From the Departments of Pharmacology,
§ Chemical Engineering, and ¶ Internal
Medicine-/Hypertension, The University of Michigan, Ann
Arbor, Michigan 48109-0632
Received for publication, August 28, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Regulators of G protein signaling (RGS) are
GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G
protein pathways. In this study, we provide experimental and
theoretical evidence that high concentrations of receptors (as at a
synapse) can lead to saturation of GDP-GTP exchange making GTP
hydrolysis rate-limiting. This results in local depletion of inactive
heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS
enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a
GTP-dependent enhancement of guanosine
5'-3-O-(thio)triphosphate (GTP A critical question in cellular signaling is what determines the
specificity of signal transduction processes. There is much recent
evidence for the formation of complexes maintained by protein scaffolds
to control signaling specificity. This contrasts with a classical model
in the G protein signaling field, the collision-coupling model (1),
which relies entirely on the structure of receptor-G protein and G
protein-effector contact sites to determine signaling specificity. The
collision coupling model also suggests that there would be significant
spread of G protein signals in a cell upon receptor activation, since
all components are freely diffusable. There have been numerous studies
indicating that such free transfer of information over long distances
may not occur for Gi or Gq mediated signals (2,
3). Thus, similar to the localized signaling by postsynaptic ionotropic
receptors via protein complex assembly (4), mechanisms to limit the
"spread" of G protein signaling appear necessary.
G protein-coupled receptors
(GPCR)1 activate cellular
signals by inducing nucleotide exchange on the G protein The maintained signaling in the face of RGS-enhanced GTPase activity
suggests that the RGS proteins somehow increase the efficiency of G
protein activation. One possible mechanism for this could be
"physical scaffolding" in which the RGS protein binds to both receptor and G protein and stabilizes a complex between them. This
could involve the diverse amino- and carboxyl-terminal domains of the
RGS proteins such as GGL, DEP, DH/PH, and PDZ domains (6, 10, 14, 15).
Indeed, RGS12 does bind to the carboxyl terminus of the IL8 receptor
through a PDZ domain (16). Alternatively, Ross and co-workers (17) have
suggested that the GAP activity of phospholipase C- In this report, we propose that RGS proteins, via their ability to
accelerate GTP hydrolysis, reduce depletion of local G Materials--
Guanosine
5'-3-O-[35S](thio)triphosphate
([35S]GTP Cell Culture and Membrane Preparation--
The TAG-L1 CHO cell
line with stable expression of an HA-epitope tagged porcine
Purification of RGS Proteins--
GST fusion proteins containing
rat RGS4, RGS7 (aa 305-453), RGS8 were prepared as described (19).
His10RGS2 was expressed in BL21/DE3 and purified as
described (20) yielding >90% purity. His6RGS4box (aa
58-177) was expressed in JM109 and purified under denaturing
conditions as described by Popov et al. (21). Following renaturation on a nickel-nitrilotriacetic acid column, bound
protein was eluted with imidazole then dialyzed against 50 mM Hepes, 1 mM EDTA, and 1 mM
dithiothreitol. Protein was >90% pure and had activity equal
to purified GST-RGS4 when measured in spectroscopic single-turnover
studies as described (19). An RGS4 NH2-terminal peptide (aa
1-51) (RGS4-(1-51)) was synthesized by the University of Michigan
Peptide Synthesis Core.
[35S]GTP [32P]GTPase Assay--
Steady state
[32P]GTPase activity was measured in a reaction mixture
(100 µl) containing 4 µg of membranes, 0.2 mM ATP, 0.2 mM AppNHp, 1 µM GDP, 50 units/ml creatine
phosphokinase, 5 mM phosphocreatine, 20 mM
NaCl, 2 mM MgCl2, 0.2 mM EDTA, 10 mM Tris/HCl, 1 mM dithiothreitol, and
0.1 µM [ Model Simulations--
A chemical kinetic model of receptor/G
protein/RGS interactions was simulated using the RK4 method in the
chemical reaction module of Berkeley Madonna (Version 8.0.2 for
Windows, Kagi Shareware, Berkeley, CA). The model (see Fig.
