From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041
Received for publication, February 20, 2001
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ABSTRACT |
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Regulator of G protein signaling (RGS)
proteins must bind membranes in an orientation that permits the
protein-protein interactions necessary for regulatory activity. RGS4
binds to phospholipid surfaces in a slow, multistep process that leads
to maximal GTPase-activating protein (GAP) activity. When RGS4 is added
to phospholipid vesicles that contain m2 or m1 muscarinic receptor and
Gi, Gz, or Gq, GAP activity
increases ~3-fold over 4 h at 30 °C and more slowly at 20 °C. This increase in GAP activity is preceded by several other events that suggest that, after binding, optimal interaction with G
protein and receptor requires reorientation of RGS4 on the membrane surface, a conformational change, or both. Binding of RGS4 is initially
reversible but becomes irreversible within 5 min. Onset of
irreversibility parallels initial quenching of tryptophan fluorescence (t1/2 ~ 30 s). Further quenching occurs
after binding has become irreversible (t1/2 ~ 6 min) but is complete well before maximal GAP activity is attained. These processes all appear to be energetically driven by the
amphipathic N-terminal domain of RGS4 and are accelerated by
palmitoylation of cysteine residues in this region. The RGS4 N-terminal
domain confers similar membrane binding behavior on the RGS domains of either RGS10 or RGSZ1.
The initial events in G protein-mediated signaling take place at
the membrane surface. G proteins are peripheral membrane proteins,
anchored to the membrane both by intrinsic hydrophobicity and by lipid
modifications of their The membrane binding behavior of RGS proteins is more diverse. Although
RGS proteins do not contain obvious membrane-spanning domains, many are
tightly membrane-associated and behave as hydrophobic molecules. At the
extreme, RGSZ12 is tightly
membrane-bound and can be extracted from natural membranes under
non-denaturing conditions only using high concentrations of Triton
X-100 at elevated temperatures (4). Members of the RGSZ family
(GAIP, RGSZ1, RGSZ2, Ret-RGS) also contain cysteine strings that
are probable sites for multiple palmitoylation (5), but RGSZ1 expressed
in Escherichia coli is hydrophobic and aggregates in the
absence of detergent even after exposure to 20 mM
dithiothreitol, which removes thioesterified palmitate (6, 7). RGSZ1
tends to form oligomers even on SDS-polyacrylamide gels. At the other extreme, RGS10 is a soluble protein found in the cytoplasm (7, but see
Ref. 8). RGS3, RGS4, and RGS16 display intermediate behavior. They are
found both in cytoplasm and in membrane fractions, their localization
is influenced by reversible palmitoylation at two or more sites, and
their N-terminal sequences can form an amphipathic helix that directs
binding to anionic lipid surfaces (7, 9-11). In addition to this
behavioral heterogeneity, the specific binding sites for individual RGS
proteins on membranes (lipids, G The GAP activities of RGS proteins are markedly influenced by their
interactions with membranes and, presumably, by the ordering influence
that membrane surfaces provide. GAP activity can be readily and
accurately measured in detergent solution under conditions where
substoichiometric GAP promotes hydrolysis of preformed G We report here that RGS protein must be able to bind membranes with
correct orientation to display optimal GAP activity. In the case of
RGS4, binding to the bilayer from solution and reorientation are
multiphasic phenomena that become essentially irreversible over a few
minutes but that goes to completion over at least 60 min at 30 °C.
The low intrinsic GAP activity of RGS10 in the vesicle-based assay can
be markedly enhanced by the addition of the N-terminal amphipathic
domain from RGS4 while maintaining the G Materials--
All RGS proteins, mutant and wild-type, were
purified from E. coli as described (6). G cDNA Constructs--
cDNA constructs used for expression
of wild-type RGS proteins (6), for the RGS box domains of RGS10
and RGS4 (18), and for RGS4 Receptor-G Protein Vesicles and Protein-free
Liposomes--
Heterotrimeric G proteins and m2AChR, with or without
added GAPs, were reconstituted in to phospholipid vesicles essentially as described previously (15, 16). Lipids (25 µg; PE:PS:CHS, 55:35:10)
were suspended in 25 µl of 20 mM NaHepes (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 1 mM
MgCl2, 0.2% sodium deoxycholate, 0.02% sodium cholate and
sonicated until translucent. The suspension was then mixed with G
protein, m2AChR, and GAP (when used) to yield a final volume of 50 µl. This mixture was then chromatographed on a 3 × 150-mm
column of Sephacryl AcA34 in the resuspension buffer, but without
detergent. The usual fraction volume was 75 µl.
