7, RGS9, and RGS11 selectively bind
G Accordingly, an emerging theme is that regulatory domains other than
the RGS core domain mediate patterns of subcellular localization and
that localization is a determinant of RGS protein function. Thus it is
of interest that many of the small RGS proteins accumulate in
compartments distinct from plasma membrane where their substrate G
proteins reside (32). This has led to the hypothesis that RGS protein
association with other factors in the cytoplasm, nucleus, and Golgi
helps to maintain an inactive pool of RGS proteins that can be
recruited to the plasma membrane in response to specific signals (32,
33). In support of this notion, G protein-mediated signals have been
shown to induce plasma membrane translocation of RGS3 and RGS4 (34,
35). However, the mechanisms and functional importance of
signal-induced recruitment to the plasma membrane of mammalian cells
remain to be determined.
A common approach to address these questions has involved heterologous
expression of mammalian RGS proteins in yeast (36-38). Such studies
have shown that several RGS isoforms can associate avidly and
constitutively with the plasma membrane and that plasma membrane
localization is required for inhibition of G
protein-dependent pheromone signaling. More recently, this
system has been used to identify putative plasma membrane targeting
domains of several RGS proteins, revealing that a conserved N-terminal
amphipathic RGS2, like RGS1, RGS4, RGS5, and RGS16, is a member of the "small"
mammalian RGS protein subfamily and contains a short N-terminal extension to the RGS box. Our previous data in yeast suggest that the
N-terminal sequences of human RGS2 may contain domains important for
its subcellular localization and function (16). Accordingly, the goals
of the present study were first to define the determinants that mediate
plasma membrane localization of RGS2 in yeast and next to determine
whether the N-terminal domain of RGS2 governs subcellular localization
and attenuation of Gq signaling in mammalian cells. Because
RGS2 has been shown to localize to the nucleus of mammalian cells (32,
33), we have also examined whether the nuclear localization is an
important determinant of RGS2 function.
Materials--
The cytomegalovirus promoter on plasmid pEGFP-C1
(CLONTECH; Palo Alto, CA) was used to express
various RGS2-GFP constructs and a constitutively active Gq
mutant (Q209L). Constitutively active Gq(R183C) construct
in pCIS was a kind gift from Dr. J. Hepler (Emory University,
Atlanta, GA). The thymidine kinase promoter-driven Renilla
luciferase reporter (pRL-TK) used as a transfection control in
transient assays was a kind gift from K. Murphy (Washington University,
St. Louis). Polyclonal GFP antibody was the kind gift of P. Silver
(Dana Farber Cancer Institute, Boston). Gq antibodies were
from Santa Cruz Biotechnology (Santa Cruz, CA). Unless otherwise stated, all other reagents and chemicals were from Sigma.
cDNA Constructs--
Mammalian and yeast expression
constructs were made by high fidelity polymerase chain reaction
(Pfu, Stratagene, La Jolla, CA) and subsequent cloning into
pEGFP-C1 or pVT102U, respectively (39). A strong translational
initiation signal (Kozak consensus; GCCACCATGGCG) was
incorporated at the initiator methionine of all clones to ensure robust
expression. RGS2 point mutants were made using the QuikChange
mutagenesis kit (Stratagene). All constructs were purified using an
Endo-Free Maxi large scale DNA purification kit (Qiagen) and verified
by DNA sequencing of the entire protein coding region.
Expression in Yeast and Halo Assays--
Expression of
GFP-tagged cDNAs in the yeast Saccharomyces cerevisiae
and determination of RGS function in halo assays were as described
previously (16).
Preparation of
Liposomes--
1,2-Dipalmitoyl-sn-glycero-3-phosphatidylocholine
(DPPC) and
1,2-dipalmitoyl-sn-glycero-3[phosphatidyl-rac-(1-glycerol)]
(DPPG) were from Avanti Polar Lipids, Inc. (Alabaster, AL), and were stored as chloroform stocks under argon at Peptide Synthesis and Circular Dichroism
Measurements--
Peptides corresponding to amino acid residues 34-57
in WT and mutant RGS2 were synthesized and purified using reverse phase high pressure liquid chromatography (PNACL, Washington University, St.
Louis). In the case of WT and each mutant peptide (K42A/R44A, W41A/L45A/F48A/L49A, L45D, and T43P) purity was confirmed using electrospray mass spectrometry. Stock solutions of the peptides were
made by dissolving 15 mg of purified peptide in 1 ml of
double-distilled water. The concentration of each peptide stock was
determined by amino acid analysis. Stock solutions were aliquoted,
stored at Tissue Culture--
HEK293 cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% (v/v) fetal calf serum,
2 mM glutamine, 10 µg/ml streptomycin, and 100 units/ml
penicillin at 37 °C in a humidified atmosphere with 5%
CO2.
