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J. Biol. Chem., Vol. 275, Issue 42, 32716-32720, October 20, 2000
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From the Department of Ophthalmology, Harvard Medical School,
Massachusetts Eye and Ear Infirmary, Boston Massachusetts 02114
Received for publication, June 28, 2000, and in revised form, September 1, 2000
The photoreceptor-specific G protein transducin
acts as a molecular switch, stimulating the activity of its downstream
effector in its GTP-bound form and inactivating the effector
upon GTP hydrolysis. This activity makes the rate of transducin GTPase
an essential factor in determining the duration of photoresponse in
vertebrate rods and cones. In photoreceptors, the slow intrinsic rate
of transducin GTPase is accelerated by the complex of the ninth member of the regulators of G protein
signaling family with the long splice variant
of type 5 G protein The phototransduction cascade of vertebrate photoreceptor cells is
a prototypic G protein-based signal transduction cascade. It is
uniquely designed to ensure both a high degree of signal amplification
and fast signal termination on the physiological subsecond time scale
(reviewed in Refs. 1-3). Vision begins upon light excitation of
rhodopsin, which activates many molecules of the photoreceptor-specific
G protein, transducin. Activated transducin binds to the inhibitory Photoresponse termination requires the timely inactivation of PDE,
which occurs when GTP bound to transducin is hydrolyzed to GDP and
inorganic phosphate by the GTPase activity of transducin. However, the
intrinsic rate of transducin GTPase is much slower than the rate of
photoresponse recovery. In the photoreceptor, this problem is solved by
the action of a powerful mechanism of transducin GTPase acceleration
(reviewed in Ref. 4). Intensive studies over the past decade have
indicated that this mechanism is based on a cooperative action between
two protein components, the RGS9·G The goal of this study was to elucidate the mechanism by which PDE In this study, we examined the mechanism by which PDE Purification of ROS and Various Photoreceptor Membrane
Preparations--
ROS were purified from frozen bovine retinas (TA & WL Lawson Co., Lincoln, NE) under infrared illumination as described
(15). To obtain membranes lacking most peripheral proteins but
retaining an active RGS9·G Preparation of Transducin, PDE Cloning and Expression of Recombinant
G GTPase Measurements--
Transducin GTPase activity was
determined by using either a multiple-turnover ([GTP] > [transducin]) or single-turnover ([GTP] < [transducin])
technique as described previously (22). GTPase assays were conducted at
room temperature (22-24 °C) in a buffer containing 10 mM Tris-HCl (pH 7.8), 100 mM NaCl, 8 mM MgCl2, and 1 mM dithiothreitol.
Washed membranes, used as the source of both rhodopsin (to activate
transducin) and RGS9·G Binding of RGS9·G The goal of this study was to evaluate the relation between PDE The unique feature of studying components of the phototransduction
cascade is that the G protein transducin can be supplied by a
practically unlimited amount of its activated receptor, bleached rhodopsin. This makes it possible to study the effects of RGS9·G First, PDE
The Effector Enzyme Regulates the Duration of G Protein Signaling
in Vertebrate Photoreceptors by Increasing the Affinity between
Transducin and RGS Protein*
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subunit
(RGS9·G5L). However, physiologically rapid GTPase is
observed only when transducin forms a complex with its effector, the
subunit of cGMP phosphodiesterase (PDE). In this study, we
addressed the mechanism by which PDE regulates the rate of
transducin GTPase. We found that RGS9·G5L alone has a significant
ability to activate transducin GTPase, but its affinity for transducin
is low. PDE acts by enhancing the affinity between activated
transducin and RGS9·G5L by more than 15-fold, which is evident
both from kinetic measurements of transducin GTPase rate and from
protein binding assays with immobilized transducin. Furthermore, our
data indicate that a single RGS9·G5L molecule is capable of
accelerating the GTPase activity of ~100 transducin molecules/s. This
rate is faster than the rates reported previously for any RGS protein
and is sufficient for timely photoreceptor recovery in both rod and
cone photoreceptors.
