 |
INTRODUCTION |
The
subunits of heterotrimeric G proteins function as
molecular switches that determine active and inactive states of
signaling pathways. The crystal structures of
G
t1 and
G
i1 in their activated, inactive, and transition state
forms have revealed the nature of the molecular switches (switches I, II, and III), which are local conformational changes in the regions around the nucleotide binding pocket depending on whether GTP or GDP is
bound (1-4). G protein
subunits are activated by seven helical
membrane receptors that catalyze the exchange of GDP for GTP by
decreasing the affinity of GDP for the
subunit. The lifetime of the
active state of an
subunit is defined by the rate of intrinsic
GTPase activity that converts GTP to GDP. Therefore, termination of the
signal response is dependent on G
GTPase activity. Purified G
subunits typically display slow (~ 4/min) GTP hydrolysis that often
cannot account for the deactivation rates of G protein-controlled
processes, for example phototransduction (5) and ion channel
regulation (6).
A large family of regulatory proteins that modulate the inactivation
rate of G
subunits by accelerating their intrinsic GTPase activity
has recently been identified (7, 8). These proteins, known as
regulators of G protein signaling (RGS), are encoded by at least 19 genes and have been identified in mammalian tissues based on homology
to the diagnostic RGS core domain of ~ 120 amino acid residues.
It is not yet certain that all RGS proteins are GAPs
(GTPase-accelerating proteins) because several very recently identified
RGS domains of D-AKAP (9), axin (10), and Lsc (11) are less conserved,
especially at the positions that correspond to the contact sites of
RGS4 with G
i1 (12). However, the RGS domain of p115,
which belongs in this less conserved RGS family, has been shown to have
GAP activity for G
12 and G
13 but not for
Gs, Gi, and Gq subfamilies of G
proteins (13), suggesting that these new RGS proteins may have GAP
activity toward other G proteins.
The crystal structure of the RGS4·G
i1 complex
identified the three conformational switch regions of G
subunits as
the major structural determinants of RGS4 binding to G
i1
(12). Unlike Ras·GAP, which contributes a catalytic Arg (14) to the
active site of Ras, the core domain of RGS4 does not contribute
catalytic residues and is thought to accomplish its GAP function
primarily by stabilizing the switch regions of G
in the transition
state. The crystal structure also suggests that RGS proteins may
down-regulate the activity of G
subunits not only by acting as GAPs
but also by competing for effector binding to G
switch regions
because these switches are involved in effector binding. Indeed, RGS4 and GAIP can block activation of phospholipase C
1 by
constitutively active G
q·GTP
S (15). Similar
observations have been made for RGS4, GAIP, and RGSr, which compete
with the G
t effector, the
subunit of cGMP
phosphodiesterase (P
), for interaction with G
t (16,
17).
Most RGS proteins studied to date show relatively little specificity
for members of the Gi subfamily of G
proteins and
discriminate minimally among them. Some RGS proteins, for example RGS4
and GAIP, are not selective among several subfamilies of G proteins, being able to act as GAPs toward members of the Gq
subfamily as well. Surprisingly, no RGS proteins accelerating
Gs GTPase activity have been identified so far, but
G
s can be converted into a substrate for RGS16 and RGS4
by a single mutation Asp229
Ser (18). Lack of RGS
specificity was studied mainly by in vitro assays using
expressed RGS domains; thus, the data do not necessarily exclude higher
specificity between particular RGS proteins and G proteins in the
cellular environment. Lipid modification and membrane association
domains can target RGS proteins to certain cellular compartments (19).
For example, RGS12 contains a PDZ domain, which could specifically
target it to certain G protein-coupled receptors (20). Additionally,
the regions flanking the core domain of RGS proteins may possess other
structural determinants for specific interaction with G
subunits.
Finally, an effector-mediated modulation of RGS function may provide
another selectivity filter.
In rod photoreceptor cells transducin GTPase activity is too slow
(1-2/min) to account for the physiologically measured light response.
The clear functional requirement for transducin inactivation promoted
biochemical studies that demonstrated that the intrinsic rate of GTP
hydrolysis of transducin is enhanced significantly by concentrated
suspensions of rod outer segment membranes (21, 22). Further studies
have identified that the inhibitory subunit of P
, together with a
membrane factor, cooperatively stimulate the GTPase rate of transducin
(23-26). The COOH-terminal 25 amino acids of P
possess the GAP
determinants (24), and Trp70 located within this region is
critical for GAP activity of P
(27). A P
Trp70
Ala mutant expressed in transgenic mouse rods caused a decrease in the
recovery rate of the flash response (28), suggesting that the normal
deactivation of transducin in vivo, similar to its
deactivation in reconstituted membranes, requires its interaction with
P
. Very recently the membrane factor was identified as RGS9 (29).
The predicted amino acid sequence of bovine RGS9 revealed a conserved
RGS domain located at the COOH terminus of the molecule. The extended
NH2 terminus of RGS9 contains approximately a 190-amino acid region that is homologous to the NH2-terminal domain
of RGS7 and Egl-10. Within this region there is an approximately
80-residue subdomain homologous to the consensus sequence of the DEP
domain of unknown functional significance, found in a number of
signaling proteins (30).
RGS9 is expressed predominantly in the retina at levels significantly
higher in cones than in rods (31). RGS9 is tightly associated with
membranes and can be solubilized at high concentrations of detergent.
