From the Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, November 26, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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The GTPase-accelerating protein (GAP)
complex RGS9-1·G Timely deactivation of G protein RGS9-1 contains multiple functional domains, including an RGS domain
that is responsible for its GAP activity (3), a G protein Additional possible mechanisms for regulation include a light- and
calcium-regulated phosphorylation (19, 20) of RGS9-1 and interactions
with the recently discovered membrane anchor protein, R9AP (7, 21).
R9AP is a 25-kDa protein that is selectively expressed in photoreceptor
outer segments. Homologues are apparent in genomic sequence from mouse
(GenBankTM accession number NW_000311), human
(GenBankTM accession number NT_011196), rat
(GenBankTM accession number AC128498), zebra fish (NCBI
trace archive numbers 15766910 and 46042404), and puffer fish
(GenBankTM accession numbers CAAB01004012 and
CAAB01001872), and in expressed sequence tags from human
(GenBankTM accession numbers AW302149 and BQ187216), mouse
(GenBankTM accession numbers BB591662 and BU506122),
Xenopus (GenBankTM accession number BG515592),
and zebra fish (GenBankTM accession number BE015922). R9AP
contains a single transmembrane Buffers--
Standard buffers were: GAPN buffer: 100 mM NaCl, 2 mM MgCl2, and 10 mM HEPES, pH 7.4; ConA buffer: 300 mM NaCl, 50 mM Tris-HCl, pH 7.0, 1 mM CaCl2, 1 mM MgCl2, and 1 mM
MnCl2; lysis buffer: 300 mM NaCl and 25 mM Tris-HCl, pH 8.0; and high-salt buffer: 10 mM HEPES, 1 M NH4Cl, and 2 mM MgCl2. For all these buffers, 1 mM dithiothreitol and ~20 mg/liter solid
phenylmethylsulfonyl fluoride were added before use.
Protein Electrophoresis and Immunoblotting--
SDS-PAGE and
immunoblotting were carried out using standard protocols (23). Rabbit
anti-RGS9-1c polyclonal antiserum was generated as described previously
and was used at a dilution of 1:1000. The secondary antibodies used
were horseradish peroxidase-conjugated (Promega) anti-rabbit antibody,
with detection by chemiluminescence using the ECL® system
(Amersham Biosciences).
Expression and Purification of R9AP--
His-tagged bovine R9AP
cytoplasmic domain (His-bR9AP- Expression and Purification of
RGS9-1·G Rhodopsin Purification--
Bovine ROS were purified as
described previously (25). Rhodopsin was purified from bovine ROS
according to the protocol described previously with modifications (26).
Briefly, ROS membranes were extracted in the dark with high salt buffer
twice to remove soluble protein contaminants and then washed with ConA
buffer and pelleted. Pelleted membranes were solubilized in 4% sodium cholate in ConA buffer, and insoluble material was removed by centrifugation. Concanavalin A immobilized on Sepharose beads was first
cross-linked in 0.05% glutaraldehyde in 250 mM
NaHCO3 at room temperature for ~2 h to prevent loss of
immobilized concanavalin A and then prepared as described previously
(26). Rhodopsin was eluted with 300 mM Vesicle Reconstitution--
Lipids used for vesicle
reconstitution were L- Electron Microscopy--
The reconstituted vesicles in solution
(vesicle concentration ~200-300 nM) were frozen across
the holes of 400-mesh carbon-coated holey grids by rapid plunging into
liquid ethane (29, 30). Electron microscopy was performed on a
JEOL1200EX equipped with a Gatan liquid nitrogen specimen holder at 100 keV. Images were recorded at a nominal magnification of ×40,000 with a
dose of ~20 electrons per Å2 onto Kodak SO-163
photographic film developed for 12 min in Kodak D19 developer at
20 °C. Micrographs were digitized using a Zeiss Phodis SCIA
microdensitometer at 14 µm/pixel.
