From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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Members of the RGS family serve as
GTPase-activating proteins (GAPs) for heterotrimeric G-proteins and
negatively regulate signaling via G-protein-coupled receptors. The
recently resolved crystal structure of RGS4 bound to
Gi1 suggests two potential mechanisms
for the GAP activity of RGS proteins as follows: stabilization of the
Gi
1 switch regions by RGS4 and the catalytic
action of RGS4 residue Asn128. To elucidate a role of the
Asn residue for RGS GAP function, we have investigated effects of the
synthetic peptide corresponding to the G
binding domain of human
retinal RGS (hRGSr) containing the key Asn at position 131, and we have
carried out mutational analysis of Asn131. Synthetic
peptide hRGSr-(123-140) retained its ability to bind the
AlF4
-complexed
transducin
-subunit,
Gt
·AlF4
,
but failed to elicit stimulation of Gt
GTPase activity. Wild-type hRGSr stimulated Gt
GTPase activity by ~10-fold with
an EC50 value of 100 nM. Mutant hRGSr proteins
with substitutions of Asn131 by Ser and Gln had a
significantly reduced affinity for Gt
but were capable
of substantial stimulation of Gt
GTPase activity, 80 and
60% of Vmax, respectively. Mutants
hRGSr-Leu131, hRGSr-Ala131, and
hRGSr-Asp131 were able to accelerate Gt
GTPase activity only at very high concentrations (>10
µM) which appears to correlate with a further decrease of
their affinity for transducin. Two mutants, hRGSr-His131
and hRGSr-
131, had no detectable binding to transducin.
Mutational analysis of Asn131 suggests that the
stabilization of the G-protein switch regions rather than catalytic
action of the Asn residue is a key component for the RGS GAP
action.
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INTRODUCTION |
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The intensity and duration of signaling via heterotrimeric
G-proteins is regulated at multiple levels. The key reaction in termination of G-protein-mediated signaling is the intrinsic GTPase activity of G subunits that convert the active GTP-bound
conformation of G-protein
subunits (G
·GTP) to the inactive
G
·GDP conformation. The GTPase activity of two G-proteins,
Gq and transducin, is stimulated by their effectors,
phospholipase C
and cGMP phosphodiesterase (PDE),1 respectively (1-3).
A novel class of GTPase-activating proteins (GAPs) for heterotrimeric
G-proteins called RGS has been identified (4-6). Strong evidence
suggests that members of this family, GAIP, RGS4, RGS1, RGS10 and
others, negatively regulate G-protein signaling by stimulating GTPase
activity of G-proteins, particularly those from Gi and
Gq families (7-9). RGS proteins from yeast to mammals
share a highly conserved RGS domain that provides relatively broad
specificity of different RGS proteins toward members of the two
G-protein classes in vitro. Tissue expression patterns and
diverse domains outside the RGS segment may play an important role in
determining specificity of RGS proteins in vivo (10-12). Precise mechanisms of RGS GAP activity are not yet clear. The transition state during GTP hydrolysis is thought to be mimicked by the
AlF4
-bound conformation of G
subunits (13, 14). It has been demonstrated that many RGS proteins
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 (8, 15, 16).
Recently, the crystal structure of RGS4 bound to
Gi1·AlF4
has been solved at a resolution of 2.8 Å (17). This structure provides
the first structural insights into the mechanism of RGS protein action.
The conserved RGS core forms three distinct sites of interaction with
the three switch regions of Gi
1 suggesting that stabilization of the switch regions and G
residues
directly involved in GTP hydrolysis may be a major component of RGS GAP activity (17). Furthermore, RGS proteins could also contribute catalytic residues to the active site and thus enhance the GTPase rate
constant. The conserved residue Asn128 of RGS4 makes a
contact with the side chain of Gln204 of
Gi
1 which stabilizes and orients the
hydrolytic water molecule in the transitional state of
Gi
1 (17). Asn128 also may be
localized within hydrogen-bonding distance of the hydrolytic water
molecule for nucleophilic attack on the GTP
-phosphate (17).