There is no evidence for lipid modification or membrane-spanning regions for RGS9, based on analysis of its primary structure. However,
Cowan et al. (31) have proposed that a strong electrostatic interaction with the membranes might be the dominant force in its
membrane localization. Several other RGSs found in the retina have been
shown to stimulate the GTPase activity of transducin: RGSr/16 (16, 32),
RGS4, GAIP (17), and RET-RGS1 (33). However, unlike other retinal RGS
proteins, the GAP activity of RGS9 is substantially (3-fold)
accelerated by P
(29). Immunodepletion of RGS9 from detergent
extracts of rod outer segments (ROS) demonstrated that RGS9 is the
predominant source of GAP activity in ROS. All of the immunological and
biochemical data (29, 31) indicate that RGS9 is the membrane-associated
G
t GAP that acts cooperatively with P
in stimulation
of G
t GTPase (24, 25, 34).
Very recently another important function has been defined for RGS9 in
photoreceptor cells. Retinal RGS9 is able to inhibit the activity of
guanylyl cyclase, thus controlling the levels of cGMP (35). This
finding suggests an additional modulatory role of RGS9 downstream of
the effector, cGMP phosphodiesterase, as the linker between
phosphodiesterase and guanylyl cyclase.
In this study we focus on further biochemical characterization of RGS9.
First, we have examined the specificity of RGS9 GAP activity using
homologous G
t and G
i1 as the substrates
and found that RGS9 is a more potent GAP for G
t. An
analysis of various G
t/G
i1 chimeras for
their ability to be substrates for RGS9 has revealed that the
GAP-responsive determinants reside within the
-helical domain of
G
t. Kinetic analysis of RGS9 binding to the
G
t·P
complex shows a distinct, nonoverlapping
pattern of a cooperative interaction of RGS9 and P
with
G
t, providing the structural basis for the acceleration
of RGS9 GAP activity by P
.
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EXPERIMENTAL PROCEDURES |
Materials--
GTP, GTP
S, GDP, deoxyribonucleotides, and
imidazole were purchased from Boehringer Mannheim. Restriction and DNA
modification enzymes were obtained from Boehringer Mannheim or Life
Technologies, Inc. Ni-NTA agarose was a product of Qiagen Inc.
[
-32P]GTP (30 Ci/mmol) was obtained from NEN Life
Science Products. All other reagents were from Sigma or other
sources described previously (36).
Preparation of ROS Membranes, Gt,
G
tGDP, G
tGTP
S,
G
1
1, G
i1, and
P
--
Gt, G
tGTP
s,
G
tGDP, G
1
1 and rhodopsin
containing ROS membranes treated with urea were prepared as described
(37). G
i1, NH2-terminally modified with
His6-tag, was expressed in Escherichia coli and
purified as described by Skiba et al. (36). Wild type
subunit of phosphodiesterase was expressed in E. coli and
purified as described (38).
Preparation of G
t/G
i1
Chimeras--
Chi6b is a derivative of Chi6 described by Skiba
et al. (36) in which amino acid residues 216-295 of
G
t are replaced with the corresponding region from
G
i1 (residues 220-299). Chi6b was generated by changing
Cys347 in Chi6 to Ser. Mutagenesis, E. coli
expression, and labeling of Chi6b with a thiol-specific fluorescent
reagent Lucifer Yellow vinyl sulfone (LY) were carried out as described
by Yang et al. (39). The stoichiometry of Chi6b labeling
with LY, calculated as a ratio of the concentration of LY and the
concentration of chimera in the labeled sample, was 1:1.
Chimera Gi/GtH is a derivative of His6-G
i1
in which residues 60-177 of G
i1 encompassing the
-helical domain are replaced with the corresponding region of
G
t, residues 56-173. The chimeric gene was constructed
by introduction of unique restriction enzyme sites flanking the DNA
fragments of G
i1 cDNA and G
t cDNA
which encode the
-helical domain. A MluI restriction
enzyme site (3'-end of the fragment) was inserted in both
G
t and G
i1 cDNA, and a BstXI site, which is present in G
i1 gene, was
inserted only in G
t cDNA (5'-end of the fragment)
using PCR-based mutagenesis with corresponding oligonucleotide
primers-mutagenes. The BstXI-MluI DNA fragment of
G
t was inserted into the G
i1 cDNA
after cutting off the corresponding fragment of G
i1 with
BstXI and MluI restriction enzymes.
Chimera Chi6/GiH contains the
-helical domain of G
i1
in the context of Chi6. The G
t
-helical domain of
this chimera (residues 56-174) was replaced with the corresponding
region of G
i1 using the same approach as described for
the construction of chimera Gi/GtH. The schematic structures of
G
t/G
i1 chimeras are shown in Table
I.
Cloning and Expression of RGS9--
Total RNA as a template for
cDNA synthesis was purified from fresh frozen bovine retinas using
RNeasy Total RNA kit (Qiagen Inc.). Random cDNA for PCR was
synthesized using Advantage RT-for-PCR kit
(CLONTECH) with an oligo(dT) primer. A DNA fragment
encoding residues 284-461 of the bovine retinal RGS9 core domain was
amplified by PCR using the specific primers
5'-AAAGGATCCCTGGTGGACATCCCAACCAAG (upstream) and
5'-TTTAAGCTTACGTGGTGGCCGCCTCCCGC (downstream) containing BamHI and HindIII restriction sites respectively
(underlined). The resulting PCR product (550 base pairs) was cut with
BamHI and HindIII and ligated with the large
fragment of the expression vector pQE30 (Qiagen) digested with the same
restriction enzymes. The DNA sequence of this construct was confirmed
by DNA sequencing over the PCR-amplified region using type III/IV and
reverse sequencing primers (Qiagen). The subcloned sequence contained
one nucleotide substitution (T
A) which resulted in the
conservative Ser400
Thr mutation. The resulting
construct (RGS9) encodes a protein where the RGS sequence is preceded
by the sequence MRGSHHHHHHGS containing a His6-tag and
RGS-His antibody (Qiagen) epitope. Recombinant protein was expressed in
E. coli JM109 and purified as described by He et
al. (29). The final yield of RGS9 ranged from 5 to 10 mg of more
than 85% pure protein/liter of bacterial culture.