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subunits of its effector, PDE,1 increasing its
catalytic activity; this leads to a decrease in the intracellular cGMP
level, the closure of cGMP-gated ion channels of the photoreceptor
plasma membrane, and progression of the photoresponse.
5L complex (5-7) and the
transducin target, PDE (8-10). In vivo experiments with
transgenic mice indicate that both components are essential to ensure
photoresponse recovery on a physiological time scale (11, 12). It is
now established that the primary catalytic role in activation of GTP
hydrolysis belongs to the RGS homology domain of RGS9·G5L. This
domain stabilizes the conformation ("transition state") of the
transducin 
subunit, which is most favorable for GTP hydrolysis.
PDE itself does not activate transducin GTPase but rather enhances
the activity of RGS9·G5L.
enhances the activation of transducin GTPase by the RGS9·G5L complex. In principle, two possibilities could be considered. First,
PDE could directly contribute to GTP hydrolysis, for example, by
further stabilizing the Gt transition state beyond the
action of the RGS domain. Second, it could act by increasing the
affinity between activated Gt and RGS9·G5L.
Previous efforts to answer this question using the expressed RGS
homology domain of RGS9 yielded contradictory results. McEntaffer
et al. (13) reported that PDE acts catalytically by
increasing the maximal GTPase rate ~2-fold and that it does not
change the apparent affinity between the RGS9 homology domain and
transducin. To the contrary, Skiba et al. (14) presented
evidence that the entire PDE effect consists of an ~3-fold
increase in the affinity between the RGS9 homology domain and
transducin. The reason for this incongruity is unknown.
Furthermore, it is unclear whether the RGS9 homology domain could serve
as a good model for studying the effects of PDE because the degree
of transducin GTPase activation by PDE observed with native
RGS9·G5L within the ROS membranes is at least one order of
magnitude higher than the effect observed with the domain alone (10,
9). This finding implies that the physiologically relevant mechanism of
PDE action should be addressed with the whole RGS9·G5L complex.
potentiates
the GAP activity of the native RGS9·G5L complex in bovine photoreceptor membranes. We used a combination of kinetic analysis and
direct binding assays and found that the major effect of PDE on the
activation of transducin GTPase is an increase in the affinity between
activated transducin and RGS9·G5L by >1 order of magnitude. This
work provides the first explanation of how an RGS protein and a G
protein effector cooperate in regulating the lifetime of G protein in
its active state.
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5L complex, ROS were washed under
infrared illumination twice with isotonic buffer containing 100 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol and 10 mM Tris-HCl (pH 7.5)
and three times by a hypotonic buffer containing 0.5 mM
EDTA and 5 mM Tris-HCl (pH 7.5). We will call this
preparation "washed membranes" throughout the rest of the text. The
amount of residual transducin in "washed membranes" did not exceed
1% of the original endogenous transducin content, as estimated from
Coomassie-stained SDS-PAGE gels. Rhodopsin concentration in all
membrane preparations was determined spectrophotometrically by
measuring the difference in absorbance at 500 nm before and after
rhodopsin bleaching (16).
, and Peptide Encompassing
PDE Residues 63-87 (PDE-(63-87))--
Transducin was
purified from bovine ROS as described (17). Transducin concentration
was first estimated by the Bradford method (18), using bovine serum
albumin as the standard, and then the precise concentration of
functionally active transducin was measured by the maximal amount of
rhodopsin-catalyzed GTPS binding (19). Transducin concentration, as
determined with GTPS, was used in all data analyses. Recombinant
bovine wild type PDE was expressed in Escherichia coli
and purified as described in (20). Peptide corresponding to residues
63-87 of PDE was synthesized and purified as described (10). The
purity and chemical formula of PDE-(63-87) were confirmed by mass
spectrometry and reversed-phase high pressure liquid
chromatography. PDE and PDE-(63-87) concentration was
determined spectrophotometrically using a molar extinction coefficient
280 of 7,100.