Assays of Gq-dependent Phosphoinositide
Hydrolysis--
Cells (7 × 106 in 10-cm plates) were
washed in serum-free media and incubated with 6.4 ml of Opti-MEM (Life
Technologies, Inc.) containing a mixture of the following DNA
constructs: 1 µg of pRL-TK; 1.5 µg of Gq(R183C); and 3 µg of RGS2-GFP or empty vector control (5.5 µg of total plasmid
DNA/well) and 10 µl of LipofectAMINE. Following a 5-h incubation, the
media were removed and replaced with maintenance media (described
above) for recovery overnight. After 17 h of recovery, cells from
each transfection were trypsinized and plated in 6-well (7 × 106 cells/well) and 10-cm dishes. For inositol hydrolysis
assays, at 22 h post-transfection, culture media were replaced
with labeling media containing 4 µCi/ml [3H]myoinositol
in the presence of 10 mM LiCl and cells were incubated overnight. Phosphoinositide hydrolysis was determined as described previously (16), and values were normalized to Renilla
luciferase levels to account for differences in transient transfection
efficiency. For determination of Renilla luciferase levels
cells at 22-25 h, post-transfections were washed twice in ice-cold
phosphate-buffered saline, harvested in 250 µl of hypotonic lysis
buffer (10 mM KH2PO4, pH 7.5, 1 mM EDTA), and spun at 12,000 × g to remove
cellular debris. Duplicate 50-µl samples of the supernatant were
measured for Renilla luciferase activity in a Optocomp II
luminometer (MGM Instruments, Hamden, CT). Renilla
luciferase substrate was diluted 1/25,000 in 10 mM
KH2PO4, pH 7.5, 0.5 M NaCl, and 1 mM EDTA for the assays. Levels of RGS and G protein
expression were monitored on Western blots using ECL detection
(Amersham Pharmacia Biotech).
Confocal Fluorescence Microscopy--
Cells (2 × 105 per well on polylysine-coated coverslips) were
transfected as described above using 1 µg of each construct to be
studied. Following overnight recovery, maintenance media were replaced
with microscopy media (maintenance media without phenol red and
bicarbonate, containing 25 mM Hepes, pH 7.5). Confocal microscopy was performed on live cells using a Zeiss Axioplan microscope coupled to an MRC-1000 laser scanning confocal microscope (Bio-Rad). Images represent single equatorial planes obtained with
a × 63 objective. Confocal images were processed with Adobe Photoshop 4.0.
Mapping the Domain of Human RGS2 Required for Plasma Membrane
Association and Function in Yeast--
The mating pheromone response
pathway in the yeast S. cerevisiae is a G protein-coupled
signaling pathway. Expression of mammalian RGS proteins in yeast cells
lacking Sst2, the RGS protein homolog that normally attenuates
pheromone signaling, permits rapid and quantitative analysis of
function in pheromone-induced growth arrest (halo) assays (40, 41). RGS
protein function in halo assays is dependent on domains that direct
plasma membrane targeting (36-38). Indeed, our previous studies
indicated that the N-terminal domain of RGS2 is necessary for plasma
membrane localization and function in yeast (16). To identify the
minimal region within the N-terminal domain required for targeting and
function, we made a series of deletions within the N terminus of RGS2
and correlated their effects on membrane localization and attenuation
of mating pheromone-induced G protein signaling in yeast (Fig.
1A). Deletion of the
N-terminal 4, 15, or 32 amino acids result did not affect membrane
localization or function. Deletion of the first 66 amino acids,
however, resulted in loss of both membrane localization and inhibitory
activity, as indicated by an increase in pheromone sensitivity
(increased halo size). This result suggested that the domain in RGS2
necessary for membrane association maps between residues 32 and 66 in
the N terminus (Fig. 2). When residues
33-67 were fused to the N terminus of GFP, the resulting protein
localized to the plasma membrane, indicating that this sequence
contained determinant(s) sufficient for plasma localization in yeast
(Fig. 1B).
Sequence alignments indicated that the apparent N-terminal membrane
targeting domain of RGS2 is conserved in a subset of the lower
molecular weight RGS family members (Fig. 2), which includes RGS1,
RGS2, RGS3, RGS4, RGS5, and RGS16 (only alignments for RGS2, RGS4, and
RGS16 are shown). Because this region of RGS2 is similar in sequence to
the amphipathic Interaction of the N-terminal Membrane Targeting Domain of RGS2
with Acidic Phospholipids--
To determine whether the apparent
membrane-targeting domain of RGS2 can adopt a
Several results indicated that, similar to RGS4, peptides derived from
the N-terminal membrane-targeting domain of RGS2 adopt an
To determine whether an amphipathic character of the RGS2 peptide is
required for helix formation and/or binding to phospholipid vesicles,
we analyzed a series of peptides with amino acid substitutions predicted to affect the amphipathic nature of the helix (Fig. 3C). The mutations used were designed to reduce the
aliphatic (W41A/L45A/F48A/L49A) or basic (K42A/ R44A/L45D) nature of
the amphipathic helix core. These mutations did not alter the ability of the resulting peptides to form a The RGS2 N Terminus Mediates Association with the Nucleus and
Plasma Membrane in Mammalian Cells--
Although previous studies have
characterized N-terminal lipid association domains in other small RGS
proteins, the functional importance of these domains in mammalian cells
has not been rigorously addressed. Therefore, we set out to define the
role of the N terminus of RGS2 in mediating localization and function
in mammalian cells. We used a GFP tagging approach to determine the
subcellular localization of RGS2 because it has not been possible to
localize the protein expressed endogenously in primary cell cultures or
cell lines. As indicated by confocal microscopy, a functional RGS2-GFP
fusion expressed transiently in HEK293 cells localized primarily to the nucleus and was also detected in the cytoplasm and on the plasma membrane (Fig. 4A). Plasma
membrane association of RGS2-GFP in these cells is indicated by
membrane structures at the periphery of the cells, pronounced GFP
fluorescence at cell-cell borders, and at the basal surface of the
cells as determined by z-sectioning during confocal
microscopy (data not shown). The subcellular localization of RGS2-GFP
appeared to be specific because RGS4-GFP or GFP did not accumulate on
the plasma membrane, and they were distributed uniformly throughout the
cytoplasm and nucleus in HEK293 cells (Fig. 4B). The
localization of RGS2-GFP in unstimulated HEK293 cells is similar to the
localization of GFP-RGS2 localization in a pre-B cell line (33) but
distinct from the exclusively nuclear localization of GFP-RGS2 in COS-7
cells (32). These differences suggest that the pattern of RGS2
localization is somewhat cell type-specific, although other
explanations are possible. Nevertheless, we found that nuclear and
plasma membrane accumulations of RGS2-GFP were reduced in the
N-terminal truncation mutant, RGS2-(aa 78-211)-GFP, suggesting a role
for the N terminus in mediating the subcellular localization of RGS2 in
HEK293 cells.