GTPase Assay--
Single turnover GTPase reactions were
performed under conditions described by He et al. (29) with
some minor modifications. Freshly illuminated urea-washed ROS membranes
(final concentration 15 µM) were reconstituted with 1 µM Gt or 1 µM G
t
and 1 µM G
1
1 in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.02 mM AMP-PNP and
incubated for 10 min in the dark at room temperature. The reaction was
started by the addition of RGS9 and 200 nM
[
-32P]GTP (~ 105 cpm/pmol) to the
reconstituted membranes and quenched by the addition of 100 µl of 7%
perchloric acid. Nucleotides were removed by activated charcoal, and
free 32Pi was measured by scintillation counting.
Fluorescent Assay--
Binding of RGS9 and/or P
to Chi6b-LY
as well as competition among G
i1, G
t,
chimeras, and Chi6b-LY for binding to RGS9 was monitored by the
fluorescent change of a single reporter group attached to
Cys210 located in the switch II region. Fluorescent
measurements were performed on an Aminco-Bowman Series 2 Luminescence
Spectrometer (SLM Aminco) at room temperature in 50 mM
Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM
MgCl2 (buffer A) using excitation at 430 nm and emission at
520 nm. In the direct binding experiment Chi6b-LY (50 nM)
was initially activated by 10 mM NaF and 30 µM AlCl3. The fluorescence of activated
Chi6b-LY in the absence of RGS9 or P
is defined as the base line.
The fluorescence increase upon the addition of an interacting protein
is expressed as a percent of the initial fluorescence of activated
Chi6b-LY after the addition of the reactant. Data were best fit to
Equation 1,
|
(Eq. 1)
|
where Y0 and Ymax
represent, respectively, the initial and maximum values of the binding
function which describes the normalized fluorescence at a given
concentration of interacting protein; X is a logarithm of
the protein concentration; and H is the Hill coefficient.
For competition measurements Chi6b-LY (50 nM) activated with AlF4
was mixed with 100 nM RGS9 in buffer A. A typical increase in the fluorescence
was 50-60% of the initial. The fluorescence of the
Chi6b-LY-AlF4
·RGS9 complex was set
to 100%. The fluorescent change (decrease) was monitored after the
addition of increasing concentrations G
t,
G
i1, or chimera and normalized as the percent of maximal fluorescence. The initial fluorescence of Chi6b-LY activated with AlF4
was set to 0%. Data were best
fit to Equation 1. Kd values for the binding of
G
t, G
i1, and chimeras to RGS9 were determined from their binding or competition curves based on calculated EC50 values, the concentration of Chi6b-LY in the assay (50 nM), and the Kd value for
Chi6-LY-AlF4
·RGS9 complex (190 nM, see Fig. 5B).
General Methods--
Protein concentration of G
subunits,
P
, RGS9, and G
were determined spectrophotometrically using
calculated extinction coefficients based on the number of Trp and Tyr
residues. The measured concentrations of
subunits were corrected
for the amount of functional protein based on a fluorescent assay
detecting an AlF4
-dependent increase in
Trp fluorescence, as described in Ref. 36. To monitor an intrinsic
AlF4
-dependent
conformational change of G
t, G
i1, and
chimeras, tryptophan fluorescence was determined with excitation at 280 nm and emission at 340 nm. The fluorescence of G
(200 nM) in buffer A was measured before and after the addition
of 10 mM NaF and 30 µM AlCl3.
SDS-polyacrylamide gel electrophoresis of proteins was performed
according to the method of Laemmli (40).
Curve fitting of the experimental data and kinetic analysis were
performed using Prism 2.01 for Windows 95 from GraphPad.
 |
RESULTS |
Expression and Purification of the RGS9 RGS Domain--
The
sequence corresponding to the RGS domain of the bovine retinal RGS9
(RGS9) (residues 284-461) was amplified from total retinal RNA using
the gene-specific primers based on its primary structure (29),
NH2-terminally modified with His6-tag, and
expressed in E. coli. It was purified on Ni-NTA resin under
denaturing conditions (8 M urea) followed by a renaturation
step using a slow stepwise dialysis to remove denaturant. Despite
significant losses of the RGS9 during renaturation (~60-70% of
total protein before dialysis) caused by reaggregation, the yield of
the remaining soluble protein/liter of the bacterial culture ranged
from 5 to 10 mg. The resulting protein migrated as a 25-kDa band
corresponding to its calculated molecular mass and was more that 85%
pure (Fig. 1).

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Fig. 1.
Expression and purification of RGS9.
SDS-polyacrylamide gel (10-20%) stained with Coomassie is shown.
Lane 1, molecular weight markers. Lane 2,
purified on the Ni-NTA agarose column and renaturated
His6-RGS9. of RGS9 (1 µM) and P at 5 s. The reaction
was stopped at 10 s by the addition of perchloric acid. Data
points represent mean percent GTP hydrolysis ± S.E.
(n = 3).
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RGS9 Is a More Potent Stimulator of GTPase Activity of
G
t than G
i1--
The effect of RGS9 on
the G
t GTPase activity was measured using a single
turnover GTPase assay. 4 M urea is known to inactivate endogenous GAP activity in the ROS membranes without its physical removal (25, 31). The rate of GTP hydrolysis by purified
G
t reconstituted with purified
G
1
1 and urea-washed ROS was 0.028 ± 0.001 s
1 (Fig.