t--
Gt cDNA, preceded by a
nucleotide sequence encoding a hexa-histidine amino acid tag, was cut
off pHis6Gt (21) with EcoRI and
PstI and inserted into the baculovirus transfer vector
pVL1393 digested with EcoRI and PstI. The
recombinant baculovirus was generated using a BaculoGold transfection
kit (PharMingen). The resulting recombinant Gt is
N-terminally modified with the His6 tag and therefore lacks
N-terminal myristoyl moiety. Sf9 cells from a 2-liter shaking
culture were collected 72 h post-infection. His6-Gt was purified from the cytoplasmic
fraction using a combination of affinity chromatography on
Ni-NTA-agarose (Qiagen) and anion exchange chromatography on MonoQ
(Amersham Pharmacia Biotech) essentially as described for
Gt/Gi1 chimeras (21). The concentration of active Gt able to interact with rhodopsin and G
was determined as the maximal amount of
rhodopsin/G-dependent [35S]GTPS
binding (19).
5L, were illuminated on ice immediately
before the experiments. The reaction was started by adding 10 µl of
[-32P]GTP at the desired concentration (approximately
105 dpm/sample) to 20 µl of washed membranes (30 µM rhodopsin) reconstituted with the proteins of choice.
The reaction was stopped by the addition of 100 µl of 6% perchloric
acid. 32Pi formation was measured with
activated charcoal as described (22). All data fitting and
statistical analyses were performed with Sigmaplot software, version 6.
5L Complex to
his6-Gt Immobilized on
Ni-NTA-Agarose--
Washed membranes containing 10 µg of rhodopsin
were solubilized in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM dithiothreitol containing 1%
lauryl sucrose (Calbiochem). Insoluble material was removed by a 10-min
centrifugation at 120,000 × g with a Beckman Airfuge,
and the supernatant was mixed with 10 µl of Ni-NTA-agarose beads
pre-bound to ~5 µg of his6-Gt in 50 µl
of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and
0.2% lauryl sucrose. 10 mM NaF, 30 µM
AlCl3, and/or 10 µM PDE was added if
needed. Samples were incubated on ice for 30 min with occasional
shaking. The agarose beads were spun down and washed twice for 5 min
with 1 ml of 20 mM Tris-HCl (pH 8.0), 300 mM
NaCl, 20 mM imidazole, 0.25% lauryl sucrose, or the same
buffer containing 10 mM NaF and 30 µM
AlCl3 (for the AlF4-activated samples). Bound
RGS9 was eluted from the resin with the SDS-gel sample buffer,
subjected to SDS-PAGE (23), and detected by Western blot analysis using
specific antibodies and an ECL substrate kit (Amersham Pharmacia Biotech).
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and the native RGS9·G5L complex in regulating transducin GTPase.
We took advantage of the fact that native RGS9·G5L is tightly
associated with ROS membranes (6), which enabled us to deplete ROS
membranes of endogenous PDE, transducin, and other extractable proteins
while preserving their entire RGS9·G5L content. Previous studies
indicate that a significant cooperation between RGS9·G5L and
PDE is observed in experiments utilizing this type of washed ROS
membrane preparation (10, 9).
5L on transducin GTPase under steady-state conditions in which hydrolysis of Gt-bound GTP is followed immediately by recycling of
transducin to the GTP-bound form (24). As illustrated in Reaction 1 below, this steady-state situation could be analyzed by the Michaelis analysis, where RGS9·G5L is considered the enzyme,
Gt-GTP the substrate, and Gt-GDP and
inorganic phosphate the reaction products (the rate of GTPase reaction
under these conditions is not dependent on the concentrations of GTP
and bleached rhodopsin to the extent that these concentrations
are saturating).
We therefore could assess directly whether PDE
influences
kcat or apparent Km in this
reaction after measuring the rate of the GTPase reaction at a fixed
concentration of RGS9·G5L and various transducin concentrations.