The N Terminus of RGS2 Mediates Gq-induced Plasma
Membrane Localization--
Studies (34, 35) on other small RGS protein
family members have suggested an important role for the N terminus in
signal-mediated translocation to the plasma membrane. The mechanism of
this translocation is currently not well understood. To test whether
RGS2 also undergoes a signal-dependent translocation, we
co-expressed RGS2-GFP with Gq(Q209L), a constitutively
active Gq The Amphipathic
To characterize the features of the plasma membrane targeting signal of
RGS2, we generated a series of truncation and point mutants in RGS2-(aa
1-67)-GFP. Deletion of the first 4 or 15 amino acids did not alter the
subcellular localization of the fusion proteins in HEK293 cells.
Surprisingly, deletion of the first 32 amino acids directed
localization of the fusion to mitochondria. Since mitochondrial
targeting signals frequently are N-terminal amphipathic RGS2 Amphipathic Helix Is Required for Tonic Plasma Membrane
Localization--
To define the boundaries of the helical domain, we
generated a series of point mutations within the context of the
RGS2-(aa 1-67)-GFP fusion. First, a series of proline substitution
mutations were constructed to determine if helix-forming ability
affects the function of the targeting signal. Introduction of proline residues between Asp-40 and Ser-46 on the putative hydrophilic face of
the helix dramatically reduced association of the RGS2-(aa 1-67)-GFP
fusion with the plasma membrane (Table
I). This appeared to reflect the
disruption of a
To determine whether the amphipathic helix is required for function of
the RGS2 plasma membrane targeting signal in mammalian cells, we
introduced point mutations into GFP-RGS2-(aa 33-67) equivalent to
those shown previously that affect association with phospholipid
vesicles (Fig. 5B). Confocal microscopy indicated that the
mutant constructs displayed dramatically decreased association with the
plasma membrane. These results suggested that positively charged and
aliphatic residues are functional determinants of the plasma membrane
targeting signal, consistent with the model that this signal functions
as an amphipathic helix.
Do the Amphipathic Helices of RGS2 and RGS4 Behave Differently in
Mammalian Cells?--
Although the N termini of RGS2 and RGS4 contain
amphipathic helical domains that can associate with phospholipid
vesicles in vitro and the yeast plasma membrane, they appear
to function differently in HEK293 cells. In contrast to what we
observed with the N-terminal domain of RGS2, the N-terminal domain of
RGS4 (residues 1-42) did not target GFP to the plasma membrane or lead
to nuclear accumulation (Fig. 4). These results are mirrored by the
observed differences in localization between full-length RGS2-GFP and
RGS4-GFP (Fig. 1).
Comparison of the apparent targeting signals of RGS2 and RGS4 was used
to suggest why the RGS4 targeting signal might not function as
efficiently as that of RGS2. We noted that the hydrophobic face of the
RGS4 targeting domain has a serine and alanine at positions equivalent
to Leu-37 and Leu-38 of RGS2 (Fig. 2). Thus, we hypothesized that the
RGS4 signal may bind membranes less avidly because the membrane-binding
surface of the helix is less hydrophobic. To test this idea, we
replaced Leu-37 and Leu-38 of RGS2(1-67)-GFP with their RGS4
equivalents (Ser-15 and Ala-16, respectively). Indeed, this mutant
displayed a defect in membrane
association.2 However, this
hypothesis appeared insufficient to explain the difference in the
apparent strength of the plasma membrane targeting signals of RGS2 and
RGS4. We suggest this because RGS4-(1-42)-GFP does not target
the plasma membrane when leucine residues were introduced at positions
14 and 15 (data not shown). This hypothesis, however, assumes the RGS4
helix extends as far toward the N terminus as the RGS2 domain. Further
studies are needed to determine whether the length of the helices are a
key determinant of the function of these two lipid association domains.
The Amphipathic Helix Is Required for Signal-induced Plasma
Membrane Association of RGS2 in Mammalian Cells--
The preceding
data indicated that the N terminus, and more specifically the
amphipathic helical domain, is sufficient to mediate tonic plasma
membrane association of an RGS2-GFP fusion in HEK293 cells. We next
determined whether the same helical domain was required for the
observed Gq-induced translocation of full-length RGS2-GFP.
Indeed, the same mutations that inhibited association with phospholipid
vesicles in vitro and association of the N terminus with the
plasma membrane in HEK293 cells also prevent Gq-induced translocation of RGS2-GFP (Fig. 6). These
data suggest the The Plasma Membrane Targeting Function of the RGS2 N-terminal
Domain Is Not Required for Attenuation of Gq
Signaling--
To determine whether stable association of RGS2 with
plasma membrane is required for inhibition of Gq signaling
in mammalian cells, we analyzed point mutations affecting the
amphipathic helical domain. We used a constitutively active
Gq mutant (R183C) whose signaling activity can be
attenuated by the ability of RGS proteins to function as a GAP or an
effector antagonist. Expression of Gq(R183C) in HEK293
cells resulted in an ~100-fold increase in phosphoinositide
hydrolysis compared with vector alone (Fig.