Vesicle Binding Assay--
Purified RGS9-1 proteins (in GAPN
buffer) were first centrifuged at 88,000 × g for 40 min to remove aggregates. Supernatants were diluted to desired
concentrations in GAPN buffer containing 0.2 mg/ml ovalbumin, mixed
with R9AP vesicles or lipid-only vesicles in a volume of 300 µl, and
incubated with gentle shaking at 4 °C for ~3 h. 150 µl of the
reaction mixture was then transferred to a new polypropylene tube
(Beckman) to separate unbound proteins from bound proteins by pelleting
the vesicles at 88,000 × g for 40 min. The pelleted
vesicles were clearly visible as a pink pellet at the bottom of the
tube because of the rhodamine tag. The final concentrations in the
binding reactions were RGS9-1 proteins
(GST-RGS9-1·G Transducin Activation Assay--
Transducin activation by
rhodopsin vesicles was determined as described previously (31).
Briefly, rhodopsin or rhodopsin and R9AP vesicles were diluted in GAPN
buffer and mixed with purified transducin in a volume of 192 µl in
dim red light. The reaction was initiated by the addition of 18 µl
of[ Single Turnover GTPase Activity Assay--
Single turnover
G Reconstitution of R9AP and Rhodopsin Vesicles--
Because R9AP
has a transmembrane domain close to its C terminus, it is expressed as
an insoluble protein in both E. coli and Sf9 cells
(data not shown). Because the yield of E. coli expression (~10 mg/liter) is much better that that of Sf9 expression
(~0.5 mg/liter), we used detergent extractions to purify R9AP
expressed in E. coli. We tested both sodium cholate and
dodecyl maltoside for their performance in extracting R9AP because of
their compatibility with rhodopsin purification (26, 33), and we found
that R9AP is readily extractable from the E. coli-expressed
proteins by both detergents with comparable efficiency (data not
shown). We therefore chose to use sodium cholate for our experiments
for its lower cost. After further purification over the
nickel-nitriloacetic acid column in the presence of detergent, R9AP was
reconstituted into lipid vesicles using a conventional dialysis method.
The composition of lipid headgroups used in the reconstitution was chosen to mimic that of mammalian rod outer segments (34). Dialysis yielded small unilamellar spherical vesicles of a fairly narrow size
distribution (r = 240 ± 50 nm; n = 150) (Fig. 1A). The
functions of rhodopsin and R9AP on the vesicles were tested by
transducin activation assays (Fig. 1B) and RGS9-1 binding
(Fig. 2), respectively, and both proteins
were found to be functional. The presence of R9AP in the vesicles had
little effect on rhodopsin activity.
Although E. coli is overwhelmingly the most common
heterologous expression system for mammalian proteins, and it generally provides the largest amounts of protein for the least expense, relatively few examples exist of successful purification and
reconstitution of mammalian transmembrane proteins from bacteria (35).
Our results provide an example of a successful strategy for the
preparation in functional form of a protein with a relatively small
cytoplasmic domain and a single transmembrane helix that may be
applicable to additional proteins of this type.
Membrane Anchoring of RGS9-1·G Membrane Anchoring of RGS9-1 by R9AP Enhances Its GAP
Activity--
Lishko et al (7) recently reported that
association of the RGS9-1·G
To determine the maximal enhancement of GAP activity by the
R9AP-containing vesicles, we performed a vesicle titration, holding the
concentrations of RGS9-1·G
Enhancement of the GAP activity of RGS9-1 by membrane anchoring was
also observed on urea-washed ROS membranes, in which the endogenous
binding sites for RGS9-1 were made accessible to the added recombinant
RGS9-1·G
In addition to establishing the ability of R9AP membrane anchoring to
stimulate GAP activity of RGS9-1, the results presented here using the
highly efficient bacterial expression and reconstitution method we have
developed will facilitate determination of the role of specific lipids
and other molecules of ROS membranes in regulating GAP activity and,
possibly, structural studies of the GAP complex.