However, before pursuing this analysis we needed to solve two
experimental problems outlined below.
has been documented to inhibit
rhodopsin-dependent transducin recycling from the GDP- to
the GTP-bound form (25, 26). This is most likely because the sites of
transducin interaction with rhodopsin and PDE overlap, allowing
competition between PDE and rhodopsin for binding to transducin
(27). As a result, transducin activation is partially blocked (27),
making it essentially impossible to know the concentration of
Gt-GTP in the steady-state Michaelis analysis when
PDE is present. We solved this problem by substituting PDE
by its C-terminal peptide, PDE-(63-87), which has been shown
to represent the GAP activity of PDE (10). This peptide essentially
lacks the ability to bind to the GDP-bound form of transducin because a
different site on PDE, PDE-(24-45), is responsible for this
interaction (21, 27). To confirm that PDE-(63-87) potentiates the
GAP activity of RGS9·G5L to the same extent as the full-length
PDE, we conducted a single-turnover GTPase assay in which only one
synchronized turnover of transducin GTPase is allowed and transducin
recycling does not take place (Fig. 1).
Our data indicate that PDE and PDE-(63-87) cause the same degree
of transducin GTPase activation at their saturating concentrations of 1 µM.
View larger version (12K):
[in a new window]
Fig. 1.
PDE and
PDE-(63-87) are equipotent in stimulating
transducin GTPase. Time courses of GTP hydrolysis were determined
in single-turnover GTPase assays as described under "Experimental
Procedures." Washed ROS membranes containing 20 µM
rhodopsin were reconstituted with 2 µM transducin in the
presence of 1 µM PDE (open circles) or
PDE-(63-87) (closed circles). In the control experiment,
no PDE or PDE-(63-87) was added (diamonds). The
reactions were started by adding 200 nM
[-32P]GTP. At indicated times the reactions were
quenched with perchloric acid. Curves were fit as single exponents with
100% hydrolysis corresponding to 200 nM GTP. The data
represent one of two similar experiments.
Second, we needed to distinguish between the basal transducin GTPase
activity and the activity stimulated by RGS9·G5L. Because it is
not possible to remove RGS9·G5L from the washed membranes under
nondenaturing conditions, we blocked RGS9·G5L activity with
specific affinity-purified sheep antibodies derived against a fragment
of RGS9 between residues 226 and 484 (5, 7). The data presented in Fig.
2 indicate that treatment of washed membranes with these antibodies blocked the ability of RGS9·G5L to
activate transducin GTPase both in the presence and absence of
PDE-(63-87). The effect was dose-dependent with
complete inhibition observed at 500 nM antibodies. The
specificity of anti-RGS antibodies to inhibit GTPase activation was
confirmed in control experiments in which a total fraction of nonimmune
sheep IgG did not affect transducin GTPase activity.
|
A typical experiment addressing the dependence of transducin GTPase
rate on transducin concentration under the steady-state conditions is
shown in Fig. 3A. The lower
data set represents the basal GTPase activity of transducin obtained
after blocking the GAP activity of RGS9·G5L with anti-RGS9
antibodies. This basal activity is a linear function of transducin
concentration. The other two data sets represent GTPase activity in the
presence of active RGS9·G5L measured with or without
PDE-(63-87). The "accelerated" GTPase activities are re-plotted
in Fig. 3B after a point by point subtraction of the basal
activity from these data sets (closed symbols). Open symbols
on the same graph show the data from an experiment carried out under
identical conditions but with another set of protein/membrane
preparations. The striking difference between the data obtained with
and without PDE-(63-87) is that the data with peptide could be
fitted by a hyperbola yielding the average value of apparent
Km of ~3.2 µM transducin, whereas
the data without peptide do not approach saturation over the range of
transducin concentrations used. Note that the Km value in the intact photoreceptor could not be derived from the experiment shown in Fig. 3 because in this experiment most of the
substrate (Gt-GTP-PDE-(63-87)) is soluble (not
shown), whereas in the intact rod, the substrate
(Gt-GTP-PDE) is membrane-attached because of the
isoprenoid modification of the PDE and subunits (28).