7). When full-length RGS2-GFP was
co-expressed with Gq(R183C), the extent of phosphoinositide
hydrolysis was reduced by ~75%. Similarly, mutant forms of RGS2-GFP
that were defective in membrane association assays (L45D;
K42A/R44A; W41A/L45A/F48A/L49A) displayed nearly wild type
activity in this assay when expressed at similar levels. We therefore
suggest that plasma membrane association mediated by the N-terminal
domain of RGS2 is not the major determinant required for attenuation of
Gq signaling in mammalian cells.
To conclude that the plasma membrane targeting function of the
N-terminal domain is not essential, we had to determine whether assays
using overexpressed RGS2 mutants were capable of detecting a loss of
function phenotype. One means of addressing this issue was suggested by
the following observations: 1) complete deletion of the RGS2 N terminus
(RGS- (aa 78-211)-GFP) resulted in mislocalization to the cytoplasm
and loss of function as an inhibitor of yeast mating pheromone
signaling (Ref. 16 and data not shown); and 2) appending a C-terminal
Ras2 CAAX box to the RGS2 core domain resulted in
plasma membrane targeting and complete restoration of its function in
yeast. We therefore analyzed the function of these RGS2 constructs in
HEK293 cells. Similar to results obtained in yeast, deletion of the N
terminus from RGS2-GFP markedly reduce its ability to inhibit
Gq(R183C)-mediated signaling in HEK293 cells (Fig.
8). However in contrast to what was
observed in yeast, appending the polybasic region and prenylation
signal of k-Ras to the C terminus of the RGS2 core domain (RGS2-(aa
78-211)-GFP-CAAX) did not completely restore
inhibitory function despite very efficient plasma membrane association
(Fig. 1B). These results therefore suggested that RGS2
function in mammalian cells may be governed by interaction of the N
terminus with other factors or by post-translational modifications that
are absent or inoperative in yeast.
Nuclear Accumulation of RGS2 Is Mediated by Its N-terminal
Domain--
The observation that several RGS proteins including RGS2
accumulate in the nucleus suggests that nuclear import and/or export could determine whether an RGS protein is available to attenuate the
activity of plasma membrane-bound G proteins. These findings also
suggest that RGS proteins may possess signals that mediate nuclear
import, export, or retention. Thus, the N terminus might direct RGS2 to
the nucleus where it is inactive (sequestered) yet poised to respond to
appropriate signals. This idea is supported by the observation that
co-expression with activated Gq resulted in a decreased
nuclear accumulation of RGS2-GFP (Figs. 1 and 6). To address these
hypotheses, we first determined whether we could identify a domain
responsible for nuclear accumulation of RGS2, and we then asked whether
exclusion of RGS2 from the nucleus affected its function as an
attenuator of Gq signaling.
The localization patterns of various RGS2-GFP fusions suggested that a
nuclear accumulation signal maps within or near the plasma membrane
targeting signal. First, deletion of the N-terminal domain of RGS2-GFP
resulted in uniform distribution of the protein throughout the
cytoplasm and nucleus, in contrast to the nuclear enrichment of the
full-length protein (Fig. 4A). Second, the N-terminal domain
of RGS2 (residues 1-67) was sufficient to direct the accumulation of
GFP in the nucleus in structures resembling nucleoli (Fig. 5). Third,
subfragments of the N-terminal domain that were active as plasma
membrane targeting signals were also active as nuclear accumulation
signals (residues 4-67, 15-67, or 33-67 appended to the C terminus
of GFP).
Analysis of additional fusions indicated that the N-terminal domain of
RGS2 does not mediate nuclear accumulation by functioning as an import
signal, arguing that the N-terminal domain functions as a nuclear
retention signal. Specifically, we found that nuclear accumulation was
blocked by appending GST to GFP-RGS2 (Fig.
9A), making an ~80-kDa
protein that is larger than the exclusion limit of nuclear pore
complexes (~60 kDa). Similarly, we found that that RGS2-(aa
78-211)-GFP-GST (data not shown) and GFP-GST were also excluded.
Rather than being actively transported, RGS2, therefore, appears to
enter the nucleus by passive diffusion where it is retained.
Because full-length RGS2-GFP-GST was excluded from the nucleus, we used
it to determine whether nuclear accumulation affects the function of
RGS2. To address this question we determined whether RGS2-GFP and
RGS2-GFP-GST differed in their ability to attenuate signaling by
constitutively active Gq(R183C). The results indicated that
these two fusion proteins were expressed at similar levels and
attenuated Gq-mediated phosphoinositide hydrolysis to a
similar extent (Fig. 9B). These results suggested that
nuclear accumulation is not required for RGS2-mediated attenuation of
Gq-directed signaling and that nuclear localization does
not appear to impair significantly RGS2 function.
Subcellular Localization of RGS2--
Determining the subcellular
localization patterns of RGS proteins in mammalian cells is required to
understand the functions of these molecules and the mechanisms that
regulate their activities. Recent studies indicate that RGS1, RGS3,
RGS4, and RGS16 are cytoplasmic; RGS10 is nuclear, and GAIP and
RGSZ associate with the Golgi apparatus (32). We have found that
RGS2-GFP localizes to the nucleus, cytoplasm, and plasma membrane of
HEK293 cells, similar to the localization of GFP-RGS2 in 3T3
fibroblasts and L1/2, a transformed pre-B-lymphocyte cell line (33). In
COS-7 cells, GFP-RGS2 localizes exclusively to the nucleus (32),
suggesting that RGS2 localization may be regulated in a cell
type-specific manner.