To prepare small, unilamellar vesicles, lipids were dissolved in
chloroform (10 mg/ml) at an appropriate molar ratio. Solvent was
evaporated under argon to form a thin lipid film, and buffer A
(50 mM NaHepes (pH 8.0), 50 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) was added to yield the desired
concentration. The lipid dispersion was vortexed and then sonicated
under argon at room temperature for 30 min until an almost clear
solution was obtained. Liposomes were analyzed by gel filtration under the same conditions used to prepare protein-lipid vesicles.
Fluorescence Measurements--
RGS4 or RGS10 (0.5 µM) was diluted in 300 µl of 50 mM NaHepes
(pH 7.5) plus 1 mM dithiothreitol in the presence or
absence of 100 µM sonicated lipid vesicles at 25 °C.
After 10 min of incubation, fluorescence was recorded either at 328 nm
or between 305 and 405 nm with excitation at 285 nm in a Hitachi F-2000
fluorescence spectrophotometer.
GAP Assays--
GAP activity was assayed in two formats. In
"single turnover" assays, G Other--
Standard procedures were used for SDS-polyacrylamide
gel electrophoresis (20) and staining with Coomassie Blue or silver (21). Protein was measured by Amido Black binding (22).
Several experimental findings initially suggested that the GAP
activity of RGS proteins depends on the extent and mode with which they
bind to phospholipid membranes. First, when RGSZ1 was assayed for its
ability to increase agonist-stimulated, steady-state GTPase activity in
receptor-G protein proteoliposomes, it was essentially inactive when
added to the vesicles in buffer; it was ~200-fold more active when
inserted into the nascent bilayer along with G protein and receptor
during formation of the vesicles (Ref. 6 and Table
I). GAIP, a close homolog of
RGSZ1, behaved similarly (data not shown). In contrast, RGS4 and
phospholipase C-
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits. G protein-coupled receptors, which activate G proteins by accelerating the binding of
GTP, span the membrane bilayer and present one relatively hydrophilic face to the cytoplasmic surface. GTPase-activating proteins
(GAPs),1 which accelerate GTP
hydrolysis and consequent deactivation, must also operate in this
interfacial environment. G protein GAPs include at least two families
of proteins, the phosopholipase C-
s and the RGS proteins (including
the RGS-related proteins such as p115 RhoGEF and G protein-coupled
receptor kinases) (1). Each group displays different modes of membrane
binding. Phospholipase C-
, which is both a
Gq-stimulated signaling protein and a Gq GAP,
is soluble in aqueous solution but binds to membranes by nonspecific
ionic interactions, with contribution from a plekstrin homology domain
that can bind to phosphatidylinositol 3,4-bisphosphate (2). The
receptor kinases are also soluble and apparently do not bind tightly to
phospholipid bilayers but rather are recruited by a combination of
palmitoylation and by binding both their receptor substrates and
G
subunits (see Ref. 3 for review).
or G
subunits, receptors, or
other proteins) remain unknown in most cases.
-GTP complexes (12). However, low GAP activities are more sensitively measured by monitoring stimulation of steady-state GTPase activity in
reconstituted phospholipid vesicles that contain trimeric G protein and
an appropriate agonist-bound receptor (12). The enhanced GAP activity
observed in this system may reflect interaction of the GAP with the
lipid bilayer. Removal of the N-terminal region of RGS4 diminishes its
activity in the membrane-based assay but not in detergent solution (9,
10, 13). RGSZ1, which is significantly more hydrophobic than RGS4, does
not incorporate into membranes from solution and is therefore
essentially inactive in the membrane-based assay unless it is
incorporated into the vesicle bilayer during the reconstitution
process (6).
selectivity characteristic
of RGS10.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
z
(14), G
q (14, 15), G
1
2 (14, 15), m2AchR, and m1AchR (16) were purified from Sf9 cells.
