(Received for publication, March 28, 1997, and in revised form, May 7, 1997)
From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
The intrinsic GTPase activity of transducin
controls inactivation of the effector enzyme, cGMP phosphodiesterase
(PDE), during turnoff of the visual signal. The inhibitory -subunit
of PDE (P
), an unidentified membrane factor and a retinal specific
member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine
retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin
-subunit,
Gt
GDPAlF4
, while its
binding to Gt
GTP
S or Gt
GDP was weak.
hRGSr and P
did not compete for the interaction with
Gt
GDPAlF4
. Affinity
of the
P
-Gt
GDPAlF4
interaction was modestly enhanced by addition of hRGSr, as
measured by a fluorescence assay of
Gt
GDPAlF4
binding to P
labeled with
3-(bromoacetyl)-7-diethylaminocoumarin (P
BC). Binding of hRGSr to
Gt
GDPAlF4
complexed with P
BC resulted in a maximal ~40% reduction of
BC fluorescence allowing estimation of the hRGSr affinity for
Gt
GDPAlF4
(Kd 35 nM). In a single turnover
assay, hRGSr accelerated GTPase activity of transducin reconstituted
with the urea-stripped rod outer segment (ROS) membranes by more than
10-fold to a rate of 0.23 s
1. Addition of P
to the
reconstituted system reduced the GTPase level accelerated by hRGSr
(kcat 0.085 s
1). The GTPase
activity of transducin and the PDE inactivation rates in native ROS
membranes in the presence of hRGSr were elevated 3-fold or more
regardless of the membrane concentrations. In ROS suspensions
containing 30 µM rhodopsin these rates exceeded 0.7 s
1. Our data suggest that effects of hRGSr on
transducin's GTPase activity are attenuated by P
but independent of
a putative membrane GTPase activating protein factor. The rate of
transducin GTPase activity in the presence of hRGSr is sufficient to
correlate it with in vivo turnoff kinetics of the visual
cascade.
In vertebrate photoreceptor cells, the signal is transduced from
light-activated rhodopsin to the effector enzyme,
cGMP-phosphodiesterase (PDE),1 via the
heterotrimeric G-protein, transducin (Gt). The
GTP-bound
-subunit of transducin (Gt
GTP) relieves the inhibition imposed by two inhibitory PDE
-subunits (P
) on the enzyme catalytic
subunits (P
). Activation of PDE leads to a closure of cGMP-gated channels in the photoreceptor plasma membranes (1-3). The inactivation of PDE is a critical component of the turnoff
mechanism in the visual transduction cascade. This inactivation is
controlled by the intrinsic GTPase activity of transducin which hydrolyzes GTP to GDP. The GDP-bound Gt
(Gt
GDP) has a substantially reduced affinity for P
and releases P
to re-inhibit P
(1, 4-6). The rate of GTP
hydrolysis by transducin measured in vitro (7, 8) is too
slow to account for the fast photoresponse turnoff in vivo
(9, 10). The P
subunit (11, 12) and a distinct membrane-associated
protein factor (13, 14) have been shown to enhance transducin GTPase
activity in the activated membrane-bound transducin-PDE complex to a
level comparable with the rate of transducin inactivation in
vivo. A recent study has shown that a retinal specific member of
the RGS family, RGSr, serves as a GTPase-activating protein (GAP) for
transducin, providing an additional dimension to an already complex
picture of the regulation of transducin GTPase activity (15).
Functional relationships between RGSr, the
-subunit of PDE, and a
putative membrane GAP factor are currently not understood.
Here, we study the interaction between transducin and a human retinal
specific RGS (hRGSr), and regulation of transducin GTPase activity by
hRGSr. We examine the effects of P and photoreceptor membrane
concentration on modulation of the GTPase activity by hRGSr.
GTP and GTPS were products of Boehringer
Mannheim. Blue-Sepharose CL-6B was obtained from Pharmacia.
3-(Bromoacetyl)-7-diethylaminocoumarin (BC) was purchased from
Molecular Probes, Inc. [
-32P]GTP (>5000 Ci/mmol) was
obtained from Amersham. [35S]GTP
S (1250 Ci/mmol) was
purchased from NEN Life Sciences Products. All other chemicals were
from Sigma.