3A) used a standard collision-coupling mechanism (22) with
the addition of RGS serving only as a GAP for GTP-bound G
Monte Carlo simulations (23) were used to model the spatial effect of
RGS proteins on the distribution of active and inactive G proteins.
Simulations were run on a 600 × 600 × 600 triangular lattice (2.5 nm per grid step) with three distinct diffusible species:
receptors, inactive G proteins, and active G proteins. Receptors and G
proteins had a diameter of two lattice spacings and could interact with
adjacent particles separated by one or fewer lattice spacings. In each
time step, receptors could activate one adjacent inactive G protein if
available, otherwise the receptor caused no reactions. Active G
proteins were allowed to revert to inactive G proteins with a
probability proportional to the GTP hydrolysis rate,
khyd. Inactive G proteins were passive. All species in the simulation were assumed to have the same diffusion rate.
The simulation was started with 600 inactive G proteins and 1 receptor
and allowed to equilibrate for 5 million iterations. Over the next 5 million iterations, data sets were gathered to determine the radial
distribution from the receptor of inactive and active G proteins.
Diffusion coefficients were 10 Data Analysis--
Data were analyzed with non-linear curve
fitting equations using GraphPad Prism 3 (San Diego, CA). Data are
reported as mean ± S.E. Statistical comparisons were conducted
using one-way ANOVA.
RGS Effects on
The RGS specificity of the GTPase stimulation was RGS4 > RGS8
With our receptor/G protein/RGS system established we wanted to test
possible mechanisms leading to the unexplained effect of RGS proteins
to increase on- and off-rates of channel kinetics without
significantly decreasing the steady state signal amplitude (11,
12). If RGS could enhance G protein activation as well as deactivation,
that might account for those results. Therefore, we tested the ability
of RGS43 to enhance
receptor-stimulated [35S]GTP
With added GTP present, RGS4 stimulated GTP Physical Scaffold Mechanism?--
One mechanism by which RGS could
stimulate GTP Kinetic Scaffolding Mechanism--
Since the RGS specificity and
concentration dependence in enhancing GTPase and
[35S]GTP
To determine whether G-GDP depletion could account for our results, we
constructed a kinetic model (Fig. 4) to
examine RGS effects on receptor-G protein interactions and GTP
hydrolysis. Simulating the presence of GTP (0.4 µM) in
the face of a strong receptor stimulus, the steady state levels of
G-GTP calculated by the model actually exceed those of G-GDP (Fig.
4B). This indicates that GDP release driven by the receptor
can exceed the basal rate of GTP hydrolysis by the G protein. As RGS is
added and the GTPase rate increases, the ratio of G-GDP/G-GTP
increases. These results show that G-GDP substrate depletion is
feasible with this set of reasonable kinetic parameters for the G
protein cycle. In addition to enhancing the G-GDP to G-GTP ratio, RGS
also increased the amount of nucleotide-free DRG (albeit still at low
levels). We then asked whether this model could also replicate our
experimental findings with steady state GTPase and GTP
To ensure that the effect of RGS to enhance [35S]GTP Spatial Implications of Kinetic Scaffolding--
The chemical
kinetic model, just described, assumes that reactions are occurring in
a homogenous three-dimensional system with free mixing of all
components. Since receptor-mediated G protein activation in cells
occurs in a two-dimensional membrane which may have diffusion
limitations, we used a Monte Carlo model similar to that developed by
Mahama and Linderman (23) to examine spatial effects of the kinetic
scaffolding mechanism. Simulations were run over a range of diffusion
coefficients (10
Fig. 6 illustrates the results of these
Monte Carlo simulations. With the lowest rate of GTP hydrolysis
(khyd 0.02 s
In ongoing work,5 we show
that three effector responses produced by the µ opioid
receptor in a C6 glioma cell line are differentially sensitive to the
influence of RGS proteins. Using an RGS-insensitive G Functional Roles of RGS--
RGS proteins play numerous roles in G
protein signaling. They reduce G protein signals via their GAP activity
and/or by competing for G protein binding to effectors (6, 10). This
inhibition of G protein signaling may be regulated either by changes in
RGS expression (7) or perhaps by post-translational modifications (34).