5 plays an important role in the
kinetics of light responses by accelerating the GTP hydrolysis of
G
t in vertebrate photoreceptors. Much, but not all,
of this complex is tethered to disk membranes by the transmembrane
protein R9AP. To determine the effect of the R9AP membrane complex on
GAP activity, we purified recombinant R9AP and reconstituted it into
lipid vesicles along with the photon receptor rhodopsin. Full-length
RGS9-1·G
5 bound to R9AP-containing vesicles with high
affinity (Kd < 10 nM), but constructs lacking the DEP (dishevelled/EGL-10/pleckstrin) domain bound
with much lower affinity, and binding of those lacking the entire
N-terminal domain (i.e. the dishevelled/EGL-10/pleckstrin
domain plus intervening domain) was not detectable. Formation of the
membrane-bound complex with R9AP increased RGS9-1 GAP activity by a
factor of 4. Vesicle titrations revealed that on the time scale of
phototransduction, the entire reaction sequence from GTP uptake to
GAP-catalyzed hydrolysis is a membrane-delimited process, and exchange
of G
t between membrane surfaces is much slower than
hydrolysis. Because in rod cells different pools exist of
RGS9-1·G
5 that are either associated with R9AP or not,
regulation of the association between R9AP and
RGS9-1·G
5 represents a potential mechanism for the
regulation of recovery kinetics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunits is a key element of
responses to the stimulation of G protein-coupled receptors. It plays
an especially important role in fast cellular responses such as those
of vertebrate photoreceptors. In the rod and cone cell outer segments,
the recovery phase of light responses depends on the presence of a
GTPase-accelerating protein
(GAP)1 complex
RGS9-1·G
5 (1-4). Whether and how the GAP activity of this complex is regulated is unknown.
subunit-like domain for G
5L binding (4, 5), an N-terminal domain that includes a DEP (dishevelled/EGL-10/pleckstrin) domain (6) and an intermediate domain (7), and a C-terminal domain that
is unique to RGS9-1 among all of the RGS proteins (8). All of these
domains have been found to participate in the regulation of GAP
activity and substrate specificity (9-11). The inhibitory PDE
subunit of the effector regulated by G
t, cGMP
phosphodiesterase (PDE6), interacts with both G
t and the catalytic core of RGS9-1 and enhances RGS9-1 GAP activity (12-14) in vitro, but it is not clear how this enhancement is
accomplished in a physiological setting in which tight PDE
binding
to PDE6 catalytic subunits blocks GAP enhancement (15-18).
helix, and sequence
analysis suggests structural similarity to the syntaxin family of
proteins involved in membrane targeting (for a review on syntaxin
family proteins, see Ref. 22). Biochemical assays and co-localization
on co-expression in cell culture indicate that it acts as a membrane
anchor for RGS9-1·G
5, which binds to the cytoplasmic
domain of R9AP (21). The molar ratio of R9AP to RGS9-1 in bovine rod
outer segments appears to be variable and was found to range from 0.4 to 0.8. Consistent with these ratios, co-immunoprecipitation
experiments revealed that whereas all detectable R9AP in the retina is
bound to RGS9-1·G
5, a distinct pool of
RGS9-1·G
5 is not associated with R9AP. Here we
describe experiments with recombinant R9AP reconstituted into
lipid vesicles, which demonstrate that binding of
RGS9-1·G
5 to membrane-anchored R9AP dramatically
enhances its GAP activity toward G
t.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C, amino acids 1-212, previously
named His-bp25-
C) and His-tagged full-length murine R9AP (His-mR9AP)
were expressed in Escherichia coli using plasmids and
procedures described previously (21), and His-bR9AP-
C was purified
as described. For His-mR9AP purification, the cells were collected and
lysed in lysis buffer by sonication on ice, and insoluble proteins
including His-mR9AP were separated from soluble proteins by
centrifugation at 24,000 × g at 4 °C in a Beckman
JA 25.50 rotor. The insoluble proteins were extracted with 4% sodium
cholate (3
,7
,12
-trihydroxy-5
-cholanic acid, sodium salt,
Sigma) in lysis buffer for ~30-60 min at 4 °C with gentle
agitation, and the proteins in the supernatant were separated from the
pellet by centrifugation. The extraction was repeated a total of 3-4
times, yielding > 70% of total His-mR9AP extracted in soluble
form. The detergent supernatants were pooled together, and His-mR9AP
was purified by nickel nitrilotriacetic (Ni-NTA superflow, Qiagen)
chromatography in 4% sodium cholate in lysis buffer according to the
manufacturer's protocol to obtain His-mR9AP of >95% purity.