Myristoylated G
i1 was purified from E. coli
(17). Brain PS and liver PE were purchased from Avanti Polar Lipids and
were used without further purification. Phosphatidylglycerol,
phosphatidic acid, CHS, dioleoylphosphatidylcholine, and
L-
-dimyristoylphosphatidylcholine were purchased from
Sigma. [
-32P]GTP was synthesized and purified as
described (15).
N57 (7) have been described. cDNAs
used for expression of other truncated and chimeric RGS proteins were
constructed in pQE60 (Qiagen) by polymerase chain reaction using as
templates the cDNAs for human RGSZ1 (6), rat RGS4 (19), and human
RGS10 (18). Sequences of the chimeras are as follows: RGS10:4,
(MGHHHHHHG)-(RGS10 1M-S26)-(MG)-(RGS4
58K-A205); RGS4:10, (MGHHHHHHG)-(RGS4
1M-S53)-(G)-(RGS10
26S-T154); RGSZ1
N82, (MGHHHHHHG)-(RGSZ1
83E-A217); RGS4:Z1, (MGHHHHHHG)-(RGS4
1M-S53)-(GS)-(RGSZ1
83E-A217); RGSZ1:4, (MGHHHHHHG)-(RGSZ1
1M-E83)-(DMG)-(RGS4
58K-A205).
is first bound to
[
-32P]GTP, and the rate of hydrolysis of
[
-32P]GTP-G
is measured in a solution that contains
25 mM NaHepes (pH 7.5), 1 mM EDTA, 6.8 mM MgCl2 (1 mM free
Mg2+; see Ref. 6), 1 mM dithiothreitol, 0.1%
Triton X-100, 5 mM GTP (12). Activity is calculated as the
increase in the rate constant for hydrolysis of the bound
[
-32P]GTP. Single turnover assays, using either 1.5 nM [
-32P]GTP-G
z at 15 °C
or 10 nM G
i1 at 8 °C, were performed as
described (12). A more sensitive and presumably more physiological
assay for GAP activity monitors the enhancement of agonist-stimulated, steady-state GTPase activity in proteoliposomes reconstituted with
receptor and heterotrimeric G proteins. Reconstitution of purified
m2AChR with either Gz or Gi and of m1AChR with
Gq were performed as described (6, 12, 15). RGS proteins
were either coreconstituted into the vesicles by mixing with the
detergent-dispersed receptor, G protein, and lipid prior to formation
of the vesicles or added to the vesicles in detergent-free solution.
Added RGS proteins were incubated with the vesicles for 1 h at
30 °C prior to assay unless otherwise indicated. Steady-state assays
were carried out at 30 °C for 5-10 min. Data are given as increases in the steady-state GTPase activity. Interpretation of GAP assay data
has been discussed elsewhere (12).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a non-RGS GAP, are active when added from solution
(6, 15). A third pattern was displayed by RGS10. It was relatively ineffective in the vesicle-based assay either when added from solution
or when coreconstituted with the other proteins and lipids, even though
its GAP activity with soluble G
i-GTP substrate is not
much different from that of the other two RGS proteins (Fig. 1, Table I).
GAP activities measured in detergent solution and in phospholipid
vesicles
-[
-32P]GTP ("Experimental
Procedures"). Assays were performed at 15 °C for G
z-GTP
(1.5 nM) or 8 °C for G
il-GTP (10 nM). GAP activity is expressed as an increase in the
hydrolytic rate constant (min
1) per pmol of RGS protein. GAP
activity was also measured according to the stimulation of steady-state
GTPase activity of phospholipid vesicles that contained m2AChR and
trimeric G protein ("vesicle"). RGS proteins were added to the
vesicles just before initiation of the assay. Steady-state GAP
activity, measured at 30 °C, is expressed as the increase in the
GTPase turnover number (min
1) per pmol of RGS protein.