RGS proteins are required for the fast kinetics of turnoff during ion
channel regulation by G proteins (12, 35). RGS can participate in many
other protein-protein interactions via amino- and carboxyl-terminal
extensions from the RGS domain (14). These likely serve to coordinate
signaling between heterotrimeric G proteins and low molecular weight
Ras superfamily G proteins (36, 37) and between pairs of
heterotrimeric G proteins (38, 39). They can also cause
signal-dependent translocation of other types of regulatory
molecules to the site of active G
To this long list of established or hypothesized functions of RGS
proteins, we present the concept of kinetic scaffolding and its
contribution to spatial focusing of G protein signals. This may occur
around a single receptor but could also play an important role in
localizing signals around small clusters of receptors in dendrites or
synaptic areas of neuronal cell bodies. Localization of ionotropic
receptors in these regions is quite exquisite (4). Thus the ability of
RGS to narrow the spatial range of signal output from G protein-coupled
receptors to the 10-100 nm scale could permit a similar fine
localization of signaling via G protein systems.
S) binding to
Gi by RGS. The RGS domain of RGS4 is sufficient for this,
not requiring the NH2- or COOH-terminal extensions.
Furthermore, a kinetic model including only the GAP activity of RGS
replicates the GTP-dependent enhancement of GTP
S binding
observed experimentally. Finally in a Monte Carlo model, this mechanism
results in a dramatic "spatial focusing" of active G protein. Near
the receptor, G protein activity is maintained even with RGS due to the
ability of RGS to reduce depletion of local G
-GDP levels permitting
rapid recoupling to receptor and maintained G protein activation near
the receptor. In contrast, distant signals are suppressed by the RGS,
since G
-GDP is not depleted there. Thus, a novel RGS-mediated
"kinetic scaffolding" mechanism is proposed which narrows the
spatial range of active G protein around a cluster of receptors
limiting the spill-over of G protein signals to more distant effector
molecules, thus enhancing the specificity of Gi protein signals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunit,
while inactivation occurs upon GTP hydrolysis by the intrinsic G
GTPase (5). Regulator of G protein signaling (RGS) proteins are a recently discovered family of proteins which act as GTPase-activating proteins (GAPs) for G
subunits (6-9). The GAP activity of RGS proteins generally reduces steady state levels of GTP-bound G
subunits and inhibits the activity of G proteins (6, 10). However, some
studies of receptor-stimulated signaling show that RGS proteins can
speed the kinetics of responses without compromising steady state
signaling strength (11-13). The mechanism and significance of this
paradoxical result is not understood.
1, which is both
a Gq GAP and its effector, serves to enhance muscarinic
receptor-Gq coupling (6, 17). In that model, the GAP
activity causes rapid hydrolysis of GTP so that the G
-GTP does not
have time to completely dissociate from receptor, which is then able to
rapidly catalyze the next round of GDP/GTP exchange.
-GDP levels to
permit rapid recoupling to receptor and maintained G protein activation
near the receptor with decreased activity farther away. This narrows
the spatial range of active G protein around a cluster of receptors by
a "kinetic scaffolding" rather than by a physical scaffolding
mechanism. While local signaling to a nearby effector is not
significantly reduced in the presence of RGS, both the kinetics and
spatial focus of signaling are sharpened, thus limiting the spill-over
of G protein signals to more distant effector molecules.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
S, 1250 Ci/mmol) and [32P]GTP
(30 Ci/mmol) were from PerkinElmer Life Sciences.
His10RGS2 in PET-19b was from Dr. John Hepler (Emory
University). His6-tagged RGS4-(58-177) in pQE60 was
from Dr. Thomas Wilkie (University of Texas Southwestern Medical Center).