5--
GST-tagged RGS9-1 (amino acids
1-484)·G
5, RGS9-1-NGD (amino acids
1-431)·G
5, and His-tagged RGS9-1-IGRC (amino acid
112-484)·G
5 complex were expressed and purified from
Sf9 cells as described previously (9, 11). GST-tagged RGS9-1-D
(amino acid 276-431) was expressed and purified from E. coli as described previously (14).
-methyl-mannoside
in 4% sodium cholate in ConA buffer. The sealed column and 1.5-column
volumes of elution buffer were gently agitated at 4 °C for 20-30
min, and then the eluted protein was drained from the column. This
procedure was repeated twice, and the eluted proteins were pooled
together and concentrated in a protein concentrator (Millipore) to
obtain a final rhodopsin concentration (determined by dark absorbance
at 500 nM) of 2-3 mg/ml.
-phosphatidylserine (brain and
porcine-sodium salt), L-
-phosphatidylcholine and (egg
and chicken), L-
-phosphatidylethanolamine (egg and
chicken) from Avanti Polar Lipids. Lipids were mixed at a ratio (w/w)
of phosphatidylchoine:phosphatidylethanolamine:phosphatidylserine = 50:35:15 in choloroform, dried under argon flow, and redissolved in
4% sodium cholate in lysis buffer to a final concentration of 20 mg/ml. In the lipid-only vesicles (see below) and R9AP vesicles that
were used in binding assays, rhodamine-labeled
phosphatidylethanolamine (Molecular Probes Inc.,
N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) was added in the lipid mixture to a final concentration of 0.5% (w/w) to facilitate the visualization of the
lipids. Detergent-solubilized lipids were then mixed with purified
proteins (rhodopsin, His-mR9AP, or rhodopsin and His-mR9AP) at a
lipid-to-protein (w/w) ratio of 20-40:1 and dialyzed against GAPN
buffer(>100× volume) for ~120 h with buffer changes every 20-24 h
(27). Under these conditions, the majority of the proteins were
incorporated into the vesicles. After dialysis, samples were collected
and centrifuged at 18,000 × g in a Beckman TL100 rotor to remove aggregated proteins and lipids, and the vesicles in the
supernatant were concentrated by one of the following two methods. For
the purpose of vesicle binding assays, R9AP vesicles were pelleted by
ultracentrifugation at 88,000 × g to collect easily
pelleted vesicles, and vesicles that stayed in the supernatant were
discarded. Pelleted vesicles were resuspended in GAPN buffer to the
desired concentration by repeated extrusion through a 26-gauge needle.
As a control for the binding assays, lipid-only vesicles without
protein were made similarly in parallel. For the purpose of GAP assays
the vesicles were concentrated in a protein concentrator (Millipore) to
achieve the maximum yield. Lipid concentrations of the samples were
determined by measuring the total phosphate concentrations using
phosphorus assays (28). The molar vesicle concentrations were
calculated using the following formula: molar concentration of
vesicles = (formal concentration of phospholipids × s)/4
r2, where s is the
average surface area of a phospholipid headgroup in Å2 (70 Å2), and r is the average vesicle radius in Å.
We used r = 272 Å, which was derived from least
squares fitting of the data in Fig. 4. This value is consistent with
the appearance of the vesicles in electron micrographs (Fig.
1A, r = 240 ± 50 Å, n = 150). The rhodopsin concentrations were determined by measuring the
differential absorbance at 500 nm (using extinct coefficient = 40,600 M
1 cm
1) of the sample
dissolved in 1.5% lauryldimethylamine oxide before and after
illumination. The R9AP concentrations were determined by densitometry
of Coomassie-stained SDS-PAGE gels using bovine serum albumin as standard.
5, His-RGS9-1-IGDC·G
5,
GST-RGS9-1-NGD·G
5, or GST-RGS9-1-D), 0.1 µM; R9AP (total concentration), 3.0 µM;
lipid (as an indication of vesicle concentration, measured by phosphate
concentration), 0.8 mM. Equal proportions of the starting
binding reactions, the supernatant after the vesicles were pelleted,
and the pelleted vesicles were loaded on SDS-PAGE, and the RGS9
proteins were detected by immunoblotting using anti-RGS9-1c antiserum.