Turnover numbers for m2-Gz vesicles were 0.11 and 1.3 in the
absence and presence, respectively, of 10 nM RGS4 vesicles
and 1.0 and 8.7 for m2-Gi vesicles in the absence or presence,
respectively, of 5 nM RGS4. Concentrations of GAIP and
RGSZ1 were varied between 2 and 16 µM to obtain
quantifiable stimulation. Vesicles used in these assays contained 1 nM Gz and 0.26 nM m2AChR or 1 nM Gi and 0.25 nM m2AChR. Note that GAP
activities for Gi and Gz cannot be compared directly
because the intrinsic GTPase activities of the two proteins (and their
interactions with receptor) vary markedly. Interpretation of these
assays has been discussed elsewhere (12).
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Fig. 1.
Coreconstitution of RGS4 or RGS10 with m2AChR
and Gi. Gi (40 pmol) and m2AChR (8 pmol)
and either RGS4 (100 pmol; ,
) or RGS10 (280 pmol;
,
) were mixed with lipids and subjected to the standard
reconstitution protocol ("Experimental Procedures"). A similar
batch of vesicles was prepared without any RGS protein. Fractions were
assayed either for m2AChR (
,
) or for Gz GAP activity
in a solution-phase single turnover assay (
,
). The pooled
vesicle peak (fractions 7-9, 250 µl) contained 28% of loaded RGS4
(110 nM; underestimated because anionic lipid inhibits
solution-phase GAP activity) and 4% of loaded RGS10 (48 nM) according to solution-phase Gz GAP assay.
The concentrations of Gi, determined by
[35S]GTP
S binding, were as follows: RGS4 vesicles,
11.4 nM; RGS10 vesicles, 13.2 nM; GAP-free
vesicles, 12.0 nM. The concentrations of m2AChR, determined
by [3H]quinuclidinylbenzilate (QNB) binding (16),
were as follows: RGS4 vesicles, 2.5 nM; RGS10 vesicles, 2.9 nM; GAP-free vesicles, 2.8 nM.
Carbachol-stimulated GTPase activities of the vesicles (fmol of GTP
hydrolyzed/min/5 µl of vesicles) were as follows: RGS4, 1370; RGS10,
190; no GAP, 60.
One explanation for the low activity of RGS10 in the vesicle-based assay is that it neither incorporates into nor binds tightly to phospholipid vesicles. To test this idea, stable incorporation of RGS proteins was monitored during preparation of receptor-G protein vesicles by gel filtration. When RGS4 and RGS10 were mixed with receptor, G protein, and phospholipids prior to reconstitution, only about 5% of the RGS10 eluted with the vesicle peak (Fig. 1). In contrast, about 80% of RGS4 coeluted with the vesicle fraction. RGSZ1 and GAIP also coeluted with the vesicles in such experiments (Ref. 6 and data not shown), although their tendency to aggregate in the absence of lipid precludes interpretation of their behavior on gel filtration.
Coreconstitution experiments do not indicate whether RGS4 binds to
phospholipids or to the receptor and G protein in the vesicles. As
shown in Fig. 2A, RGS4
spontaneously binds to protein-free phospholipid vesicles when added
after their formation (see also Ref. 9). Association is stable over the
time course of gel filtration, suggesting that the affinity of RGS4 for
phospholipids is high. In contrast, RGS10 showed no evidence of stable
binding to preformed phospholipid vesicles (Fig. 2). These data support the idea that the major determinant of the low GAP activity of RGS10 in
vesicle-based assays is its inability to bind to the vesicle surface.
This idea is supported by our previous observation that palmitoylation
of RGS10 at Cys66 markedly increases its GAP activity in
vesicle-based assays (7) and allows it to bind to phospholipid vesicles
with about the same efficiency as seen for RGS4 (data not shown). The
importance of membrane binding is emphasized by the fact that
palmitoylation of RGS10 at Cys66 increases net GAP activity
in the vesicle-based assay even though it inhibits the intrinsic GAP
activity of the RGS10 protein (7).