2aAR adrenoreceptor (
2aAR-CHO, 10-20
pmol/mg) was cultured and cell membranes prepared as described
(18).
S
Binding--
[35S]GTP
S binding was determined in 100 µl of reaction mixture containing 50 mM Tris (pH 7.6), 5 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, 1 µM GDP, 400 nM
GTP2 unless
otherwise indicated. Reactions also contained 4 µg of CHO cell
membrane and 0.2 nM [35S]GTP
S with or
without the full
2 agonist UK 14,304 (10 µM). The reaction was started at 30 °C by adding
[35S]GTP
S and was stopped at 10 min with ice-cold
washing buffer (20 mM Tris, 25 mM
MgCl2, 100 mM NaCl (pH 7.7)) using a Brandel cell harvester. For the kinetic study in CHO membranes (Fig. 4), 50 nM [35S]GTP
S was used with 1 µM GTP, but no GDP was added. The reactions were
initiated at 25 °C in reverse order 10-60 s prior to simultaneous filtration on a Brandel harvester.
-32P]GTP (pH 7.6). Reactions
were started by addition of [32P]GTP containing mixture
to the incubation mixture in the presence or absence of RGS proteins
and UK 14,304 (10 µM) and incubated at 30 °C for 10 min. Reactions were then terminated by adding ice-cold charcoal slurry
as described previously (19). Release of
[32P]Pi was linear with time up to 15 min.
proteins
(G-GTP). No stable R-G-RGS complex was included. Rate parameters listed
in Table I and initial reactant concentrations were designed to closely approximate the conditions used
in our assays. Receptor concentrations were estimated by [3H]yohimbine binding (~20 pmol/mg of protein or ~0.8
nM final), and G protein concentrations were similar (1 nM). Other model parameters were derived from measured
literature values in the indicated references (Table I).
Parameters used in simulating the RG model
10, 10
9, and
10
8 cm2/s, and khyd
values were 0.02, 0.2, 2, 20, and 200 s
1.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2a Receptor-coupled G Proteins in CHO
Cell Membranes--
To study RGS function in the presence of receptor
we examined CHO cell membranes expressing high levels (10 pmol/mg) of
2a adrenergic receptor (
2aAR). Since RGS
does not affect steady state GTPase activity of
purified G proteins because GDP release is rate-limiting,
any effect on GTPase in these membranes should be attributable to
receptor-stimulated G protein. Surprisingly, the
2aAR
membranes without added RGS showed only a modest agonist-induced increase in GTPase activity (from 2.3 to 3.3 pmol/mg/min) but RGS4
further increased the
2aAR-stimulated GTPase activity
3.8 ± 0.1-fold (Fig. 1A)
with an EC50 for RGS4 of 0.46 ± 0.06 µM
(n = 3). As expected, the basal (or non-agonist
stimulated) GTPase activity was only marginally increased by RGS4
(1.2-fold). Also, membranes expressing lower amounts of receptor gave
proportionally smaller increases in GTPase activity (data not shown).
Thus the RGS-mediated enhancement of GTPase is
receptor-dependent and requires that the rate of
receptor-stimulated GDP release exceed that of the unstimulated GTP
hydrolysis, making hydrolysis rate-limiting in the G protein cycle.
This raises the interesting possibility that at these receptor
densities, the rate of receptor activation of G protein may become
limited by depletion of the G-GDP receptor substrate in the absence of
RGS activity.
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Fig. 1.