2A), which is in agreement
with previously published data (0.022 s
1) (29, 32). The
addition of the RGS9 resulted in a dose-dependent stimulation of transducin GTPase activity with an approximate 10-fold
increase at the maximal dose (10 µM RGS9,
k = 0.30 ± 0.007, Fig. 2A). A similar
stimulatory effect of RGS9 on GTPase activity of G
t was
observed when holo Gt was reconstituted with urea-washed ROS membranes (data not shown). To evaluate the specificity of RGS9 for
different G
s we have determined its effect on the GTPase activity of
G
i1, a close structural homolog of G
t. It
is known that rhodopsin can catalyze GDP/GTP exchange on
G
i1 in the presence of G
1
1
with a rate similar to that of G
t (36, 41). Indeed, G
i1 GTPase activity in the presence of urea-washed ROS
membranes and
1
1 (k = 0.031 ± 0.05 s
1, Fig. 2B and Table I)
was in good agreement with the kinetic parameters determined in the
single turnover GTPase assay using a nucleotide autoexchange assay (42,
43). Surprisingly, RGS9 produced only a 2-fold enhancement
(k = 0.058 ± 0.004 s
1) of
G
i1 GTPase activity at 10 µM, the
concentration that maximally stimulated the GTPase activity of
G
t (Fig. 2B). However, at higher concentrations, stimulatory effects of RGS9 on G
i1
increased, reaching approximately 5-fold at 40 µM (Fig.
2B and Table I). This finding demonstrates a high
GTPase-activating specificity of RGS protein for G
from the
Gi subfamily of heterotrimeric G proteins.

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Fig. 2.
Stimulation of GTPase activity of
G t (panel A) and
G i1 (panel B) by
RGS9 and effect of P (panel
C). Time courses of GTP hydrolysis were determined in a
single turnover GTPase assay as described under "Experimental
Procedures." Urea-washed ROS membranes (15 µM
rhodopsin) were reconstituted with 1 µM
 t and 1 µM G t or 1 µM G i1 in the presence of different
concentrations of RGS9. The reaction was started at time 0 with 200 nM [ -32P]GTP. At the indicated times the
reaction mixtures were quenched with perchloric acid. The GTPase
reactions were analyzed using an exponential function: % GTP
hydrolyzed = 100(1 e kt), where k is a rate
constant of GTP hydrolysis. Panel C shows the effect of P
on activation of G t GTPase by RGS9.
Urea-washed ROS membranes (15 µM) were reconstituted with
1 µM G t and 1 µM
 t. The GTPase reaction was initiated at time 0 by the
addition of 200 nM [ -32P]GTP followed by
the addition
|
|
The Switch III Region of G
t Is Not Involved in the
Selective Interaction with RGS9--
The crystal structure of the
RGS4·G
i1 complex combined with the data on mutational
analysis of G
s indicate that the three conformational switch regions
of G
are the major structural determinants of the RGS-G
interface. Although there is a high degree of conservation between
G
i1 and G
t in the switch regions, switch
III is the most divergent. To analyze the role of the switch III region
in the G
t GTPase-activating specificity for RGS9, we
measured the ability of RGS9 to stimulate the GTPase activity of the
functional analog of G
t (Chi6) in which the switch III
region of G
t was replaced with the corresponding region
of G
i1 (residues 216-295). Functional analysis of Chi6
has revealed its similarity to G
t in interaction with
rhodopsin and G
t (36). RGS9 stimulated the GTPase
activity of Chi6 to an extent similar to that of G
t (approximately 8-fold) (Table I), suggesting that the switch III region
of G
t plays little if any role in defining the
specificity of G
t GTPase acceleration by RGS9. Chi6
expressed in E. coli lacks an NH2-terminal
myristoyl group. The ability of RGS9 to stimulate the GTPase activity
of Chi6 to an extent similar to that of myristoylated G
t
indicates that the lipid group does not play a critical role in this process.
The Molecular Determinants of Specific GTPase Stimulation by RGS9
Reside within the Helical Domain of G
t--
Besides the
switch III region of G
t, which does not contribute to
its sensitive response to RGS9, switches I and II can be considered as
the main GAP-responsive regions of G
subunit because they have a
number of contacts with RGS protein according to the crystal structure
of the G
i1·RGS4 complex (44). The nearly identical
conformation of the switch regions for activated and transition state
forms of G
t and G
i1 (1, 4, 45) suggests that only a difference in the primary structure of the switches could
account for the different GTPase stimulation effects of RGS9 on
G
t and G
i1. However, residues in the
switch II region are identical between G
i1 and
G
t, whereas in the switch I region only
Val185 of G
i1, which contacts RGS4, is
replaced with Ile in G
t. To probe the role of other
regions of G
t in specifying the interaction with RGS9 we
have replaced the
-helical domain of G
i1 with the corresponding domain of G
t and vice versa and
evaluated the ability of RGS9 to stimulate GTPase activity of the
resulting proteins. Replacement of the
-helical domain of
G
i1 with the
-helical domain of G
t
(chimera Gi/GtH) resulted in an increased stimulation of its GTPase
activity by RGS9. The intrinsic rate of GTP hydrolysis for chimera
Gi/GtH was 0.032 s
1. A maximal stimulation effect by RGS9
for this chimera was approximately 8-fold at 10 µM RGS9
(Table I), similar to the RGS9 effect on G
t and Chi6
(Fig. 2A and Table I). Thus, the presence of the
-helical
domain of G
t in the G
i1 context is
sufficient to provide the G
t-like specificity for RGS9.