Bovine ROS membranes were prepared as described
previously (16). Urea-washed ROS membranes (uROS) were prepared
according to protocol in Ref. 17. Hypotonically washed ROS membranes
were prepared as described in Ref. 14. Transducin,
Gt, was extracted from ROS membranes using GTP as
described in Ref. 18. The Gt
GTP
S was extracted from
ROS membranes using GTP
S and purified by chromatography on
Blue-Sepharose CL-6B by the procedure described in Ref. 19.
Gt
GDP was prepared and purified according to protocols
in Ref. 20. P
BC was obtained and purified as described in Ref. 6.
The purified proteins were stored in 40% glycerol at
20 °C or
without glycerol at
80 °C.
A BLAST search at NCBI
(Bethesda, MD) to compare the mouse mRGSr cDNA sequence against DNA
sequence data bases revealed a human homologue, A28-RGS14p (the GenBank
accession number U70426), which is 85% identical to mRGSr. DNA
prepared from the amplified human retinal cDNA gt10 library
(kindly provided by J. Nathans, Johns Hopkins University) was used as a
template for the polymerase chain reaction amplification with primers
that were synthesized based on the A28-RGS14p sequence. The polymerase
chain reaction was performed in 30 µl of reaction mixture containing
100 ng of the template DNA and 0.5 µM of the following
primers: ATACTCTAGACATGTGCCGCACCCTGGC (5
) and
ATGCCTCGAGACTCAGGTGTGTGAGG (3
). The polymerase chain reaction product (620 bp) was digested with XbaI and
XhoI (the restriction sites are underlined) and subcloned
into the pGEX-KG vector (21) for GST-hRGSr fusion protein expression.
The DNA sequence was verified by automated DNA sequencing at the
University of Iowa DNA Core Facility using the 3
-pGEX sequencing
primer (Pharmacia) and the 5
-primer shown above. The subcloned
sequence was different from A28-RGS14p at two nucleotide positions of
the open reading frame (C125
T and A160
G), leading to substitutions of amino acid residues Ser42
Phe and Asn54
Asp. Typically, expression host
E. coli DH5
cells were grown on 2 × TY medium and
induced at OD600 = 0.5 by addition of
isopropyl-1-thio-
-D-galactopyranoside (0.4 mM final concentration). After a 4-h induction at 37 °C, cells were harvested and sonicated in 20 mM Tris-HCl (pH
8.0) buffer containing 100 mM NaCl, 2 mM
MgCl2, 6 mM
-mercaptoethanol, and 5%
glycerol (buffer A). The supernatant (100,000 × g,
1 h) was loaded on a glutathione-agarose column. GST-hRGSr was
eluted from the column using 50 mM Tris-HCl buffer (pH 8.0)
containing 10 mM glutathione. GST-hRGSr was then passed
through a PD-10 column (Pharmacia) to separate glutathione and digested
with thrombin (0.25 NIH units/mg) for 90 min at room temperature. hRGSr
was reapplied on a glutathione-agarose column to remove GST.
GtGDP or
Gt
GTP
S (6 µM final concentration) were
mixed with glutathione-agarose retaining ~10 µg of hRGSr in 40 µl
of 20 mM HEPES buffer (pH 7.6), 100 mM NaCl,
and 2 mM MgCl2 (buffer B). Where indicated, the
buffer contained 30 µM AlCl3, 10 mM sodium fluoride, and 10-30 µM P
. After
incubation for 20 min at room temperature, the agarose beads were spun
down, washed with 1 ml of buffer B, and the bound proteins were eluted
with a sample buffer for SDS-polyacrylamide gel electrophoresis.
Fluorescence assays were performed on a
F-2000 Fluorescence Spectrophotometer (Hitachi) in 1 ml of buffer B
essentially as described in Ref. 6. Where indicated, the buffer
contained 30 µM AlCl3 and 10 mM
sodium fluoride. Typically, hRGSr was added to PBC prior to addition
of transducin. The assays were carried out at equilibrium which was
reached in less than 3 s after mixing of the components except
when Gt
GDP and
AlF4
were used. In the latter
experiments the equilibrium due to Gt
GDP activation by
AlF4
was reached in less than
15 s. Fluorescence of P
BC was monitored with excitation at 445 nm and emission at 495 nm. Concentration of P
BC was determined using
445 = 53,000.