RGS4 enhanced receptor-stimulated
[32P]GTPase activity. A, steady state
GTPase activity CHO membranes expressing high levels of the
2aAR was measured at 30 °C for 10 min in buffer
containing 1 µM GDP and 100 nM
[
-32P]GTP as described under "Experimental
Procedures." The effect of RGS4 was determined in the absence
(open circles) or presence (filled circles) of
the
2 agonist UK 14,304 (10 µM). Data were
fit to sigmoidal dose-response curves using GraphPad Prism 3. B, the dependence of UK 14,304-stimulated
[32P]Pi release on the type of RGS was also
determined. One µM GST-RGS4, GST-RGS8,
His10RGS2, or GST-RGS7 (box domain, aa 305-453) were added
with boiled GST-RGS4 or GST alone as controls. Data shown are mean ± S.E. of three experiments. Statistical signficance was determined
using one-way ANOVA with Dunnett's post test. ***, p < 0.001; *, p < 0.05.
RGS7 = RGS2 (Fig. 1B). This is expected, since
G
i2 and G
i3 subunits are activated by the
2aAR in CHO cells (24). RGS4 and RGS8 are good GAPs for
G
i2 and G
i3, while RGS2 and RGS7 are
specific for Gq and Go (19), respectively,
which are either not activated by
2aAR
(G
q) or not expressed in CHO (G
o). This ability of RGS4 and RGS8 to increase steady state GTPase of
receptor-stimulated G proteins is similar to recent data from Milligan
and co-workers (25) using receptor-G
fusion proteins.
S binding. As expected,
the full
2aAR agonist UK 14,304 stimulated
[35S]GTP
S binding (4.1 ± 0.3-fold) (Fig.
2A), indicating efficient receptor-G protein coupling in the absence of RGS. Consistent with the
possibility that RGS could enhance activation, RGS4 caused a small
(~50%) increase in UK 14,304-stimulated [35S]GTP
S
binding with an EC50 of 0.4 µM (Fig.
2A). In attempting to optimize the magnitude of the
RGS-stimulated [35S]GTP
S binding we tested the ability
of GDP and GTP to enhance the effect of RGS4. Unlike
receptor-stimulated GTP
S binding for which the -fold increase is
enhanced by GDP, the RGS effect was strongly increased only upon
addition of GTP (Fig. 2, B and C, insets). The small increase (~30%) in GTP
S binding in
the absence of added nucleotide probably results from endogenous GTP
present in the membrane preparations. Furthermore, the micromolar GTP concentrations used in the subsequent studies are appropriate, since
cellular GTP levels are in the high micromolar range.
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Fig. 2.
RGS4-stimulated
[35S]GTP S binding: nucleotide
dependence. [35S]GTP
S binding to CHO membranes
expressing
2aAR was measured at 30 °C for 10 min in
buffer containing 1 µM GDP as described under
"Experimental Procedures." A, with no added GTP, RGS4
produced a modest, concentration-dependent stimulation of
[35S]GTP
S binding in the absence (open
circles) and presence (solid circles) of 10 µM UK 14,304. B and C, increasing
concentrations of GDP (B) or GTP (C) were added
to the 1 µM GDP in the binding mixture in the absence
(open circles) and presence (solid circles) of 1 µM RGS4. Data plotted are receptor-stimulated
[35S]GTP
S binding calculated by subtracting from
binding with 10 µM UK 14,304, the basal binding with 10 µM yohimbine (which represented 15-20% of the total
binding). Insets show the RGS-stimulated -fold increase in
[35S]GTP
S binding at 0, 1, and 10 µM
added GTP and 0, 10, and 100 µM added GDP, to permit a
comparison at similar degrees of inhibition with each nucleotide. Data
show mean ± S.E. values from three experiments each conducted in
triplicate. Curves are non-linear least squares fits to a sigmoid
function.
S binding 3.0 ± 0.1-fold with an EC50 of 1.0 µM (Fig.
3A). There was also a small increase in [35S]GTP
S binding in the absence of UK
14,304 (EC50 of 1.3 µM and a maximum effect
of 2.3-fold), possibly due to constitutive receptor activity or
GTP-bound G
subunits. To ensure that the effect of RGS4 was
specific, we also tested the protein buffer (phosphate-buffered saline), boiled RGS4, and GST alone (Fig. 3B), which
had no effect. Also, thrombin-cleaved purified RGS4
protein4 gave effects similar
to GST-RGS4 (data not shown). We examined the specificity of the
different RGS proteins and found that it was identical to that for
GTPase stimulation (compare Figs. 1B and
3B).