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Although the binding of RGS4 to lipid vesicles appears to be necessary
for efficient GAP activity, a second and slower event is apparently
also required. When RGS4 is added to receptor-G protein vesicles before
a steady-state GTPase assay, some GAP activity is displayed
immediately, but prolonged incubation of RGS4 with vesicles before
assay further increases GAP activity up to 3-fold (Fig.
3). The rate of activation depends on
temperature. Full activation required several hours at 30 °C (Fig.
3) but was not complete even after 10 h at 20 °C (data not
shown). The extent of the increase in GAP activity was not altered by
temperature, however. Slow activation of GAP activity is not limited to
Gi. A similar slow increase in GAP activity of RGS4 was
observed with vesicles that contained Gz. The activation
process was not influenced by the presence of agonist. The increased in
the GAP activity of RGS4 was accelerated by N-terminal palmitoylation,
measured using the C95V mutant to avoid inhibiting GAP activity (see
Ref. 7). Palmitoylated C95V RGS4 displayed initially high GAP activity when assayed with m2AChR-Gi vesicles, and activity did not
increase with prolonged incubation. RGS10 did not display a
time-dependent increase in GAP activity even when added at
a high enough concentration to produce an easily measured effect (Fig.
3).
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Functional activation of RGS4 upon vesicle binding is preceded by a
slow change in either its conformation or orientation, as determined by
quenching of its intrinsic tryptophan fluorescence (Fig. 2,
B and C). Phospholipid binding causes ~30%
quenching in the fluorescence of RGS4 and a slight blue shift in the
emission maximum. This process is biphasic; about 40% of the
observed quenching takes place rapidly (t1/2 ~ 30 s at room temperature), and the remainder occurs over about 30 min (Fig. 2C). We do not know the physical basis of the
observed quenching, which would not be predicted if the two tryptophan
residues in RGS4 were simply inserted into the hydrophobic environment
of the membrane. Both tryptophan residues, Trp59 and
Trp92, are in the conserved RGS GAP domain, and neither
faces the exterior of the molecule (23). Trp59 is almost
completely buried among helices 1, 2, 3 and 9; Trp92 lies
in the cleft between the two bundles of helices opposite the site of
G binding (23). It seems most likely therefore that fluorescence
quenching observed upon binding to a phospholipid bilayer reflects a
conformational change of the RGS4 protein, probably in the environment
of Trp92. The fluorescence of RGS10 was also quenched
slightly upon exposure to lipid vesicles (~10%; Fig. 2), probably
reflecting the same physical phenomenon observed for RGS4, but with
weaker binding.
Based on the results above, we used competition for RGS binding between
receptor-G protein vesicles and protein-free liposomes to study the
lipid binding behavior of RGS4 in greater detail. Mixing RGS4 with
receptor-G protein vesicles and empty vesicles causes net inhibition of
GAP activity (Fig. 4). Inhibition
apparently reflects competition for RGS4 between the two populations of
vesicles, because GAP activity is inhibited half-maximally when the
concentration of both vesicles is equal (Fig. 4A). Maximal
inhibition is essentially complete at high concentrations of added
liposomes, as is also consistent with competitive binding of RGS4 to
available lipid surfaces. Binding of RGS4 to lipid vesicles depends
significantly on the presence of anionic lipids (Fig. 4B;
see also Ref. 9). Although we have not attempted to analyze the
selectivity of RGS4 among phospholipids in any detail, individual or
mixed neutral lipids such as PE (or phosphatidylcholine; data not
shown) do not by themselves bind RGS4. Phosphatidylglycerol and
phosphatidic acid are approximately as effective, as is PS. A
dispersion of cholesteryl hemisuccinate alone also does not bind RGS4
tightly (Fig. 4B).
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As suggested by the gel filtration experiments (Figs. 1 and 2), binding
of RGS4 is at best poorly reversible. If RGS4 is first allowed to bind
to receptor-G protein vesicles, subsequent addition of liposomes
decreased GAP activity only slightly (Fig.