-35S] GTP (specific activity = 400-500
Ci/mol) to the mixture, and 18 µl of the reaction was removed and
quenched on the filter membrane at the specified time points. The
reactions were kept in dim red light until t = 3.0 min
and were exposed to room light starting from t = 3.0 min until the end of the assays. The final concentrations in the assays
were 1 nM rhodopsin, 0.5 µM transducin, and
2.5 µM [
-35S]-GTP.
t GTPase assays were carried out as described
previously (9), and full-length His6-tagged PDE
(expressed and purified from E. coli) was added to enhance
RGS9-1 GAP activity. Briefly, rhodopsin, rhodopsin and R9AP vesicles,
or urea-washed ROS membranes were mixed with purified transducin and
RGS9-1 proteins in GAPN buffer and incubated on ice for ~30 min. GTP
hydrolysis was initiated by the addition of [
-32P] GTP
to the above mixture and was quenched by adding 5% trichloroacetic acid at various times. Phosphate released from hydrolyzed GTP was
determined by activated charcoal assay. Final concentrations in the
reactions using the reconstituted vesicles were 0.1 µM RGS9-1 proteins and 0.2 µM recombinant
His6-PDE
; final concentrations in the reactions using
urea-washed ROS were 72 µM rhodopsin, 1.0 µM transducin, and 1.0 µM recombinant
His6-PDE
. The first order rate constant for GTP
hydrolysis (kinact) was obtained by the fitting
of data to single exponentials, and the rate constants were plotted as
mean ± S.D. In the reactions with urea-washed ROS, we used ROS
membranes washed with either 4 M urea (32) or 6 M urea (7). Similar results were obtained on these two membranes. The difference was a slightly decreased
kcat/Km value of
RGS9-1·G
5 on the membranes treated with 6 M urea that was probably caused by denaturation of some
endogenous R9AP.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Vesicle reconstitution. A,
electron cryomicroscopy of reconstituted vesicles in vitreous ice.
R9AP, R9AP-only vesicles; Rhodopsin+R9AP,
rhodopsin and R9AP vesicles. B, rhodopsin activity on
reconstituted vesicles measured by Gt
[ -35S] GTP uptake on light illumination. Closed
circles, rhodopsin-only vesicles; open circles,
rhodopsin and R9AP vesicles. Each sample contained 1 nM
rhodopsin. The curves shown for data points after
illumination are fits to single exponentials.
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Fig. 2.
Membrane anchoring of RGS9-1 by R9AP.
The binding of GST-RGS9-1·G 5 (A),
GST-RGS9-1-NGD·G
5 (B), GST-RGS9-1-D
(C), and GST-RGS9-1-IGDC·G
5 (D)
to R9AP vesicles or lipid-only vesicles is shown. Equal proportions of
the starting reaction containing the RGS9-1 proteins and the vesicles
(input), the supernatant after pelleting the vesicles
(Sup't), and the pelleted vesicles collected by
resuspension (Bound) were separated by SDS-PAGE, and RGS9-1
proteins were detected by immunoblotting with chemiluminescence
detection. Total GST-RGS9-1·G
5 in the lipid-only
samples is lower because of the loss of GST-RGS9-1·G
5
to the walls of the reaction tubes.
5 Complex by
R9AP--
We have shown previously (21) that R9AP directly
interacts with the RGS9-1 N-terminal domain. Here we tested whether
RGS9-1 can be anchored to lipid vesicles by R9AP through this
interaction (Fig. 2) by incubating recombinant RGS9-1 domain proteins
with R9AP vesicles and removing the vesicles by centrifugation. We found that both the RGS9-1-NGD (amino acids 1-431)·G
5
complex and RGS9-1 (amino acids 1-484)·G
5 complex
bound to the R9AP vesicles, whereas RGS9-1-IGDC (amino acids
112-484)·G
5 bound much more weakly, and RGS9-1-D
(amino acids 276-431) did not bind at all. The binding to lipid
vesicles (Fig. 2A, lane 6) was very weak. Therefore, R9AP is sufficient to anchor RGS9-1 to the membranes by
interacting with its N-terminal domain, and both the
dishevelled/EGL-10/pleckstrin (DEP) domain and the intermediate domain
of RGS9-1 are important for the binding, consistent with our previous
findings (21). By titrating in full-length RGS9-1·G
5
over a fixed concentration of R9AP vesicles in binding assays, we
further determined that ~40% of the total R9AP on the vesicles was
able to bind RGS9-1·G
5 (data not shown), suggesting
that R9AP assumed a nearly random orientation in the vesicles during reconstitution.