Functional Domains Governing Subcellular Localization of
RGS2--
By identifying features of RGS2 involved in plasma membrane
and nuclear localization, we have addressed whether subcellular distribution governs the function of this molecule as an inhibitor of
Gq signaling. This approach has indicated that RGS2
possesses an N-terminal domain that targets the protein to the plasma
membrane. Similar to RGS4 and RGS16, the plasma membrane targeting
signal of RGS2 appears to be an amphipathic
RGS2 and several other RGS proteins have been suggested to possess a
basic bipartite motif in their RGS domain that functions as a nuclear
import signal (32). However, our studies indicate that this apparently
is not the case as follows: 1) an RGS2 fusion protein larger than the
exclusion limit of the nuclear pore complex does not enter the nucleus
of HEK293 cells, suggesting that the RGS core domain or other regions
of the molecule lack a functional nuclear import sequence; 2) RGS2
lacking its N-terminal domain but possessing its RGS domain does not
accumulate preferentially in the nucleus; and 3) a GFP fusion bearing
the N-terminal domain of RGS2 and lacking an RGS domain preferentially
accumulates in the nucleus. Therefore, it appears that RGS2 enters the
nucleus by passive diffusion. Whether other RGS proteins enter the
nucleus by active transport or passive diffusion remains to be determined.
Does RGS2 Require Stable Association with the Plasma
Membrane?--
Although RGS2 possesses an N-terminal domain capable of
targeting the molecule to the plasma membrane, stable association of
with plasma membrane may not be necessary for the ability of RGS2 to
attenuate G protein signaling. Plasma membrane association does not
appear to be essential because point mutations in the N-terminal domain
that impair basal or Gq-induced plasma membrane localization nonetheless preserve the ability of RGS2 to attenuate Gq signaling in this system.
Whereas our results indicate that plasma membrane localization is not
essential for attenuation of activated Gq by overexpressed RGS2 in mammalian cells, it remains possible that plasma membrane targeting activity is important for function of RGS2 expressed at
endogenous levels. Addressing this issue would require analysis of RGS2
mutants expressed at wild type levels. However, the inability to detect
RGS2 protein expressed endogenously in mammalian cells thus far has
precluded such investigations.
Other lines of evidence indicate that the N-terminal domain of RGS2 has
an essential function. Deletion of this domain inactivates the ability
of RGS2 to attenuate Gq signaling, and forced targeting of
this truncated protein to the plasma membrane fails to restore function
in mammalian cells. Therefore, we speculate that the N-terminal domain
of RGS2 may associate with receptors or other components of G protein
signaling pathways at the plasma membrane, as has been proposed for
RGS4 (19).
Signal-induced Recruitment of RGS Proteins to the Plasma
Membrane--
Although RGS2 associates inefficiently with the plasma
membrane in unstimulated cells, it is targeted more efficiently when activated Gq is expressed, similar to what has been
reported for RGS3 and RGS4. Presumably, signal-induced recruitment of
these RGS proteins to the plasma membrane provides a negative feedback loop that enables cells to desensitize. Once the signal decays, RGS
proteins could dissociate from the plasma membrane, facilitating resensitization.
How Do Activated G Protein Roles of RGS2 in the Nucleus--
Our studies have begun to
address potential functions of RGS proteins localized to the nucleus.
First, it is unlikely that RGS2 transports cargo into the nucleus
because it enters the nucleus by passive diffusion and is subject to
the exclusion limit (~60 kDa) of nuclear pore complexes. Second,
sequestration of RGS2 in the nucleus does not appear to limit the
ability to attenuate Gq signaling, because exclusion of
RGS2 from the nucleus does not increase its inhibitory potency.
Although there is no evidence that RGS2 has a novel function in the
nucleus, this is an intriguing hypothesis. Indeed, effectors of
Gq such as phospholipase C
The redistribution of RGS2 out of the nucleus in response to activation
of a G protein signaling pathway recalls a similar trafficking pattern
for Ste5 (46), the MAP kinase pathway scaffold of the mating pheromone
response pathway of yeast. However, unlike what has been proposed for
Ste5, our results suggest that overexpressed RGS2 does not require
nuclear entry and exit for activity as an inhibitor of Gq
signaling. Nevertheless, nuclear entry and exit conceivably could
modulate the function of RGS2 expressed at normal levels or impact
novel functions of RGS2 yet to be discovered.
-helix that binds vesicles containing acidic
phospholipids. However, the plasma membrane targeting function of the
amphipathic helical domain did not appear to be essential for RGS2 to
attenuate signaling by activated Gq. Nevertheless,
truncation mutants indicated that the N terminus is essential,
potentially serving as a scaffold that binds receptors, signaling
proteins, or nuclear components. Indeed, the RGS2 N terminus directs
nuclear accumulation of GFP. Although RGS2 possesses a nuclear
targeting motif, it lacks a nuclear import signal and enters the
nucleus by passive diffusion. Nuclear accumulation of RGS2 does not
limit its ability to attenuate Gq signaling, because
excluding RGS2 from the nucleus was without effect. RGS2 may
nonetheless regulate signaling or other processes in the nucleus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits leading to
dissociation of GTP-bound G
subunits from G
heterodimers and
activation of downstream effector pathways. Signals are terminated
following G
-catalyzed hydrolysis of GTP and reformation of G protein
heterotrimers. Thus, G proteins act as molecular switches to coordinate
the wide range of responses elicited by extracellular stimuli.
subunits (3-6). The GAP activity of RGS proteins shortens the
half-life of active, GTP-bound G
subunits and leads to attenuation of a response during prolonged stimulation or accelerated termination of the signal following removal of agonist (7-11). Binding of RGS
proteins to active G
subunits can also interfere with effector binding, thereby blocking activation and downstream signaling (12).