5, upper curve). Whereas
binding appeared to be complete by 5 min, stabilization of binding was
incomplete at 15 s. Adding liposomes at this time produced almost
as much inhibition as did mixing all three components simultaneously.
In the converse experiment, prior exposure of RGS4 to liposomes before
addition of receptor-G protein vesicles increased inhibition over the
same 5-min period (Fig. 5, lower curve). In the experiments
shown in Fig. 5, mixtures of receptor-G protein vesicles and liposomes
were held only 1 min before GAP assay, and there would be little
opportunity for exchange of RGS4 among vesicles. To test for possible
exchange, we also incubated liposomes for up to 60 min with added
vesicles that had been pre-equilibrated with RGS4. The extended
incubation did not further inhibit GAP-stimulated GTPase activity (data
not shown). These data confirm the idea that once RGS4 binds to an
anionic lipid bilayer there is an initially freely reversible
interaction that is stabilized over the course of a few minutes. This
stabilization correlates with the rapid phase of quenching of Trp
fluorescence and precedes the conformational or orientational event
that increases GAP activity.
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The N-terminal region of members of the RGS4 family (RGS1-4, 16) is
important for maintaining both correct subcellular localization (7,
9-11) and cellular interactions with receptors and G proteins (13,
24). This region, usually defined as the first 53-57 amino acid
residues for RGS4, contains sites for covalent palmitoylation (7, 11)
and behaves as an amphipathic helix (9). To study the contribution
of the N-terminal region of RGS4 to GAP activity in vesicle-based
assays, we prepared and assayed several truncated and chimeric RGS
proteins with distinct N-terminal domains. As shown in Fig.
6, the N-terminal domain of RGS4 is both
necessary and sufficient for most of the interactions with membrane
lipids described above for RGS4. Replacement of the N-terminal domain of RGS10 with that of RGS4 increased its potency as a Gq
GAP by about 1000-fold in the receptor-coupled, vesicle-based assay
relative either to RGS10 itself or the RGS10 box. This effect can be
accounted for by the ability of the RGS4:10 chimera to associate stably with receptor-G protein vesicles (Fig. 6B). RGS4:Z bound to
liposomes, as did RGS4 or RGS4:10, and incubation of these chimeras
with the vesicles increased GAP activity, as was seen for RGS4 itself (Fig. 3). RGS10:4 did not bind vesicles, and RGSZ:4 aggregated in
aqueous solution such that vesicle binding could not be measured (data
not shown). Each case mimicked the behavior of the RGS protein that
donated the N terminus.
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The experiments shown in Fig. 6 were performed with vesicles that contained Gq and m1AChR, instead of m2AChR and Gi or Gz, to demonstrate the generality of the effect of the N-terminal domain and to confirm a brief report that truncation of the N terminus of RGS4 decreases Gq GAP activity (13). However, similar results were obtained with the m2AChR and either Gi or Gz (Table II). Truncation of the N-terminal domain of RGS4 resulted in loss of GAP activity in the vesicle-based, steady-state assay with either G protein as target. Conversely, activity was significantly restored when the N-terminal region of RGS4 replaced that of RGS10, which does not drive bilayer attachment, or that of RGSZ1, which causes aggregation in aqueous buffer. GAP activities of intact RGS10, the isolated RGS domain of either RGS10 or RGSZ1, or an N-terminally truncated RGS4 were all similarly low. For RGS10, which is already relatively inactive in this assay (Fig. 3, Table I), removal of the short N- and C-terminal domains had little effect. Similarly, replacement of the N-terminal domain of RGS4 with that of RGS10 did not support activity in this assay.