The GTPase activity of the complementary chimera (Chi6/GiH), where the
-helical domain of G
t in Chi6 was replaced with the
corresponding region of G
i1, was G
i1-like
in its interaction with RGS9 (Table I). These data indicate that the
-helical domain of G
t possesses the determinants of
RGS9 specificity.
P
Potentiates G
t GTPase Stimulation by
RGS9--
P
is known to cooperate with another protein to stimulate
the GTPase rate of G
t (24, 34). We have shown previously
that the
subunit of cGMP phosphodiesterase inhibits the ability of RGS4 and GAIP to stimulate the GTPase activity of G
t,
suggesting overlapping binding sites on G
t. In contrast,
RGS9 acts cooperatively with P
to stimulate GTP hydrolysis (29),
thus potentially implicating P
in the physiological regulation of
the active lifetime of G
t in rods. The effect of P
on
the G
t GTPase stimulation by RGS9 was examined in the
single turnover GTPase assay in the presence of a concentration of RGS9
which gives intermediate GTPase acceleration (1 µM). P
noticeably enhanced G
t GTPase acceleration by RGS9 (Fig.
2C). The maximal effect of P
observed at a concentration of 500 nM was approximately a 2.5-fold enhancement
(k = 0.11 s
1). In the absence of RGS9,
P
did not accelerate G
t GTPase up to a concentration
of 5 µM when reconstituted with urea-washed ROS membranes.
The Affinity of RGS9 for G
t, G
i1, and
Chimeras--
To compare the functional effects of RGS9 with its
affinity for different substrates we have developed a sensitive
fluorescent assay. As we reported recently (39), Cys210
located at the distal end of the switch II region of G
t
can be labeled selectively with the thiol-specific fluorescent reagent Lucifer Yellow. G
t and Chi6 have only two cysteine
residues accessible for modification with LY, located at positions 210 and 347. We have replaced Cys347 of Chi6 with Ser. The
resulting mutant (Chi6b) was labeled selectively at the only accessible
cysteine (Cys210) with the fluorescent group. LY at
Cys210 of Chi6b was a reporter of the activating
conformational change in the switch II region. The addition of
AlF4
to the labeled protein resulted
in a profound increase in LY fluorescence (200 ± 20%; Fig.
3). The addition of RGS9 increased the
fluorescence of Chi6b-LY in a dose-dependent manner (see
Fig. 5B). The maximal fluorescence increase of
AlF4
-activated Chi6b-LY was 108 ± 5% in the presence of 2 µM RGS9 (Fig. 3). The binding
was completely reversible by adding an excess of G
t
(Fig. 4A) or Chi6 (not shown).
In the absence of AlF4
, the addition
of 2 µM RGS9 to Chi6b-LY caused no detectable
fluorescence change (data not shown). The Kd of the
Chi6b-LY·RGS9 complex calculated from the binding curve was 190 ± 9 nM (Fig. 5B).

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Fig. 3.
Binding of RGS9 to Chi6b-LY. Sequential
fluorescence increase of Chi6b-LY (50 nM) (1 above the arrow) upon the addition of 10 mM NaF
and 30 µM AlCl3 (2), followed by
the addition of 2 µM RGS9 (3). The
AlF4 -dependent increase of
Chi6b-LY fluorescence was 200 ± 20%. The maximal fluorescent
increase of AlF4 -activated chimera in
the presence of 2 µM RGS9 was 104 ± 5%. The
fluorescence trace represent one of five independent similar
experiments.
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Fig. 4.
Binding of
G t,
G i1, and chimeras to RGS9.
Panel A, competition between
G tGDP-AlF4 or
G tGTP S and Chi6b-LY for binding to RGS9. The
fluorescence of the complex of RGS9 (100 nM) with Chi6b-LY
(50 nM) in the presence of
AlF4 was measured before and after the
addition of increasing concentrations of
G tGDP-AlF4
(squares) or G tGTP S
(triangles). Panel B, competition between
G i1 or chimeras for binding to RGS9. The fluorescence of
Chi6b-LY-AlF4 (50 nM) in
the presence of 100 nM RGS9 was measured before and after
the addition of increasing concentrations of G i1
(squares), chimera Gi/GtH (triangles), or
Chi6/GiH (inverted triangles). The fluorescence change is
expressed as a percent of maximal change (100% was fluorescence
Chi6b-LY-AlF4 -RGS9 complex before adding G
subunit, 0% was the initial fluorescence of Chi6b-LY before adding
RGS9) and plotted against the G concentration using a four-parameter
logistic function (sigmoidal curve) as described under "Experimental
Procedures." Data points represent the mean percent fluorescent
change ± S.E. (n = 3).
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Fig. 5.
Interaction of P and
RGS9 with Chi6b-LY. Panel A, the increase in
fluorescence of AlF4 -activated
Chi6b-LY (50 nM) was measured after the addition of
increasing concentrations of P in the absence (triangles)
or in the presence (squares) of 500 nM RGS9. The
initial fluorescence of the
Chi6-LY-AlF4 or
Chi6b-LY-AlF4 ·RGS9 complex, before
the addition of P , was set at 0. Fluorescence change, expressed as a
percent of the initial fluorescence, is plotted against the P
concentration. The solid lines represent the best fit to the
four-parameter logistic (sigmoidal) equation as described under
"Experimental Procedures." The best parameter values for the curve
1 (triangles) are: Kd = 100 ± 11 nM; Fmax, 99%; Hill slope, 1.2;
for curve 2 (squares): Kd = 70 ± 8 nM; Fmax, 104%: Hill slope, 1.3, p = 0.31. Panel B, the increase in
fluorescence of AlF4 -activated
Chi6b-LY (50 nM) was measured after the addition of
increasing concentrations of RGS9 in the absence (triangles)
or in the presence (squares) of 500 nM P .