5 complex with high
affinity sites on ROS membranes dramatically enhanced its GAP activity.
To test the enhancement of GAP activity caused by membrane anchoring of
the RGS9-1·G
5 complex by R9AP, we measured the GAP
activity of RGS9-1·G
5 complex on reconstituted
vesicles with defined protein compositions. We reconstituted purified
rhodopsin with and without recombinant full-length R9AP in lipid
vesicles and compared the GAP activity of RGS9-1 on these two vesicles.
We found that the activity of RGS9-1 increased ~3-4-fold on the
rhodopsin and R9AP vesicles above that on the rhodopsin-only vesicles.
The increase in activity occurred on the full-length
RGS9-1·G
5 complex that binds to R9AP but not on the
RGS9-1-D protein that does not bind R9AP (Fig. 3A). Furthermore, the increase
in activity was not merely because of the interaction between the R9AP-
soluble domain and RGS9-1, because the addition of R9AP-soluble domain
to rhodopsin-only vesicles had no effect on RGS9-1 activity (Fig.
3B). Therefore, we conclude that membrane anchoring of
RGS9-1 by R9AP enhances RGS9-1 GAP activity, probably at least in part
simply by localizing and orienting RGS9-1 on the vesicles.
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Fig. 3.
Enhancement of RGS9-1 GAP activity by R9AP
membrane anchoring. A, GTP hydrolysis rates for
Gt reconstituted on vesicles containing rhodopsin only or
rhodopsin and R9AP, in the presence of GST-RGS9-1·G 5
(RGS9) or GST-RGS9-1-D (RGS9-1-D), were measured
as described in the text. Final concentrations in the assays were:
rhodopsin, 4.0 µM; Gt, 0.5 µM;
RGS9-1 proteins, 0.1 µM; R9AP, 1.2 µM;
vesicles, 96 nM (formal molar concentration of phospholipid
divided by average number of phospholipids per vesicle). RGS9-1 GAP
activity (
kinact) on each vesicle was
calculated as
kinact = kinact (of Gt in the presence of
RGS9-1)
kinact (of Gt).
B, GTP hydrolysis rates on rhodopsin-only vesicles in the
absence or presence of 0.1 µM
GST-RGS9-1·G
5 (RGS9) and in the absence or
presence of 0.5 or 2.0 µM His-bR9AP-
C
(R9AP-
C) were measured by single turnover assays. Other
protein and vesicle concentration were the same as in
A.
5 constant. Under these
conditions, the GAP activity of RGS9-1·G
5 is expected
to increase when the fraction of RGS9-1·G
5 associated
with R9AP increases. However, as the vesicle concentration increases at
a constant RGS9-1·G
5 concentration, a potential
counteracting effect also occurs when the number of vesicles approaches
and exceeds the number of RGS9-1·G
5 complexes. In
these conditions, an increase in the number of vesicles increases the
probability of G
t-GTP formation on a vesicle lacking RGS9-1·G
5. Therefore, if G
t-GTP
exchange between vesicles is very slow on the time scale of GTP
hydrolysis, then at higher vesicle concentrations the apparent GAP
activity must decline. Moreover, if Gt and
RGS9-1·G
5 bind to rhodopsin- and R9AP-containing vesicles independently of one another, the apparent GAP activity should
decline in a way predicted by Poisson statistics. As shown in Fig.