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Fig. 3.
RGS specificity and concentration dependence
in enhancing receptor-stimulated
[35S]GTP S binding.
A and B, RGS-enhanced
[35S]GTP
S binding to
2aAR-CHO membranes
was measured at 30 °C for 10 min as described under "Experimental
Procedures" in the presence of 1 µM GDP and 400 nM GTP. Reagents used, data analysis, and statistical tests
were the same as for GTPase measurements in Fig. 1. C and
D, full-length RGS4, RGS4 catalytic domain (RGSbox aa
58-177), or the amino-terminal amphipathic helix (aa 1-51) were
tested at 1 µM each for stimulation of
[35S]GTP
S binding in the presence of the
2 adrenergic agonist UK 14,304 (10 µM).
Statistical significance was determined using one-way ANOVA with
Dunnett's post test. **, p < 0.01.
S binding is by enhancing receptor-G protein coupling.
The simplest scheme would be for RGS to bind directly to both proteins
(R and G) forming a physical scaffold perhaps enhancing receptor-G
protein pre-coupling (26, 27). Indeed, the amino-terminal amphipathic
sequence of RGS4 confers receptor specificity in regulation of
Gq signaling, and it has been suggested that it may
directly interact with receptors (28). Thus that region would be a
logical candidate to engage in the formation of a
receptor/RGS/Gi protein complex. To determine whether the
amino-terminal sequence of RGS4 was necessary or sufficient for
enhancing receptor-stimulated GTP
S binding to Gi, we
prepared the catalytic domain fragment of RGS4 (RGS4box, aa 58-177,
His6-tagged) and an amino-terminal synthetic peptide
(1-51), which has previously been shown to enhance RGS regulation of
Gq in cells (28). The catalytic domain alone
(i.e. RGSbox) stimulated GTP
S binding to Gi
in a manner identical to that of full-length RGS4 (Fig. 3, C
and D). In addition, the amino-terminal fragment alone had no effect (Fig. 3, C and D). Furthermore the
peptide did not potentiate or inhibit the effects of the RGSbox
construct (data not shown). These results rule out a physical
scaffolding model that depends on the amphipathic amino-terminal
sequence of RGS4. While we cannot rule out a physical scaffold
mechanism mediated by the RGS domain itself, these observations taken
together prompted us to consider other mechanisms.
S binding were strikingly similar and only the
RGS GAP domain was required, we reasoned that these two phenomena might
depend only on the GAP activity. Furthermore, the dependence of
RGS-stimulated GTP
S binding on GTP versus GDP in the
reaction mixture suggested a mechanism involving GTP hydrolysis. If a
strong receptor stimulus caused sufficient accumulation of activated
G
-GTP to deplete the receptor substrate (i.e.
heterotrimeric G-GDP) in the vicinity of receptor, then the GAP
activity of the RGS could: restore local G-GDP substrate levels, permit
receptor to stimulate more GDP release, produce more "empty" DRG
state, and permit more GTP
S binding per unit time. Such a mechanism
has been proposed for muscarinic receptors and Gq by Ross
and co-workers (6, 17). In that case the effector, phospholipase C-
,
serves as the GAP to maintain a complex of receptor/G protein/effector,
but RGS4 can also enhance the rate of GTP binding and GTPase in that
system (29).
S binding.
Fig. 4C shows that agonist-simulated GTP hydrolysis
increased from 12 to 20 pmol/mg/min, and RGS was also able to increase
GTP
S binding from 105 to 241 fmol/mg (Fig. 4D).
Interestingly, this effect was dependent on the GTP concentration,
since modeling with a GTP concentration of 4 nM
(i.e. the amount present endogenously in the membranes) showed a marginal effect on GTP
S binding (527-539 fmol/mg). Thus the kinetic model predicts that the ability of RGS to increase GTP
S
binding should only be evident when GTP is included in the assay (Fig.