These activities are mediated by the ~120-amino acid RGS domain, a
conserved feature of this protein family.
5 (22-29); and a Dbl-like domain in
p115RhoGEF activates Rho GTPases (30, 31). Thus, modular
domains of RGS proteins may act as scaffolds to recruit specific
classes of signaling proteins or alternatively as motifs that target
these molecules to specific subcellular compartments.
-helical domain is required for plasma membrane
targeting and inhibition of G protein signaling (37, 38). However, it
has not been determined in mammalian cells whether these targeting motifs mediate stable or signal-induced association of RGS proteins with plasma membrane, and whether they are required for RGS proteins to
attenuate G protein signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Small
unilamellar liposomes were prepared as described previously (38). Lipid ratios of the resulting liposomes were monitored by high performance thin layer chromatography. Vesicle preparations were used on the same
day that they were prepared.
20 °C, and thawed only once prior to CD spectrometric
analysis. Far UV CD spectra of peptide samples were collected on a
Jasco J600 Spectropolarimeter using a 1-mm path length quartz cell at ambient temperature. Spectra were recorded from 250 to 180 nm in 0.4 nm
steps at 50 nm/min. Data represent the average of five independent
spectra. Peptide and liposomes were diluted in 20 mM sodium
phosphate, pH 7, to final concentrations of 30 µM and 4 mM, respectively. Background signals for the buffers and
lipids were not significant and were subtracted from peptide spectra prior to analysis. Data are expressed as mean molar ellipticity (degrees cm2/dmol). The percentage
-helical
contents of RGS peptides was estimated from the molar ellipticity at
222 nm as described previously (38).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Localization and attenuation of yeast mating
pheromone response by GFP-tagged N-terminal deletion mutants of
RGS2. A, yeast cells (SWY518) that carry plasmids
expressing GFP-tagged RGS2 or the indicated N-terminal truncations were
analyzed by confocal microscopy (right-hand panels). All
images are shown at the same magnification. The ability of the
indicated GFP fusions to inhibit the pheromone response
(left-hand panels) was measured using growth arrest
(halo) assays. For halo assays, a mutant yeast strain
(BC180) that lacks Sst2, the RGS protein that normally regulates the
mating response, was transformed with the indicated RGS2 constructs.
Shown are the responses elicited by 0.5 nmol of pheromone; other doses
were used to quantify the extent of inhibition by the various
constructs. The data shown are representative of three transformants
assayed in two independent experiments. B, the yeast strain
BC180 carrying the indicated RGS2-(aa 33-67)-GFP fusion was analyzed
as in A.
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Fig. 2.
Sequence and helical net comparisons
of RGS2 with membrane association domains of RGS4 and RGS16. Shown
is the schematic representation of the RGS2 membrane association domain
(red), proximal to the N-terminal end of the RGS box
(green). The corresponding sequence for this region was
aligned to RGS4 and RGS16 by the CLUSTAL method, and the consensus is
shown below. Shading indicates a hydrophobic core of
aliphatic residues found along one face of the putative amphipathic
-helix identified in RGS4 and RGS16. Red sequences are
conserved in all three sequences, and blue indicates
conservation in two out of three sequences. The same region was
represented as a helical net with residues flanking the
shaded regions (hydrophobic core) shown in color.
Aliphatic and nonpolar aromatic residues are shown as black,
and basic residues are yellow.
-helical domains of RGS4 and RGS16 implicated in
plasma membrane localization and function in yeast cells, we compared
helical net views of these domains (Fig. 2). This analysis indicated
that the apparent membrane-targeting domain of RGS2 also has the
potential to form an amphipathic
-helix. Indeed, RGS2, RGS4, and
RGS16 share a hydrophobic (aliphatic) core along one face of the
putative helix that is flanked at the N-terminal end of the helix by
two or more basic residues. These observations suggested that the
N-terminal domain of RGS2 may function as an amphipathic
-helical
membrane-targeting domain similar to those of RGS4 and RGS16.
Furthermore, putative N-terminal amphipathic helices of RGS2, RGS4, and
RGS16 are positioned immediately proximal to the RGS core domain,
suggesting a similar organization of functional domains among this
subset of RGS proteins.
-helical conformation
and interact with membranes, we analyzed the helical content of
synthetic peptides corresponding to this region by CD spectroscopy in
the presence or absence of phospholipid vesicles. Bernstein et
al. (38) used a similar approach to show that the N-terminal
membrane-targeting domain of RGS4 adopts a
-helix upon binding to
negatively charged phospholipid vesicles.
-helical
conformation upon binding to vesicles containing anionic phospholipids
(Fig. 3). A peptide corresponding to
residues 34-57 of RGS2 adopted a random coil conformation in aqueous
solution, indicated by the single minimum at 200 nm (Fig.
3B). The helix-forming potential of this peptide was
characterized by minima at 208 and 222 nm in the presence of the
helix-stabilizing solvent trifluoroethanol (data not shown).
Addition of vesicles made from neutral lipids (DPPC) did not induce a
helical conformation of the peptide (Fig. 3A). However,
addition of phospholipid vesicles made with anionic (DPPG) and neutral
(DPPC) phospholipids at a mole ratio of 10:90 caused the peptide to
adopt a more
-helical conformation. Apparent
-helical content
increased upon increasing the mole fraction of DPPG, reaching a maximum
helical content at a 40:60 ratio of DPPG:DPPC (Fig. 3, A and
B).
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Fig. 3.