Data were plotted as described in panel A. The calculated
kinetic parameters are: curve 1 (triangles)
Kd = 190 ± 9 nM;
Fmax, 114%; Hill slope, 1.12; curve 2 (squares) Kd = 67 ± 5 nM; Fmax, 103%; Hill slope,
1.15; p = 0.035. Data points represent the mean percent
fluorescent increase ± S.E. (n = 3).
|
|
To determine the affinity of RGS9 for different G
s we used a
competition approach. Fig. 4A shows that unlabeled
G
t completely displaces the Chi6-LY from its complex
with RGS9 in a dose-dependent manner. The
Kd of the
G
t-AlF4
·RGS9 complex
calculated from the competition curve was 185 ± 8 nM.
Chi6 activated with AlF4
had a similar
affinity for RGS9 (Kd 174 ± 11 nM,
Table I), closely corresponding to the affinity of this complex
calculated in the direct binding experiment (Fig. 5B).
However, G
tGTP
S was less potent in its ability to
compete with Chi6b-LY for binding with RGS9 compared with
G
tGDP-AlF4
. The
Kd of the G
tGTP
S·RGS9 complex
calculated from the competition curve was 0.9 µM (Fig.
4A). These data provide an accurate measurement of the
difference in affinity of RGS protein for G
s in GTP-bound and
transition state analog forms, which was reported earlier based on
immunoprecipitation (46, 47) and bead precipitation (32) of the
G
·RGS complexes.
G
i1 was also able to compete with Chi6b-LY for binding
to RGS9 and displaced the labeled protein from the complex. The
affinity of G
i1 for RGS9 calculated from the competition
curve was more than 10-fold lower (Kd 2 ± 0.25 µM, Fig. 4B) than that of G
t
(185 nM). The decreased affinity of G
i1 for
RGS9 is consistent with its decreased ability to stimulate
G
i1 GTPase activity.
To determine the region of G
t which is responsible for
the increased affinity to RGS9 we used
G
t/G
i1 chimeras with exchanged helical
domains (Table I) in the fluorescent competition assay (Fig.
4B). Replacement of the
-helical domain of
G
i1 with the corresponding region of G
t
(chimera Gi/GtH) resulted in a more than 10-fold increase in its
affinity for RGS9 (Kd 170 ± 7 nM)
compared with G
i1 (Kd 2 ± 0.25 µM). On the other hand, the reciprocal chimera where the
-helical domain of G
t was replaced with the
corresponding domain of G
i1 (chimera Chi6/GiH) exhibited
decreased affinity for RGS9 (Kd 1.6 ± 0.1 µM) compared with G
t or Chi6, but similar
to G
i1 (Table I). Comparison of the functional and
binding data indicates that the GTPase stimulation activity of RGS9
correlates well with its affinity for the substrate.
Effect of RGS9 on the Interaction of P
with Chi6b--
P
potentiates the RGS9-mediated stimulation of G
t GTPase.
However, the mechanism responsible for this effect is not yet known.
Different structural events may cause this effect. First, P
could
participate in the trimeric complex by binding directly to RGS9.
Alternatively, P
could induce a conformational change on
G
t resulting in a higher affinity for RGS9. Third, P
could participate directly in stabilizing the transition state of the G
t·GTP complex. To understand how P
potentiates the
GAP effect of RGS9, we have studied the interaction of P
with the
Chi6b-LY·RGS9 complex in the fluorescent assay.
We first studied P
interaction with Chi6b-LY. P
increased the
fluorescence of Chi6b-LY in the presence of
AlF4
in a dose-dependent
manner. The binding was specific and completely reversible by the
addition of the unlabeled chimera or trypsin-activated phosphodiesterase (data not shown). The maximal fluorescence increase at saturation was 104 ± 5%. The Kd of the
P
·Chi6b-LY·AlF4
complex
calculated from the binding curve was 100 ± 11 nM
(Fig. 5A). Thus, LY at Cys210 in the switch II
region of G
t is a sensitive reporter of P
binding.
The binding of P
to Chi6b-LY was activation-dependent, since no appreciable change in fluorescence was observed in the absence
of AlF4
(data not shown).
To determine the effect of RGS9 on P
binding to Chi6b-LY in the
fluorescent assay, we first formed the RGS9·Chi6b-LY complex by
mixing RGS9 (500 nM) with the labeled chimera (50 nM). The fluorescence increase was an indicator of complex
formation. Under these conditions more than 90% of the chimera was in
complex with RGS9 as determined from the binding curve in Fig.
5B (triangles) and, therefore, the effect of P
binding to free Chi6b-LY (less than 10%) is negligible. In the
presence of RGS9, P
further increased the fluorescence of LY-labeled
Chi6b in a dose-dependent manner reaching a maximal effect
(
Fmax) of 99% from the initial fluorescence of Chi6b-LY-AlF4
similar to the
Fmax of P
binding to free Chi6b-LY (Fig.
5A, 104%). RGS9, prebound to Chi6b-LY, did not change the
affinity of P
for the chimera significantly (Kd
70 ± 8 nM, p = 0.31, Fig.
5A). The similar affinity of P
for Chi6 with or without
RGS9 present indicates as well that P
and RGS9 binding sites on
G
t do not overlap.