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To confirm that manipulation of the N termini of the RGS proteins had
not inactivated them in some way, we also assayed the GAP activities of
the truncated and chimeric proteins in single turnover assays in
detergent solution (Table III). As
suggested by the work of Popov et al. (18), alteration of
the RGS4 N-terminal domain in the constructs shown here had little
effect on their activities with
Gi-[
-32P]GTP as substrate. Structural
determinants of Gz GAP activity were more complex and
indicate that the N-terminal regions of RGS4 contribute to interaction
with G
z. Full-length RGS4 displayed far more GAP
activity toward G
z-GTP than did RGS4
N57 (or RGS10), indicating the importance of the N-terminal region in addition to the
RGS domain. The RGS4 N-terminal domain thus contributes to interaction
with Gz, as is true for Gq (13). Consistent
with this positive role, the RGS4 N-terminal domain in the RGS4:10 chimera potentiated the G
z GAP activity of RGS10, which
has slight activity with or without its N-terminal region. The RGS4:10
construct was reproducibly more active than RGS4. Replacement of the
N-terminal domain of RGSZ1 with that of RGS4 had relatively little
effect. Indeed, RGSZ1
N82, which lacks the entire N-terminal region
up to the RGS, retained substantial activity in the solution-based assay. Thus, some interaction of RGS proteins with G
z is
contributed by the N-terminal domain as well as by the RGS box. This
situation is qualitatively similar to that described for
G
q, where removal of the N-terminal region of RGS4 also
decreased activity in solution (13).
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DISCUSSION |
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To modulate a membrane-bound G protein signaling pathway, an RGS protein must act at the surface of a cellular membrane, but patterns of membrane attachment vary widely among RGS proteins. Some RGS proteins are hydrophobic and essentially integral membrane proteins (6). For others, such as RGS10, membrane binding has been hard to demonstrate convincingly except when the protein is palmitoylated (7). RGS4 also behaves as a soluble protein when purified but clearly binds to cellular membranes in a process that is dependent on its N-terminal domain and that is apparently driven by direct binding to anionic lipids (Ref. 9 and Fig. 4B). Although RGS4 is also palmitoylated, palmitoylation is not required for its binding to natural membranes or phospholipid bilayers (6, 7, 9, 11).
RGS4 binds tightly to receptor-G protein vesicles or to protein-free liposomes such that, after a few minutes, binding is essentially irreversible. Vesicle binding is not appreciably influenced by receptor or G protein, because receptor-G protein vesicles and protein-free liposomes bind RGS4 at similar rates (Fig. 5) and compete equally for limiting RGS4 (Fig. 4). The interaction of RGS4 with membranes consists of several steps that occur after initial contact. Quenching of Trp fluorescence is observed essentially immediately upon mixing RGS4 with liposomes and occurs with a t1/2 of about 30 s at room temperature. Additional quenching occurs more slowly and roughly correlates with the loss of reversibility of binding. Onset of irreversibility of binding was measured as the ability of protein-free liposomes to sequester RGS4 and thus inhibit its interaction with subsequently added receptor-G protein vesicles. The coelution of RGS4 with lipid vesicles during gel filtration also supports the idea that binding becomes poorly reversible.
The most interesting of these slow phenomena is the time- and
temperature-dependent increase in GAP activity that is
observed after initial binding of RGS4 to receptor-G protein vesicles. Increased GAP activity kinetically follows both irreversible binding and the initial quenching of Trp fluorescence (Fig. 2C).
Activation is apparently dependent on these prior events, because
N-terminal palmitoylation increases the rates of onset of all the
binding-related phenomena described. Slow activation may result from a
change in the orientation of RGS4 with respect to the bilayer or from a
change in its conformation. Either effect could be consistent with the
slow increase in fluorescence quenching. Enhanced GAP activity might
also result from slow association with receptor or G protein. In the
latter case, binding could be either to G or G
. G
interacts functionally with RGS proteins (6, 25), and they
appear to bind directly to each other
(26).3 We consider
this possibility less likely, because the activation process remains
even when high concentrations of RGS4 are added to the vesicles. For
this same reason, slow activation does not reflect redistribution of
RGS4 among vesicles or from an aggregated state to vesicles.
Essentially all of the membrane association behaviors described here
for RGS4 are dependent on the contribution to net lipid binding of the
N-terminal region. This region binds directly to anionic lipid
bilayers, apparently as an amphipathic helix (9), and contains
sites for palmitoylation that further increase hydrophobicity (7, 11).