Residues 34-57 in RGS2 adopt an
amphipathic -helix in the presence of anionic
liposomes. A, values indicating the %
-helix for a
peptide corresponding to RGS2 residues 34-57 were calculated from CD
data collected at 222 nm as described previously (Bernstein et
al. (38)) and under "Experimental Procedures." Values were
determined in the presence of liposomes containing the indicated ratios
of DPPC (nonpolar) and DPPG (anionic) lipids. B, CD spectra
for the wild type RGS2 peptide was recorded as described above. Control
spectrum for WT peptide in buffer alone is shown in black.
Spectra for the WT peptide in buffer with liposomes containing 20 and
40% DPPG are shown as red and green,
respectively. C, CD spectra for RGS2 mutant peptides were
recorded as above. Spectra for WT (square), L45D
(circle), W41A/L45A/F48A/L49A (triangle) and
K42A/R44A (inverted triangle) were compared for peptides in
buffer (black curves) and anionic liposomes (colored
curves WT, red; L45D, cyan;
W41A/L45A/F48A/L49A, green; K42A/R44A, purple).
Each curve is the average of at least two independent spectra.
-helix since each mutant peptide
showed similar helix-forming potential as the wild type in the presence
of trifluoroacetic acid (data not shown). However, the mutant peptides
did display an impaired ability to adopt an
-helical conformation in
the presence of vesicles containing anionic phospholipids.
Substitutions predicted to decrease or disrupt the aliphatic nature of
the hydrophobic face decreased lipid-induced helix formation.
Similarly, introducing a negatively charged residue along the putative
membrane-binding interface resulted in reduced helix formation, whereas
changing positive residues in the putative amphipathic helix to alanine
had little effect in this assay. These data indicated that in the case
of the RGS2 helix, the aliphatic interface is a primary determinant of
its ability to interact with lipid vesicles.
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Fig. 4.
Definition of the subcellular localization of
full-length and N-terminally truncated RGS2-GFP with and without
co-expressed Gq(Q209L). A, localization of
the indicated GFP fusion constructs was examined by transient
transfection with and without Gq(Q209L) in HEK293 cells
followed by confocal microscopy of live cells. Pictures are of cells
with low to intermediate fluorescence intensity and are representative
of at least 50 cells transfected with the same construct. Shown are GFP
fluorescence images of the basal side (relative to nuclear equator) of
the cell, as determined by a z axis series. B,
localization of GFP and similarly tagged control proteins RGS4 and
RGS2-(aa 78-211) were examined as in A.
mutant (Fig. 4A). Indeed, RGS2-GFP
accumulated more markedly at the plasma membrane when co-expressed with
Gq(Q209L). This apparent subcellular redistribution in the
presence of activated Gq was also indicated by a decrease in RGS2-GFP accumulation in the nucleus. To examine the role of the N
terminus in RGS2-GFP translocation, we also studied an N-terminally truncated construct. Since translocation was not observed when the
RGS2-(aa 78-211)-GFP fusion was co-expressed with
Gq(Q209L), it appeared that the N terminus of RGS2 rather
than RGS2-G protein binding is required for Gq-induced
plasma membrane localization.
-Helical Domain of RGS2 Mediates Plasma Membrane
Targeting in Mammalian Cells--
The preceding data show the
N-terminal domain of RGS2 is necessary to mediate tonic and
signal-induced plasma membrane association in HEK293 cells. To identify
the domains that confer localization in mammalian cells amino acid
residues 1-67 of RGS2 were fused to the N terminus of GFP (Fig.
5). As shown by confocal microscopy, this
construct localized to the plasma membrane in HEK293 cells indicating
that these residues directed plasma membrane association. Notably, this
is the first demonstration of an RGS protein domain that can mediate
tonic association with the plasma membrane in mammalian cells.
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Fig. 5.
Characterization of the N-terminal plasma
membrane targeting motif of RGS2 in HEK293 cells. A,
localization of N-terminal-GFP fusions of RGS2-(aa 1-67) and RGS4-(aa
1-42) and the indicated RGS2 truncation mutants were examined by
confocal microscopy as described above. B, wild type and
mutant (K42A/R44A; W41A/ L45A/F48A/L49A) RGS2 domains
corresponding to residues 33-66 were cloned as fusions at the
C-terminal end of GFP and analyzed as described above.
-helices
(42), these data were consistent with the model that residues 33-67 in
RGS2 form such a structure. To preclude mitochondrial localization and
assess plasma membrane targeting by this fragment of RGS2, we fused
residues 33-67 of RGS2 to the C terminus of GFP (Fig. 5B).
As expected, the GFP-RGS2-(aa 33-67) fusion did not associate with
mitochondria, but it did associate with the plasma membrane indicating
that this region contains a functional targeting signal. We also noted
that GFP-RGS2-(aa 33-67) accumulated in the nucleus, an observation
that will be explored at the end of the "Results."
-helix because introduction of an alanine residue at
any of these positions did not affect plasma membrane association.
Second, a series of negatively charged amino acid substitutions were
generated to determine if association of the targeting signal with
phospholipids is important, as predicted by the amphipathic helix
model. Introduction of acidic residues along the hydrophobic face of
the presumptive helix between Leu-37 and Leu-49 resulted in severely
decreased association with the plasma membrane (Table I). In contrast,
introduction of acidic residues on the hydrophilic side of the helix
had little effect on membrane association, further substantiating the
model. Together these results suggest that an amphipathic membrane
targeting helix extends from Leu-37 to Leu-49.
Subcellular localization of point mutations affecting the putative
amphipathic -helical domain in RGS2(aa 1-67)-GFP
that of WT. ± indicates detectable
fluorescence less than that for WT.
indicates no detectable
fluorescence.