Effect of P
on the Interaction Between RGS9 and
Chi6b-LY--
To evaluate whether P
can modulate the binding of
RGS9 to G
, we determined the affinity of RGS9 to free and
P
-complexed Chi6b-LY in the fluorescent assay. The fluorescence
experiment was set up similarly to that described in the previous
section. This time, we preformed the complex of Chi6b-LY (50 nM) with P
(500 nM). The increase in the
fluorescence of Chi6b-LY after the addition of P
indicated that more
than 90% of the chimera was in complex with P
. The addition of
increasing concentrations of RGS9 enhanced the fluorescence of the
chimera·P
complex (Fig. 5B). The affinity of RGS9 for
the chimera·P
complex, as calculated from the binding curve, was
67 ± 5 nM, nearly 3-fold higher than the affinity of
RGS9 for the chimera alone (Kd 190 ± 8 nM, p = 0.035, Fig. 5B). The
maximum increase in fluorescence of Chi6b-LY upon binding of RGS9 in
the presence of P
(104 ± 2%) was similar to its effect on the
chimera alone (maximal fluorescent change 114 ± 4%). This
indicates that the environmental change around the LY group at
Cys210 of Chi6b as a result of RGS9 binding is the same
regardless of whether P
is in the complex or not.
 |
DISCUSSION |
Visual stimuli produce very rapid activation of rod
photoreceptors, and the inactivation must be very rapid as well for
perception of movement. Early biochemical measurements of the GTPase
rate of the rod G protein transducin showed the same slow GTPase rate as other G proteins. Because of the clear functional requirement for
rapid turn-off, transducin was the first heterotrimeric G protein whose
GTPase activity was shown to be regulated. The intrinsic rate of GTP
hydrolysis of transducin is enhanced significantly by concentrated
suspensions of ROS membranes, providing the initial evidence for GAPs
in the ROS (21, 22). Biochemical analysis showed that the inhibitory
subunit (P
) of the visual cascade effector, cGMP phosphodiesterase,
could accelerate the GTP hydrolysis rate of transducin (23). Further
studies demonstrated that P
alone is not the transducin GAP and
requires another unknown membrane protein to activate GTP hydrolysis by
transducin (24, 26, 34). Together P
and the membrane factor
cooperatively accelerate the GTPase rate of transducin. Very recently
this unknown membrane protein was identified to be a member of a large
family of RGS proteins, RGS9 (29).
Retinal RGS9 is a unique GAP in its ability to act synergistically with
P
. Stimulation of the G
t GTPase activity by RGS9 is
potentiated by P
(29), unlike other RGS proteins found in the retina
RGSr, RET-RGS1, RGS4, GAIP (17, 32, 33). Does this imply that the
effector-mediated mechanism of GTPase stimulation by RGS9 is different
from that for other effector-independent RGS proteins highlighted by
the crystal structure of the RGS4·G
ia1 complex? To
answer this question we first studied the specificity of GTPase
activation by retinal RGS9. The majority of mammalian RGS proteins
studied to date can stimulate GTP hydrolysis of several members of the
G
i family proteins. However, accurate comparisons of
kinetic parameters of a particular RGS protein interaction with
different G proteins have been difficult because of a lack of methods
to compare binding and functional effects. Comparison of the affinity
of RGS for structurally homologous proteins may provide important
information on the molecular principles of this interaction.
Specificity Determinants of RGS9 Interaction with
G
t--
Our data indicate that the core domain of the
RGS9 is a more potent accelerator of the G
t GTPase than
of its close structural homolog G
i1. The same
stimulation effect of RGS9 on G
t was detected at 10-fold
lower concentrations than on G
i1. The difference in the
maximal effect by RGS9 on G
t (10-fold) and
G
i1 (5-fold) is also evident. The less potent
G
i1 GTPase stimulation by RGS9 compared with
G
t is a result of its decreased affinity for
G
i1. What is the structural basis for such a specificity
for RGS9? The crystal structure of the RGS4-G
i1 complex
combined with the data on mutational analysis of G
s (12, 43, 49, 50)
indicates that the three conformational switch regions of G
are the
major structural determinants of the RGS-G
interface. These switch regions are highly homologous in G
i1 and
G
t, and we showed that they are not responsible for the
specificity. We found instead that the helical domain determines the
specificity of RGS-G
interaction. We can switch the specificity by
switching the helical domains. This region is less homologous between
G
i1 and G
t.
There are several structural differences in the
-helical domain of
G
t and G
i1 which potentially could
participate in contacts with RGS9. One of the local differences is
evident in the conformation of residues 108-120 (G
i1),
which correspond to the distal end of helix B and the following loop
(4, 45) (Fig. 6). It is noteworthy that
Glu116, the only contact of the helical domain of
G
i1 with RGS4, is located in this region.
Glu116 is conserved in G
i1 and
G
t and interacts with Glu161 and
Arg166 of RGS4 which correspond to Lys387 and
Ala392 of RGS9. Therefore, the different conformation of
Glu116 in the
-helical domain of G
i1 and
G
t (Fig. 6) may result in abolishing this contact with
RGS9 for G
i1. Alternatively, the slight difference in
the packing of the helical and GTPase domains of G
t and
G
i1 may result in extended orientation of the switch regions and potential RGS9 contact forming residues in the helical domain for G
i1 compared with G
t. However,
conserved interdomain contacts for G
i1
(Asp150-Lys270 and
Arg178-Glu43) and G
t
(Asp146-Lys266 and
Arg174-Glu39) most likely assure a similar
domain packing in both chimeras.