4A, the behavior observed
corresponds precisely to these expectations. The effect of titration in
R9AP vesicles is biphasic, with GAP activity enhancement increasing at
low concentrations and decreasing at higher vesicle concentrations. We
were able to model the experimental results by theoretical calculations on the basis of the above considerations and another assumption that
the dissociation coefficient (Kd) between RGS9-1 and
R9AP on the vesicles is much lower than the concentrations used in the experiments (i.e. all of the RGS9-1 could be
anchored by available R9AP). The calculated -fold enhancement agreed
very well with the experimental data (Fig. 4A, fitted
curve), supporting the validity of the assumption and giving a
maximum GAP enhancement of 4-fold. The declining activity at higher
vesicle concentrations supports the notion that activated
Gt must interact with RGS9-1 on the membrane on which it
was formed for rapid GTP hydrolysis, or, equivalently, that
intervesicle exchange of G
t-GTP is a slow process
relative to the intravesicle encounter of G
t-GTP with RGS9-1 and consequent rapid GTP hydrolysis.
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Fig. 4.
Maximum enhancement on RGS9-1 GAP activity by
R9AP membrane anchoring. A, RGS9-1 GAP activity
( kinact) on rhodopsin-only or rhodopsin and
R9AP vesicles measured similarly as described for Fig. 3 under a
series of different vesicle concentrations (nM): 9.0, 18, 36, 72, 144, 288, and 576. Corresponding rhodopsin concentrations were:
0.38, 0.75, 1.5, 3.0, 6.0, 12.0, and 24.0 µM, and
corresponding R9AP concentrations were 0.12, 0.24, 0.48, 0.96, 1.9, 3.8, and 7.7 µM. Final GST-RGS9-1·G
5
concentration was 0.1 µM, and Gt
concentration was 0.2 µM for vesicle concentrations from
9.0 to 36.0 nM and 0.5 µM for vesicle
concentrations from 72.0 to 576.0 nM. Enhancement on RGS9-1
GAP activity by R9AP membrane anchoring (closed squares) was
defined as -fold enhancement =
kinact
(on rhodopsin and R9AP vesicles)/
kinact (on
rhodopsin-only vesicles) and was plotted as mean ± S.D. against
vesicle and R9AP concentrations (on a log scale). Theoretical values
were predicted based on the following three assumptions. 1) The binding
of Gt
to the vesicles was independent of the binding of
RGS9-1, and the numbers of RGS9-1 per vesicle (x) followed
the Poisson distribution P(x,µ) = µxe
µ/x,
where P(x,µ) is the probability of a vesicle
having exactly x molecules of RGS9-1 when the average number
of RGS9-1 molecules per vesicle is µ. 2) The dissociation
constant Kd between RGS9-1 and R9AP on the vesicles
was much lower than the concentrations used in the experiments so that
the concentration of the complex RGS9-1·R9AP is equal to the product
of the total formal concentration of R9AP and the fraction exposed on
the outside of vesicles when the value of that product is less than the
total formal concentration of RGS9-1 and is equal to the total formal
concentration of RGS9-1 when the concentration of surface-exposed R9AP
exceeds the total RGS9-1. 3) Each G
t-GTP stays on the
vesicle in which it was originally activated until its bound GTP is
hydrolyzed. Thus, the expected -fold-enhancement (smooth
line), E, is given by: E
1 = (Emax
1) × ((total concentration of
accessible R9AP)/(total concentration of RGS9)) × [1
exp(
µ)] when accessible R9AP
RGS9 and by
E
1 = (Emax
1) × [1
exp(
µ)] when accessible R9AP
RGS9. The values of parameters determined from least-squares fitting
were Emax, 4.0; average radius of the vesicles
(used in calculating µ), 272 nm, consistent with the
number 240 ± 50 nm calculated from vesicle radii determined by
electron microscopy; percent of accessible R9AP (with its soluble
domain facing outside), 40%, which is consistent with our result from
the binding assays and also suggests that nearly 100% of the R9AP was
functional. B, RGS9-1 GAP activity
(
kinact) on urea-washed ROS membranes was
measured by single turnover assays as described under "Experimental
Procedures." The final concentrations of RGS9-1 were 10, 20, 30, 50, 100, 200, and 400 nM. Data were plotted as mean ± S.D. and fit to two linear functions (straight lines) with
slopes of 1.36 µM
1·s
1 and
0.34 µM
1·s
1.