Based on structural similarity and the behavior of RGS16 (10), this
lipid binding function of the N-terminal domain is probably conserved
through the RGS4 subfamily. This domain appears to function
autonomously for lipid binding (9) and functions independently of the
RGS domain to which it is attached. The RGS4:10 and RGS4:Z chimeras
also bound to lipid vesicles, became irreversibly bound, and underwent
slow activation in a manner similar to that of RGS4 (data not shown).
The RGS4 N terminus also functioned in the context of the m2AChR and
either Gi or Gz or of the m1AChR and
Gq. Taken together, these data suggest that the N-terminal
region of RGS4 acts primarily to support adsorption to the bilayer, the
first step in positioning a GAP for regulating the receptor-stimulated,
steady-state GTPase reaction. The N-terminal domain may also be
involved with subsequent reorientation of RGS4, but such an effect
cannot readily be distinguished.
Independent of its effect on lipid binding, the RGS4 N-terminal domain
also contributes significantly to its GAP activity with
Gz and G
q, although not with
G
i. Removal of the N terminus does not interfere with
Gi GAP activity (Ref. 18; confirmed in Table III), but
N-terminal truncation markedly reduces activity with G
z
(Table III) and G
q (13). This probably reflects direct interaction between the N terminus and G
z or
G
q, because addition of the RGS4 N-terminal domain to
N-terminally truncated RGS10 yielded a chimeric protein, RGS4:10, that
displayed about 20-fold more Gz GAP activity than did
intact RGS10.
The data presented here point to the complexity of the process whereby
RGS proteins must become oriented in their membrane environment to
display optimal GAP activity in a receptor-coupled system. They are
consistent with previous results that indicated that the amphipathic
and cationic RGS4 N terminus and its palmitoylation are important to
the energetics of membrane binding (Ref. 9; see also Refs. 27 and 28)
but argue that proper orientation or alignment with receptor or G
protein subunits or both is a necessary subsequent step. This
conclusion agrees with the idea that relatively nonspecific hydrophobic
or ionic interactions are frequently the major energetic components of
binding of peripheral membrane proteins and that protein-protein
binding is needed for specificity (27). The energetic contribution of
lipid binding to affinity may in part explain the very high affinities
of binding of RGS proteins to multiple partners recently described by
Dowal et al. (26). We still need to understand how
peripheral domains of RGS proteins contribute to these specific
interactions, and the ability of the RGS4 core domain to distinguish
Gi from Gz or Gq may help us answer
the question. The N-terminal regions may be generally used by RGS
proteins to determine the mode of membrane attachment, as seems true
for the RGS4 and RGSZ families. In addition, though, the presence of
diverse N-terminal functional domains in RGS proteins indicates that
membrane binding is only one of many roles for this region (1, 29, 30).
The present work also leads to the next question of how RGS proteins,
which are naturally expressed at levels below those of their G targets, are themselves directed to the specialized sites of receptor-G protein signaling.
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ACKNOWLEDGEMENTS |
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We thank Suchetana Mukhopadhyay for providing m1AChR-Gq vesicles and for advice on their use, Jun Wang for constructing some of the cDNA constructs, and other members of our group for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM30355 and R. A. Welch Foundation Grant I-0982.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.
To whom correspondence should be addressed. Tel.: 214-648-8717;
Fax: 214-648-2994; E-mail: ross@utsw.swmed.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M101599200
2 Ret-RGS (31) and RGSZ1 (6) are products of alternatively spliced mRNAs derived from the RGS20 gene (S. A. Barker, J. Wang, D. A. Sierra, and E. M. Ross, submitted for publication). Ret-RGS has an extended N-terminal domain predicted to contain a membrane-spanning segment (31).
3 Y. Tu, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
GAP, GTPase-activating protein;
RGS, regulator of G protein signaling;
AChR, muscarinic acetylcholine receptor;
PS, phosphatidylserine;
PE, phosphatidylethanolamine;
CHS, cholesteryl hemisuccinate;
GTPS, guanosine 5'-3-O-(thio)triphosphate.
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