-helical domain is required for signal-induced
translocation of RGS2. The same principle may hold for RGS proteins
with related domains such as RGS4, RGS5, and RGS16.
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Fig. 6.
Determination of
Gq(Q209L)-mediated plasma membrane translocation potential
for RGS2 proteins with mutations in the amphipathic
-helix domain. Localization of GFP-tagged wild
type or the indicated RGS2 mutants was studied in cells with and
without a co-expressed constitutively active Gq mutant
(Gq(Q209L). Confocal microscopy was performed as described
in the legend of Fig. 4.
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Fig. 7.
Ability of RGS2 amphipathic helix domain
mutants to attenuate Gq-mediated phosphoinositide
hydrolysis. HEK293 cells were co-transfected with control or
constitutively active Gq(R183C), the indicated GFP control
or GFP-tagged RGS2 wild type and amphipathic -helical domain mutant
constructs, and a Renilla luciferase control plasmid.
Triplicate wells containing transfected cells (1 × 106) were labeled with [3H]inositol and 10 mM LiCl. Inositol 1,4,5-trisphosphate
(IP3) levels were measured as described under
"Experimental Procedures." Values represent the average of
triplicate samples normalized for luciferase activity and were
expressed as the percentages of soluble inositol 1,4,5-trisphosphate
relative to total soluble inositol-containing material; S.E. are
indicated by error bars. These data are representative of
three independent experiments. The levels of RGS2-GFP constructs were
determined by immunoblotting (inset).
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Fig. 8.
Ability of RGS2 core and plasma
membrane-targeted core domains in to inhibit Gq-mediated
phosphoinositide hydrolysis. HEK293 cells were co-transfected with
control or constitutively active Gq, the indicated GFP
control or RGS2 constructs, and a Renilla luciferase
control. Phosphoinositide hydrolysis was measured as described in the
legend to Fig. 7. IP3, inositol
1,4,5-trisphosphate.
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Fig. 9.
Localization and function of RGS2-GFP-GST
constructs. A, confocal microscopy of GST-tagged
control and RGS2 constructs. The indicated GST-tagged GFP control or
RGS2 constructs were transfected into HEK293 cells and analyzed by
confocal microscopy as described above. B, function of
GST-tagged RGS2 in phosphoinositide signaling assays. The GST-tagged
full-length RGS2-GFP construct was compared with RGS2-GFP in
Gq(R183C)-mediated phosphoinositide hydrolysis assays as
described in the legend to Fig. 7. IP3, inositol
1,4,5-trisphosphate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical domain located at or near the N terminus of the molecule. Indeed, the targeting signals of RGS2, RGS4, and RGS16 function similarly when analyzed by
heterologous expression in yeast (36-38) and/or by their association with lipid vesicles in vitro. However, the targeting signals
of these RGS proteins may not function identically. Whereas full-length RGS2 or its N-terminal domain alone can associate with the plasma membrane of unstimulated HEK293 cells, RGS4 and its N-terminal domain
apparently do not. These differences suggest that RGS2 and RGS4 may
have somewhat different functions. For example, in cells that
constitutively express RGS2 and RGS4, the threshold of agonist needed
to elicit a response in naive cells may be set in part by RGS2 because
it can pre-associate with the plasma membrane, whereas RGS4 apparently
does not.
Subunits or Other Signals Induce
Plasma Membrane Targeting of RGS Proteins?--
Although the
mechanisms are not understood, it appears that the N-terminal
non-catalytic domains of RGS2, RGS3, and RGS4 mediate signal-induced
plasma membrane accumulation. Because the N-terminal domains of RGS
proteins are not believed to interact with G protein
subunits,
signal-induced plasma membrane recruitment could be caused by
regulating the availability or affinity of these domains for the lipid
bilayer. We suggest that for RGS2 and other amphipathic helix-containing RGS proteins, the translocation observed in the presence of activated G protein
subunits is a downstream
consequence of G protein signaling. This conclusion stands in contrast
to that suggested by analyzing RGS2 function in yeast, possibly because in mammalian cells the N-terminal domain of RGS2 interacts with other
cellular factors that are absent or whose homologs are not recognized
in yeast. For example, signal-induced recruitment of RGS proteins to
the plasma could be caused by binding or release of accessory proteins,
such as 14-3-3 (43) or calmodulin (44) which have been shown to bind
RGS3, RGS4, or RGS7, and allow the plasma membrane targeting signals of
these RGS proteins to function efficiently.
-isoforms are present in the
nucleus (45), suggesting that RGS2 or other RGS proteins in the nucleus
have the potential to regulate novel intracellular signaling pathways
or other processes that control gene expression, nucleocytoplasmic
trafficking, chromosome replication, or nuclear architecture.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Andrew Grillo-Hill for consultation on CD spectroscopy experiments, Sharon Chinault for RGS2 constructs, and an anonymous reviewer for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Institutes of Health, National Science Foundation, American Heart Association, and Monsanto.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.
Fellow of the American Heart Association. To whom
correspondence should be addressed: Dept. of Cell Biology and
Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-1662; Fax:
314-362-7463; E-mail: sheximer@cellbio.wustl.edu.
§ Established Investigator of the American Heart Association.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009942200
2 S. P. Heximer and K. J. Blumer, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: G protein, guanine nucleotide binding regulatory protein; GAP, GTPase-activating protein; aa , amino acid; GFP, green fluorescent protein; RGS, regulator of G protein signaling; DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3[phosphatidyl-rac-(1-glycerol)]; WT, wild type.
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