|
) and Km = 3.3 µM, Vmax = 1.2 µM·s
) with PDE
) and Km = 94 µM, Vmax = 1.09 µM·s
). Panel C shows
the best hyperbolic fits to the data without PDETo emphasize that the rate of GTP hydrolysis did not saturate without
PDE-(63-87), we re-plotted the data without peptide from both
experiments in Fig. 3C and compared the best hyperbolic fits
of these data (same as in Fig. 3B) with the fits forced to the Km value of 3.2 observed with the peptide
(dotted lines). Whereas the coefficients of determination
(R2) of the best fits were equal to 0.99 and
0.97, the corresponding parameters for the forced fits were only 0.75 and 0.54. Although we were not able to concentrate the components of
this reconstituted system sufficiently to obtain a highly reliable
determination of both Km and
Vmax values in the absence of PDE, the difference in the apparent Km values with and
without PDE63
87 in these experiments was
at least 15-fold. Hyperbolic fits of the data obtained without PDE
yielded Vmax values within an ~25% range of
the values observed with PDE-(63
87); this result should be treated
cautiously, however, because only ~30% of the calculated
Vmax was achieved at the highest transducin
concentrations used. The finding that PDE primarily affects the
value of the apparent Km rather than the
Vmax is further illustrated in Fig.
3D where the data from Fig. 3B are re-plotted in
double-reciprocal coordinates. Thus, we conclude that the major effect
of PDE in potentiating the GAP activity of RGS9·G5L is to
increase the affinity between Gt-GTP and
RGS9·G5L.
Another important parameter calculated from the data presented in Fig.
3B is the kcat value for
RGS9·G5L observed in the presence of PDE-(63-87). Based on the
1:1640 ± 620 RGS9·G5L:rhodopsin ratio measured by He
et al. (5) in bovine ROS, we calculated that the
RGS9·G5L concentration in this experiment was 0.012 ± 0.005 µM, and therefore the average kcat
value was equal to 98 ± 40 s1, which
makes RGS9·G5L the most efficient RGS protein studied so far.
The next set of experiments illustrated in Fig.
4 provides further proof for our
conclusion that PDE serves as an "affinity enhancer" between
GtGTP and RGS9·G5L. We monitored the binding of
RGS9·G5L to the His6-tagged Gt-GDP
immobilized on the Ni-NTA-agarose in the absence or presence of PDE.
Native RGS9·G5L was solubilized from washed ROS membranes by the
mild, non-ionic detergent lauryl sucrose and incubated with the
Ni-NTA-agarose beads in the presence of the same detergent. The beads
were rinsed, and bound proteins were extracted with the SDS-PAGE sample
buffer (see "Experimental Procedures"). The amount of RGS9·G5L
in the extract was then detected by immunoblotting with anti-RGS9
antibodies. We chose to use full-length PDE in this experiment
because its higher affinity for transducin than the PDE-(63-87)
peptide allowed better protein retention upon agarose washes. The
leftmost lane shows the amount of RGS9 loaded on the beads. The next
two lanes provide a control showing that RGS9·G5L did not bind to
inactive GDP-bound Gt both with and without PDE. As
evident from the last two lanes, transducin activation by
AlF4, which mimics the
transition state for GTP hydrolysis most favorable for interacting with
RGS proteins (29-31), results in RGS9·G5L binding to transducin,
and significant binding was observed only in the presence of
PDE.
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The dual requirement for an RGS protein (RGS9·G5L) and an
effector (PDE) for the timely inactivation of G protein is a unique feature of the vertebrate phototransduction cascade. It is now established that the major catalytic role in activating transducin GTPase belongs to the RGS homology domain of RGS9 and that PDE acts
as a facilitator of RGS activity (5, 14). Although PDE was
historically the first identified protein capable of activating G
protein GTPase activity (8), its exact role in this process remained
unknown. The data reported in this study indicate that PDE acts as
an enhancer of the affinity between Gt-GTP and
RGS9·G5L. What remains to be elucidated in the future is whether
PDE acts allosterically by optimizing the conformation of
Gt-GTP switch regions, suggested to bind to the RGS9
homology domain or whether it interacts directly with
RGS9·G5L.