Single turnover GTPase activity
measurements were carried out essentially as described in Ref. 22. The
reaction was initiated by mixing bleached ROS membranes with 200 nM [-32P]GTP (~5 × 104
dpm/pmol) in a total volume of 20 µl. The reaction was quenched by
addition of 100 µl of 7% perchloric acid. Nucleotides were then
precipitated using charcoal, and 32Pi formation
was measured by liquid scintillation counting. Concentrations of
transducin in different preparations of ROS membranes were determined
using the [35S]GTP
S binding assay. ROS membranes were
incubated with 2 µM GTP
S (105 dpm/pmol)
for 10 min at room temperature in 20 µl of buffer B, and the mixture
was applied onto GF/B filters (Millipore). The filters were washed with
3 ml of 40 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl and 4 mM MgCl2 and counted
in a liquid scintillation counter. To determine if hRGSr or P
may
affect the nucleotide binding to Gt
, 200 nM [35S]GTP
S was added to uROS membranes
(5 µM rhodopsin) reconstituted with 0.4 µM
Gt
and 1 µM hRGSr (and/or 1 µM P
) in 20 µl of buffer B. The mixture was
immediately (<2 s) diluted into 3 ml of cold 40 mM
Tris-HCl buffer (pH 8.0) containing 100 mM NaCl, 4 mM MgCl2, and 2 µM GTP
S. The
GTP
S binding was then measured as described above. The PDE activity
was measured using the proton-evolution assay as described in Ref. 23.
Protein concentrations were determined by the method of Bradford (24) using IgG as a standard or using calculated extinction coefficients at
280 nm. SDS-polyacrylamide gel electrophoresis was performed by the
method of Laemmli (25) in 12% acrylamide gels. Rhodopsin concentrations were measured using the difference in absorbance at 500 nm between "dark" and bleached ROS preparations. The
Kd and IC50 values were calculated as
described in Ref. 26. The results are expressed as the mean ± S.E. of triplicate measurements.
The sequence encoding
for the human homologue of mouse retinal RGSr protein was polymerase
chain reaction amplified from the retinal cDNA library, subcloned
into the pGEX-KG vector, and expressed as a GST fusion protein as
described under "Experimental Procedures." Eluate of GST-hRGSr from
a column with glutathione-agarose is shown in Fig. 1
(lane 2). GST-hRGSr migrated on SDS gels with the expected
mobility of a 52-kDa protein. Appearance of an additional doublet at
~28-30 kDa, which most likely contains GST polypeptide, is typical
for the GST fusion expression system (27). hRGSr, purified after
cleavage of the fusion protein GST-hRGSr with thrombin, migrated as a
25-kDa protein (Fig. 1, lane 3). The yield of hRGSr was
~6 mg/liter of culture.
Binding of GST-hRGSr to Gt
A number of
RGS proteins (RGS1, RGS4, and mouse RGSr) have been shown to interact
preferentially with a transitional
GGDPAlF4
conformation of G
subunits (15, 28, 29). We examined the
ability of GST-hRGSr bound to glutathione-agarose to co-precipitate Gt
in different conformations. Fig.
2A shows that GST-hRGSr precipitated
stoichiometric amounts of
Gt
GDPAlF4
, while
amounts of Gt
GTP
S and Gt
GDP that
co-precipitated with GST-hRGSr were significantly lower. In control
experiments, Gt
GDPAlF4
,
Gt
GTP
S, and Gt
GDP did not
co-precipitate with glutathione-agarose that contained no bound
GST-hRGSr (not shown). The results suggest that the affinity of hRGSr
for the Gt
conformations decreases in the following
order: Gt
GDPAlF4
Gt
GTP
S > Gt
GDP.
Effects of P
To
determine if P can compete with hRGSr for the interaction with
Gt
GDPAlF4
, we
initially tested effects of P
on
Gt
GDPAlF4
binding to GST-hRGSr. Even at high concentrations (up to 30 µM) P
did not affect binding of
Gt
GDPAlF4
to
GST-hRGSr immobilized on glutathione-agarose (Fig. 2B).
We next investigated effects of hRGSr on the interaction between P
and Gt
GDPAlF4
or
Gt
GTP
S using a fluorescence assay. Addition of
Gt
GDPAlF4
to a
fluorescently labeled P
, P
BC, produced an approximately 7.5-fold
maximal increase in the BC fluorescence (Fig.