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|
Fig. 6.
Superposition of
-helical domains of
G tGDP-AlF4
(45) and
G i1GDP-AlF4
(4). Blue shows G i1, red
indicates G t. The side chains depicted as ball-and-stick
models represent Glu112 of G t
(yellow) and Glu116 of G i1
(cyan). The image was generated using WebLab ViewerLite 3.1 from Molecular Simulation Inc.
|
|
P
Accelerates G
t GTPase Activity Stimulated by
RGS9 by Increasing Its Affinity for G
t--
Among the
many RGS proteins found in the retina only RGS9 can stimulate the
GTPase activity of G
t synergistically with P
. Unlike
RGS9, other retinal RGSs are either not responsive to (33) or are
inhibited by P
(17, 32). In some cases it was shown that P
competes with RGS proteins for binding to G
t, indicating that P
and RGS binding sites on G
t may overlap (16,
17). Our data demonstrate that RGS9 did not significantly change the binding of P
to the labeled derivative of G
t
(p value = 0.31, t test), indicating that
P
and RGS9 binding sites on G
t do not overlap. The
lack of effect of RGS9 on P
binding to G
t also indicates that there is no direct interaction of P
with RGS9 in the
G
·P
·RGS9 complex. On the other hand, P
in complex with G
increases the binding of RGS9 to the complex approximately 3-fold
(p value = 0.035, t test). The increased
affinity of RGS9 to the Chi6b·P
complex closely corresponds to the
stimulatory effect of P
on enhancement of G
t GTPase
by RGS9. The two distinct effects of P
and RGS9 on the binding of
each other to G
suggest an allosteric effect of P
on binding of
RGS9 to G
t.
The crystal structure of the G
i1·RGS4 complex
complemented by the mutational analysis suggests a mechanism by which
RGS proteins stimulate GTPase reaction by G
. According to this
mechanism RGS binds to the switch regions of G
and stabilizes the
transition state of the G
GTP. Unlike for Ras·GAP, no residues of
RGS4 contribute catalytically to the active site of G
. It is
appropriate to assume based on the sequence similarity (35% identity,
58% homology) that the core domain of RGS9 has a similar fold to that
of RGS4 and analogous to the G
i1·RGS4 interface with
G
t. What is the structural basis for the P
effect on
the RGS GAP activity? It is known that P
contacts G
t
at
-helices 3 and 4 as well as
3-
5 and
4-
6 loops and
switch II and III regions of the
subunit (36, 51, 52). Two residues
from the switch II region (Trp207, Ile208) have
been identified to interact directly with P
(49, 53). The increased
fluorescence of the reporter group attached to Cys210
indicates that RGS9 contacts the switch II region as well. Our data
indicate, however, that there is no steric conflict in the trimeric
G
t·RGS9·P
complex. Earlier we demonstrated that
the last 25 COOH-terminal amino acid residues of P
are critical for this GTPase activation (24) and that this region binds to the switch
regions of G
t (36). Trp70 located within
this region plays a critical role in the P
activation of the
transducin GTPase rate (27). This suggests that the structural basis
for enhancement of RGS9 GAP activity by P
could be a conformational change in the vicinity of the switch II region induced by the COOH
terminus of P
which increases the affinity of RGS9 for
G
t.
Natochin et al. (49) showed that P
and RGS16 binding
sites on G
t do not overlap. However, P
does not
synergize with RGS16. Thus, a nonoverlapping pattern of RGS and P
interaction with G
t is not sufficient for cooperative
stimulation of the transducin GTPase function. We speculate that the
determinants of RGS9 specificity located in the
-helical domain of
G
t distinguish the mechanism of cooperative interaction
of P
and RGS9 with transducin (Fig. 6).
Arshavsky et al. (24) showed that phosphodiesterase-depleted
ROS membranes, even at high concentrations, cannot accelerate the
GTPase activity of transducin. Thus, full-length RGS9 present in ROS is
not a transducin GAP in the absence of P
. On the other hand, the RGS
domain of RGS9 can on its own stimulate GTPase activity of
Gt reconstituted with the membranes (29, this work). These seemingly contradictory observations may suggest a role of the NH2-terminal domain of RGS9 in attenuation of GAP function.
One of the possible roles of P
in cooperating with RGS9 in
vivo might be to relieve the inactive state of RGS9. The inactive
state of RGS9 in the physiological environment thus may assure
transducin interaction with P
and phosphodiesterase activation
leading to the photoresponse before the rapid inactivation of
transducin by RGS. Further characterization of full-length RGS9 and its
NH2-terminal domain in vitro and in
vivo will provide valuable information for understanding the
unique mechanism of effector-potentiated GAP activity of RGS protein.
Snow et al. (54) have very recently demonstrated that RGS11
specifically binds to G
5 in a region homologous to G
protein
subunits. This domain was defined as the GGL domain (G
protein
subunit-like domain) and is found in RGS11, 9, 5, 7 and
Egl-10. This RGS11·G
5 complex may exist in
vivo because the expression of mRNA for RGS11 and
G
5 in human tissues overlaps. Finding the RGS7·G
5
complex in cytosolic fractions of photoreceptor cells supports this
idea (48). The RGS11·G
5 complex functions as a GAP
that selectively stimulates GTPase activity of G
o. It is not clear whether only G
5 can form complexes with RGS
proteins or whether this is a common property for many G
subunits.
It is also important to understand if G
5 is associated
with G
in its complex with RGS. Analysis of the functional
properties of different G
·RGS complexes will allow us to
understand possible roles of G
interaction with RGS proteins in
signaling processes.