Our finding that PDE enhances the affinity between
Gt-GTP and RGS9·G5L provides a solid mechanistic
basis for our hypothesis of why both RGS9·G5L and PDE are
needed for timely transducin inactivation in photoreceptor cells (4,
11). We noted that when a rod photoreceptor is hit by a photon of
light, it needs to accomplish two nearly opposite tasks. First, it must
transduce the signal from excited rhodopsin to PDE with high
efficiency. Second, it has to inactivate the whole cascade within a
fraction of a second. If transducin were allowed to be discharged by
RGS9·G5L before it formed a complex with PDE, then some transducin
molecules would never activate PDE and signal amplification would be
diminished. Therefore, the dependence of GTPase activation on
transducin association with PDE ensures both high efficiency of
signal transmission between transducin and PDE and timely photoresponse
recovery. It is tempting to speculate that similar mechanisms may be
utilized in other G protein-based signal transduction cascades to solve the same general problem of preserving both response sensitivity and
time resolution.
Another important finding of this study is that the PDE-enhanced
activity of RGS9·G5L reaches the rate of ~100
s1, which is at least one order of magnitude
higher than most reported rates for various RGS proteins
(cf. Refs. 24 and 30). However, this rate is on the same
order of magnitude as the ~25 s1 rate
constant reported by Mukhopadhyay and Ross (32) for the RGS4-stimulated
GTP hydrolysis by Gq. It is interesting to consider the
RGS9·G5L kcat value of 100 s1 in the context of photoresponse recovery
kinetics. This number sets the upper limit for the rate of PDE
inactivation. It appears to be not only sufficient but even excessive
when compared with the ~7 s1 rate of the
recovery from single-photon responses of dark-adapted mammalian rods
(33, 34). Although the slowest rate-limiting step in the photoresponse
recovery may not be transducin GTPase (35), the rate of 100 s1 is too high when compared with the
quenching rates of any phototransduction step derived from
physiological experiments (36). This implies that transducin
GTPase in rods does not work at its maximal rate, either because each
RGS9·G5L has to sequentially stimulate the GTPase activity of
several transducin molecules and/or because the concentration of
activated transducin remains below saturation for RGS9·G5L during
the course of the photoresponse.
Remarkably, the GTPase rate of 100 s1 is in
the same range as the photoresponse recovery observed in mammalian
cones. For example, the flicker fusion frequency of human cone vision,
which reflects the speed of the photoresponse, exceeds 60 s1 and is ~5-fold higher than in rod vision
(37). In this context, Cowan and colleagues (6) reported that bovine
cones contain a significantly larger amount of RGS9 than rods and
suggested that this difference underlies one of the biochemical
mechanisms contributing to faster recovery kinetics of cones. Our
finding that RGS9·G5L has the potential to stimulate transducin
GTPase at the rate of ~100 s1 provides a
strong support for their idea. Higher RGS9·G5L concentration in
cones should result in a higher rate of transducin GTPase and photoresponse recovery by providing a higher frequency of RGS9·G5L encounters with Gt-GTP-PDE.
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ACKNOWLEDGEMENTS |
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We thank Drs. V. I. Govardovskii and P. D. Calvert for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY-12859 and a grant from the Massachusetts Lions Eye Research Fund (to V. Y. A.).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.
Recipient of the Jules and Doris Stein Professorship from Research
to Prevent Blindness Inc. To whom correspondence should be addressed:
Howe Laboratory of Ophthalmology, Harvard Medical School/MEEI, 243 Charles St., Boston, MA 02114; Tel.: 617-573-4371; Fax: 617-573-4290;
E-mail: vadim_arshavsky@meei.harvard.edu.
Published, JBC Papers in Press, September 5, 2000, DOI 10.1074/jbc.C000413200
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ABBREVIATIONS |
|---|
The abbreviations used are:
PDE, type 6 cyclic
nucleotide phosphodiesterase from ROS;
ROS, rod outer segments;
PDE, the inhibitory subunit of PDE;
RGS9, the ninth member of the
regulators of G protein signaling family;
G5L, the long splice
variant of type 5 G protein subunit;
Gt, subunit
of transducin;
GAP, GTPase-activating protein;
GTPS, guanosine 5'-O-(thiotriphosphate);
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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REFERENCES
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