5 by denaturation of the endogenous RGS9-1
with urea. We titrated RGS9-1·G
5 at a fixed
concentration of urea-washed ROS membranes and measured its GAP
activity. As reported previously (7), the activity of
RGS9-1·G
5 increased steeply until the binding sites on
the membranes were saturated, and then at higher concentrations the
slope decreased (Fig. 4B). The RGS9-1 concentration at the
breakpoint was 100 nM, close to the estimated R9AP
concentration on the membranes of 60 nM R9AP, assuming a
ratio of rhodopsin:R9AP of 1250:1 (3, 21), consistent with the status
of R9AP as the endogenous factor providing the binding sites. The
enhancement of the GAP activity by membrane binding on these membranes,
as calculated from the slopes of the two straight lines in Fig.
4B, was approximately 4-fold, consistent with our results
using the reconstituted vesicles. The 4-fold enhancement we observed is
considerably lower than the ~70-fold enhancement Lishko et
al. reported (7), for experiments using similarly prepared
membranes. Assuming the Km value of 3.2 µM reported previously (36), the higher ratio apparently results from a 5-fold higher kcat value for
membrane-enhanced RGS9-1·G
5, and a ~3.5-fold lower
kcat value for RGS9-1·G
5 in solution relative
to the values we observed. Two differences in the experiments, which
may in part account for these discrepancies, are our use of single
turnover assays here rather than the steady-state assays used
previously (7) and our use of full-length recombinant PDE
in a 1:1
stoichiometric ratio with G
t rather than a peptide fragment (amino acid 63-87) derived from it used in high molar excess
(7). The use of full-length PDE
necessitates the use of
single-turnover assays rather than steady-state assays, because PDE
binds to GDP-form G
t tightly enough to inhibit
R*-catalyzed nucleotide exchange (37). In our hands peptides can give
very different results from full-length
PDE
.2 It is interesting to
consider whether the maximally enhanced GAP activity we observe is
sufficient to account for in vivo inactivation kinetics.
From our experiments (Figs. 4B and 3A), we deduce
that the kcat/Km value of
RGS9-1·G
5 when its GAP activity is maximally enhanced
either by ROS membranes or reconstituted R9AP-containing vesicles is
~1.3 µM
1·s
1 at room
temperature. Given the in vivo RGS9-1 concentration of 3-6
µM in mammalian rods (assuming the rhodopsin
concentration to be 3-6 mM (38, 39) and the
rhodopsin:RGS9-1·G
5 ratio to be 1:1000), our
kcat/Km value would provide
recovery times of 160-320 ms, very close to the observed ~200-ms
recovery time of mouse rod photoresponses measured in
electrophysiological experiments (1). Because of higher temperatures
and lower concentrations of chloride ion (an inhibitor of GTP
hydrolysis), GTP hydrolysis kinetics likely are more rapid than those
observed under our conditions. However, if GTP hydrolysis in rods were
10-fold faster than our results suggest, it would be hard to understand
why mammalian cones, which recover less than 10-fold more rapidly than
rods, require 10 times higher levels of the RGS9-1·G
5
complex (24, 40, 41). Thus, our estimate of maximal RGS9-1 GAP
activity, when appropriately corrected for differences in conditions,
seems likely to reflect the value attained in intact rod cells.
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ACKNOWLEDGEMENTS |
---|
We thank the National Center for Macromolecular Imaging for the use of electron microscopy facilities, Dr. Vadim Arshavsky of the Massachusetts Eye and Ear Infirmary for supplying baculovirus encoding RGS9-1-IGRC, and Mathew E. Sowa and Su Gu for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by NEI, National Institutes of Health and the Welch Foundation.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 Biochemistry
and Molecular Biology, Baylor College of Medicine, One Baylor Plaza,
Houston, Texas 77030. Tel.: 713-798-6996; Fax: 713-798-1625; E-mail:
twensel@bcm.tmc.edu.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M212046200
1
The abbreviations used are: GAP,
GTPase-accelerating protein; RGS, regulator of G protein signaling;
ROS, rod outer segments; R9AP, RGS9-1 anchor protein; PDE,
phosphodiesterase
subunit; ConA, concanavalin A; GST, glutathione
S-transferase.
2 M. E. Sowa and T. G. Wensel, unpublished data.
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