3A), while Gt
GTP
S enhanced
the fluorescence of P
BC by more than 6-fold (not shown). The
Kd values for the
Gt
GDPAlF4
and
Gt
GTP
S binding to P
BC were 2.8 ± 0.1 and
2.1 ± 0.1 nM, respectively. The affinity of
Gt
GDPAlF4
binding to P
BC was somewhat higher in the presence of 100 nM hRGSr (Kd 1.2 ± 0.1 nM), suggesting that hRGSr and P
bind to
Gt
GDPAlF4
noncompetitively (Fig. 3A). Addition of hRGSr had no effect
on the fluorescence of P
BC alone (not shown), but resulted in a dose-dependent decrease in the fluorescence enhancement of
P
BC caused by the latter binding to
Gt
GDPAlF4
(Fig.
3B). The fluorescence was decreased maximally by ~40%
with an IC50 of ~35 nM. Since hRGSr and P
interact with
Gt
GDPAlF4
noncompetitively, this IC50 value may serve as an estimate
for the affinity of hRGSr interaction with
Gt
GDPAlF4
. In
control experiments, hRGSr did not affect the fluorescence of the
Gt
GTP
S·P
BC complex (not shown).
Effects of hRGSr on Transducin's GTPase Activity
Single
turnover measurements of GTPase activity were carried out as described
under "Experimental Procedures." Under these conditions ([GTP] < [Gt]) the GTPase reaction can be analyzed using an
exponential function: % GTP hydrolyzed = 100(1-e-kt),
where k is a rate constant for GTP hydrolysis. First,
transducin GTPase activity was measured in the reconstituted system
with uROS membranes. According to previous reports, uROS membranes lack
the GAP activity of a membrane factor, and the P subunit did not
affect the GTPase activity of transducin when uROS membranes were used
(14). The calculated rate of GTP hydrolysis by transducin (0.4 µM) reconstituted with uROS containing 5 µM
rhodopsin was 0.022 ± 0.001 s
1 (Fig.
4A). Addition of 1 µM hRGSr
resulted in acceleration of the GTPase activity by more than 10-fold
(k = 0.23 ± 0.01 s
1). The basal
GTPase activity of transducin was not significantly altered in the
presence of 1 µM P
(k = 0.019 ± 0.003 s
1). However, P
substantially reduced the
accelerated GTPase activity of the Gt
·hRGSr complex
(k = 0.085 ± 0.005 s
1). In control
experiments, hRGSr, P
, or the two proteins combined had no notable
effect on the binding of [35S]GTP
S to transducin under
similar conditions (not shown). The reaction was complete in less than
2 s.
The basal GTPase activity of transducin in the suspension of untreated
native ROS membranes containing 5 µM rhodopsin and 0.41 µM transducin was higher (k = 0.052 ± 0.002 s1) than in the reconstituted system with uROS
(Fig. 4B). However, the elevation of transducin GTPase
activity by hRGSr was only ~3.3-fold (k = 0.17 ± 0.01 s
1). Perhaps, the presence of PDE containing the
P
subunit in native ROS preparations prevented a larger enhancement
of the GTPase activity by hRGSr as seen using uROS. To test this idea
we have prepared hypotonically washed dROS membranes depleted of PDE. The GTPase activity in suspensions of dROS containing 5 µM rhodopsin and 0.38 µM transducin
(k = 0.031 ± 0.001 s
1) was
accelerated by ~7.5-fold (k = 0.23 ± 0.02 s
1) with addition of hRGSr (Fig. 4C). The P
subunit increased transducin's GTPase activity by more than 2-fold to
a rate of 0.075 ± 0.003 s
1. Effects of hRGSr and
P
on transducin were not additive. Moreover, the GTPase activity of
transducin was lower in the presence of both hRGSr and P
(k = 0.18 ± 0.01 s
1) than in the
presence of hRGSr alone. Interestingly, hRGSr enhanced the GTPase rates
in suspensions of untreated ROS to levels similar to those in
suspensions of dROS membranes reconstituted with P
.
The GTPase activity of transducin has been shown to elevate with
increasing concentrations of ROS membranes presumably due to the action
of a putative membrane GAP factor (11-14). At low concentrations of
ROS membranes the GtGTP·P
complex dissociates from
P
and is mainly soluble (30, 31). Increasing the membrane concentration shifts the equilibrium toward the membrane bound complex
(Gt
GTP)2P
2 (32-34)
allowing the interaction of Gt
GTP with a putative GAP
factor. The calculated rate for the GTP hydrolysis in the suspension of
native ROS membranes containing 30 µM rhodopsin was
~3-fold higher (k = 0.16 ± 0.02 s
1) than that seen using 5 µM rhodopsin
(Fig. 4D). hRGSr was at least as effective at higher
concentrations of ROS membranes as it was in diluted ROS suspensions.