4D), which is consistent with our data. Thus, the structural
data (Fig. 3D) and model predictions (Figs. 2C
and 4D) are all consistent with the kinetic model in which
enhanced GTP
S binding is caused simply by the accelerated GTP
hydrolysis in the face of a strong receptor stimulus.
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Fig. 4.
Modeling a receptor-mediated G protein
activation cycle with RGS. A, kinetic model of
receptor-mediated G protein activation with RGS acting only as a GAP of
GTP-bound G subunit. Parameters for the individual steps are shown
in Table I. Components include: D, drug or agonist; R, receptor; G, G
protein. Reversible reactions are indicated as
double-headed arrows. Molecular complexes are represented by
concatenated names of individual components. Initial reactant
concentrations (M) were: R, 8 × 10
10;
G-GDP, 1 × 10
9; D, 10
5; GDP,
10
6. B, the concentrations of G
complexes
(DRG, open circles; GGTP, filled circles; and
GGDP, open squares) were simulated at steady state (10 min)
using Berkeley Madonna (Version 8.02). For this simulation, the initial
GTP was 400 nM with the indicated concentrations of RGS.
C, simulated GTPase activity was determined at 10 min with
an initial GTP concentration of 0.1 µM and varied RGS
concentrations. Pi release is plotted with values converted
to units relevant to our experimental measurements (4 µg of membrane
protein in a reaction volume of 100 µl with receptor and G protein
concentrations at values in Table I). D, GTP
S binding. To
simulate GTP
S binding an extra step was added to the model in which
GTP
S binds irreversibly to DRG (steps 10 and 11 in Table I). The
initial reactant concentrations were GTP
S, 0.2 nM and
GTP, 4 nM (open circles) or 400 nM
(filled circles). The GTP
S bound after a 10-min
simulation was transformed into fmol/mg as in C.
S
binding was not dependent on the long incubation times and very low
GTP
S concentration used in these experiments, we also tested the
effect of RGS4 on binding of 50 nM
[35S]GTP
S (Fig. 5). In
the presence of agonist, RGS4, and 1 µM GTP, [35S]GTP
S binding occurred very fast
(t1/2 ~20 s), and a substantial fraction of the G
protein pool was occupied (~2 pmol/mg, about 20% of total
Gi present in CHO cell membranes (24)). Under these
conditions, the RGS stimulation of agonist-induced [35S]GTP
S binding was even more striking.
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Fig. 5.
Time course of RGS-enhanced
GTP S binding.
2aAR
membranes were preincubated for 15 min on ice with 10 µM
UK14,304 with (filled circles) or without (open
circles) 3 µM GST-RGS4 and 1 µM GTP as
the only added nucleotide. The reaction mixture was prewarmed to
25 °C, then [35S]GTP
S binding was initiated by
adding 50 nM nucleotide. Samples were filtered at the
indicated times and bound [35S]GTP
S measured. Data are
the mean ± S.E. of three separate experiments. Curves are linear
regression (
RGS) or non-linear least squares fits to a
single exponential curve (+RGS).
10-10
8 cm2
s
1) (30, 31) and GTP hydrolysis rates (from the intrinsic
G
GTPase rate of 0.02 s
1 to 200 s
1, a
value exceeding measured RGS-stimulated rates of 5-30
s
1) (19, 29).
1) and
D = 10
9 cm2 s
1
(Fig. 5A), a single receptor can activate the entire pool of 600 G proteins over the simulation area of 1 µm2 leading
to profound and extensive G-GDP depletion (total G protein density is
constant so G-GDP is almost fully depleted throughout the membrane
system). As the RGS concentration and the rate of GTP hydrolysis
increase, the envelope of active G protein (and depleted G-GDP)
narrows. One can define the range of activity by the radius at which a
given concentration of active G* is reached. Fig. 6B
illustrates, for different diffusion coefficients and GTPase rates, the
distance from receptor at which the active G* level is 200 µm
2 or G protein is 40% active. The zone of active G*
can range from less than 20 nm to over 450 nm depending on the RGS
activity (i.e. khyd) and the
diffusion coefficient. At any given diffusion coefficient, RGS
dramatically narrows the range of G protein activation around a single
receptor or a cluster of receptors. Interestingly, the amount of active
G protein immediately adjacent to the receptor (i.e. within
about 10-20 nm) is not reduced significantly even at very high RGS
concentrations, so an effector in close proximity would not show an
RGS-dependent decrease in activity. Such an effect could
explain the ability of RGS to speed the kinetics of G protein-coupled
inwardly rectifying K+ channel responses without
altering steady state activity (11, 12).
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Fig. 6.
Monte Carlo simulation of spatial effects of
RGS on receptor-stimulated G protein activation. A,
effect of RGS on the distribution of active G protein around a single
receptor (or cluster of receptors). A Monte Carlo simulation of active
G proteins was performed as described under "Experimental
Procedures." B, effect of diffusion coefficient and rate
of G deactivation (khyd) on the spatial
distribution of active G protein around a single receptor.
C, illustration of the effects of RGS on the two-dimensional
distribution of active G
subunit. If G protein activation is
sufficiently rapid to convert all G
into the GTP-bound form (thus
depleting local G-GDP) then RGS will reduce active G protein at a
distance from receptor, but the concentration of active G-GTP in the
local vicinity of receptor will be maintained.
o
(19) it is shown that inhibition of adenylyl cyclase and activation of ERK are greatly enhanced in the absence of RGS effect while the increase in intracellular calcium is not. Thus different effectors may be differentially modulated by RGS action. Those results
are consistent with the RGS-mediated kinetic scaffolding model proposed
here (for example if the Ca2+ response effectors were more
closely associated with µ opioid receptor and
G
o than those for adenylyl cylcase and ERK responses). Clearly other models may also account for the differential RGS effects,
but kinetic scaffolding is one possible mechanism.
subunits (40) and may play a role
in nuclear processes (33).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. John Hepler (Emory University)
and Dr. Thomas Wilkie (University of Texas Southwestern) for providing
RGS expression constructs. We thank Dr. Stephen Ikeda (Guthrie Research
Institute) for providing the pertussis toxin-insensitive
Go constructs. We thank Marianne Bowker and Dr. Donna
Shewach (University of Michigan) for the high performance liquid
chromatography measurements of endogenous nucleotide
concentrations in
2aAR-CHO membranes. We also thank
Masakatsu Nanamori, William Lim, Duane Chung, and Leighton Janes for
assistance in some of the experiments.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM 39561 (to R. R. N.), GM 062930 (to J. J. L.), and DA 04087 and DA 00254 (to J. R. T.). Peptide synthesis was supported by the Michigan Diabetes Research and Training Center Grant NIADDK P60 DK20572.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: Dept. of
Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Tel.: 734-763-3650; Fax: 734-763-4450; E-mail:
RNeubig@umich.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208819200
2
The 400 nM added GTP was necessary
to obtain the maximal enhancement of [35S]GTPS binding
by RGS proteins (see Fig. 4B, inset).
3 All RGS proteins were prepared and used as GST fusion proteins unless indicated otherwise.
4 The thrombin-cleaved RGS4 construct has an amino-terminal extension of GSPGIRL and was >90% pure by Coomassie staining on SDS-PAGE.
5 M. J. Clark, C. Harrison, H. Zhong, R. R. Neubig, and J. R. Traynor, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptors;
AppNHp, 5'-adenylylimidodiphosphate;
AR, adrenergic receptor;
GAP, GTPase-activating protein;
GST, glutathione
S-transferase;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
RGS, regulator of G protein
signaling;
CHO, Chinese hamster ovary;
ERK, extracellular
signal-regulated kinase;
aa, amino acid(s);
ANOVA, analysis of
variance;
D, drug or agonist;
R, receptor;
G, G protein.
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