We estimate that the rate of GTP hydrolysis in ROS suspensions
containing 30 µM rhodopsin in the presence of 1 µM hRGSr is >0.7 s
1.
We measured PDE
inactivation in suspensions of bleached native ROS membranes using the
proton evolution assay (23). The activation and inactivation of PDE was
monitored after addition of GTP under single turnover conditions using
a pH microelectrode. The PDE activity was maximal in less than 1 s
after addition of GTP. Therefore, we were unable to resolve the
activation phase. As shown earlier, the rate of PDE inactivation can be
well approximated by fitting the inactivation phase with a single
exponential decay function (14). The PDE activity in the assay is
proportional to the concentration of active GtGTP. The
change of pH due to hydrolysis of cGMP under single GTP turnover
conditions can be described using an exponential function: pH =
pHmax(1-e-kt) or [cGMP]hydrolyzed =
[cGMP]max(1-e-kt). The PDE activity represents
a derivative of this function, and it decays with the inactivation
constant k from the equation above. In diluted suspensions
of native ROS (5 µM rhodopsin), PDE was inactivated with
a rate of 0.096 s
1. In the presence of hRGSr, PDE
inactivation was enhanced to a rate of 0.31 s
1 (Fig.
5A). The increase in
kinact (>3-fold) correlated well with the
decrease in maximal amounts of cGMP hydrolyzed in the presence of hRGSr
(Fig. 5A). Furthermore, the acceleration of PDE inactivation caused by hRGSr was proportional to the elevation of transducin GTPase
activity. We next tested effects of ROS membrane concentration on the
modulation of PDE inactivation by hRGSr. The PDE inactivation rate
(0.26 s
1) was significantly higher in suspensions of ROS
membranes containing 30 µM rhodopsin than in diluted ROS
suspensions (Fig. 5B). However, this enhanced rate was not
accompanied by an equivalent decrease in the maximal amount of
hydrolyzed cGMP because the initial PDE activity after addition of GTP
was higher. It has been shown previously that PDE activation by
transducin is more efficient at higher concentrations of photoreceptor
membranes (32-34). Addition of 1 µM hRGSr to ROS
membranes containing 30 µM rhodopsin resulted in
~3-fold increase in the PDE inactivation rate. The PDE inactivation rate was as high as 0.75 s
1 and correlated well with the
GTPase rate (>0.7 s
1) measured under the same
conditions. To determine if hGRSr can serve as an antagonist for PDE
and block the enzyme activation, we have measured effects of hRGSr on
GTP
S-induced PDE activity in suspensions of ROS membranes containing
5 µM rhodopsin. The rates of the GTP
S-induced cGMP
hydrolysis were not significantly affected by hRGSr. In the presence of
relatively high concentrations of hRGSr (5 µM) PDE
activity was suppressed by only ~15% (not shown).
Recent findings have established that members of a new family of
RGS proteins serve as GAPs for heterotrimeric G-proteins and attenuate
G-protein-mediated signal transduction (28, 35, 36). Evidence suggests
that RGS proteins accelerate the rate of GTP hydrolysis by
G subunits but do not affect GDP/GTP exchange induced by
activated G-protein-coupled receptors (35). Precise mechanisms of RGS
GAP activity are not yet clear. It has been demonstrated that at least
some RGS proteins (RGS1, RGS4, GAIP, and mRGSr) interact preferentially
with the AlF4
bound conformation of
G
subunits and thus may accelerate GTP hydrolysis
through stabilization of the transitional state of G-proteins (15, 28,
29). In several signaling systems the GTPase activity of G-proteins is
enhanced by their effector enzymes (11, 37). Regulation of the visual
G-protein appears to be even more complicated. The
subunit of PDE
in concert with an unidentified membrane factor accelerate GTPase
activity of transducin (11-14). The mouse retinal specific RGS (mRGSr)
protein has been recently identified and preliminarily characterized. mRGSr was shown to enhance GTPase activity under steady-state conditions (15).
We have investigated the regulation of transducin GTPase activity by
human RGSr and examined effects of P and photoreceptor membrane
concentration on the functional activity of hRGSr. Our results support
the data that retinal RGS binds tightly to the transitional
conformation of transducin,
Gt
GDPAlF4
(15),
and extend this observation by showing that it also weakly interacts
with Gt
GTP
S and Gt
GDP. Using a
fluorescence assay of interaction between P
BC and
Gt
GDPAlF4
, we
demonstrated that P
and hRGSr interact with
Gt
GDPAlF4
noncompetitively. Furthermore, the affinity of the
P
/Gt
GDPAlF4
interaction was modestly enhanced in the presence of hRGSr. This increase in affinity may reflect a stabilization of the
Gt
GDPAlF4
conformation which interacts with P
with high affinity. Binding of
hRGSr to
Gt
GDPAlF4
affected the Gt
conformation resulting in a decrease of the maximal fluorescence enhancement caused by
Gt
GDPAlF4
binding to
P
BC. We used this effect to estimate the affinity of the
hRGSr/Gt
GDPAlF4
interaction (~35 nM). Interestingly,
Gt
GTP
S and
Gt
GDPAlF4
had
similar high affinities for P
and significantly different affinities
for hRGSr, supporting the conclusion that
Gt
GDPAlF4
has
distinct interfaces for interaction with P
and hRGSr. Comparison of
the crystal structures of Gt
GTP
S and
Gt
GDPAlF4
shows
limited conformational differences in the switch I and II regions of
transducin (38). This suggests that hRGSr interacts with the switch I
and/or switch II regions of
Gt
GDPAlF4
.
Indeed, a crystal structure of RGS4 bound to
Gi
1AlF4
, that
has been published during the review of this paper, shows that the
RGS4-binding site is formed by the three switch regions of
Gi
1 (39). The switch III,
3/
5, and
4/
6
regions of transducin have been earlier implicated in Gt
interaction with P
(5, 40-43). The P
and hRGSr-binding sites on
Gt
may possibly overlap, particularly, at the switch III
region. Because the interactions between the switch III region and RGS protein are not extensive (39), such potential overlap would not cause
a significant effect of hRGSr on apparent affinity of P
binding to
Gt
.
Binding of RGS proteins to the switch regions of G-proteins supports
the idea that RGS proteins may serve as antagonists for some effectors
(39). RGS4 has been shown to block activation of phospholipase C by
Gq
GTP
S (44). In our experiments, initial rates of
cGMP hydrolysis in suspensions of ROS membranes after addition of GTP
were unaffected in the presence of hRGSr (Fig. 5). Likewise, hRGSr has
little effect on PDE activity in ROS membranes stimulated by GTP
S.
It appears that the main mode of RGS action in the transducin/PDE
signaling system is to accelerate G-protein and effector inactivation
rather than to block effector activation.
Measurements of GTPase activity under single turnover conditions
presented in this study demonstrate that hRGSr can dramatically increase the kcat of GTP hydrolysis by
transducin. Effects of hRGSr on transducin's GTPase activity were
attenuated but not eliminated by P. Even in the presence of P
,
hRGSr accelerated the GTPase activity by ~3-fold. Similar inhibitory
effects of P
on stimulation of transducin GTPase activity by mouse
RGSr have just been reported (45). However, our data indicate that P
attenuates effects of RGSr allosterically rather than competitively as
suggested by Wieland et al. (45). In agreement with earlier observations, the rates of GTP hydrolysis were significantly higher in
more concentrated suspensions of photoreceptor membranes (11-14). hRGSr was equally potent as a GAP for transducin regardless of the
membrane concentration, indicating that: (a) a putative
membrane GAP factor for transducin represents a distinct non-RGS-like
protein, and (b) the membrane factor does not compete with
hRGS for binding to transducin. A newly discovered second
retina-specific RGS protein (RET-RGS1) is a membrane-bound protein and
therefore is a potential candidate for a putative GAP factor (46). Our
study indicates that RET-RGS1 and an unidentified GAP factor are likely
to be different proteins.
The PDE inactivation rates were increased by hRGSr proportionally to
the acceleration of the GTPase rates. The evidence that transducin's
GTPase activity represents a major mechanism for inactivation of PDE in
the turnoff of visual signals (11, 13, 47) has been disputed (48, 49).
Our data support the conclusion that transducin's GTPase activity
controls PDE inactivation. The rates of GTP hydrolysis and PDE
inactivation (>0.7 s1) observed at relatively high
concentrations of photoreceptor membranes in the presence of RGS are
adequate to explain fast turnoff kinetics in the visual transduction
cascade in vivo.