In this study we evaluate a potential catalytic role of the Asn residue
for G GTPase acceleration by RGS proteins using the
interaction between human retinal RGS (hRGSr) protein and transducin as
a model system and mutational analysis of Asn131 of hRGSr
which is equivalent to Asn128 of RGS4.
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EXPERIMENTAL PROCEDURES |
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Materials--
GTP and GTPS were products of Boehringer
Mannheim. Blue-Sepharose and PD-10 Sephadex G-25 columns were obtained
from Pharmacia Biotech Inc. [
-32P]GTP (>5000 Ci/mmol)
was purchased from Amersham Corp. [35S]GTP
S (1250 Ci/mmol) was obtained from NEN Life Science Products. All other
chemicals were acquired from Sigma.
Preparation of Rod Outer Segment (ROS) Membranes,
Gt·GTP
S, Gt
·GDP, and
hRGSr--
Bovine ROS membranes were prepared as described previously
(18). Hypotonically washed ROS membranes (dROS) depleted of PDE subunits were prepared as described in Ref. 2. Transducin, Gt
, was extracted from ROS membranes using GTP as
described in Ref. 19. 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. 20.
Gt
·GDP was prepared and purified according to
protocols in Ref. 21. hRGSr was prepared and purified as described
previously (22). The purified proteins were stored in 40% glycerol at
20 °C or without glycerol at
80 °C.
Site-directed Mutagenesis of hRGSr--
Mutagenesis of
Asn131 residue of hRGSr was performed using PCR
amplifications from the pGEX-KG-hRGSr template (22) with 3'-antisense primer ATGCCTCGAGACTCAGGTGTGTGAGG (unique XhoI
site is underlined) and the 5' primers:
XXXATTGACCATGAGACCCGCGAGC. XXX indicates
nucleotides that generate substitutions of Asn131 (AAC) in
hRGSr cDNA by the following amino acid residues: Ala (GCG), Asp
(GAT), His (CAT), Leu (CTG), Gln (CAG), Ser (AGC), and deletion mutant
(). PCR reactions were performed in 100 µl of reaction mixture
containing 1 ng of the pGEX-KG-hRGSr plasmid, 3 units of AmpliTaq DNA
polymerase (Perkin-Elmer), 25 mM
Tris-(hydroxymethyl)-methylaminopropane sulfonic acid, pH 9.3, 2 mM MgCl2, 1 mM 2-mercaptoethanol,
200 µM of dNTPs, and 0.5 µM primers.
Conditions for PCR were as follows: 94 °C for 3 min, 30 cycles of
94 °C for 1 min, 64 °C for 30 s and 72 °C for 30 s,
and a final extension at 72 °C for 3 min. The PCR products (~220
base pairs) were blunt-ended with Klenow fragment and digested with
XhoI. Wild-type hRGSr cDNA was subcloned into
XbaI/XhoI sites of pBluescript polylinker. The
resulting construct was digested with HincII and
XhoI and ligated with the XhoI-digested PCR
products carrying mutations. The mutant sequences were verified by
automated DNA sequencing at the University of Iowa DNA Core Facility
using the T7 primer and subcloned into the
XbaI/XhoI sites of pGEX-KG vector for protein
expression. Mutant GST-hRGSr proteins were expressed in DH5
Escherichia coli cells, and the GST portion was removed as
described earlier (22). Typical yields of purified hRGSr and hRGSr
mutants, except for a mutant with deletion of Asn131, were
5-6 mg/liter of culture. Deletion of Asn131 led to an
~4-5-fold reduction in expression of soluble recombinant protein
suggesting that the residue at position 131 may be important to the
stability and proper folding of RGS proteins.
Binding of Transducin to GST-hRGSr and
Mutants--
Gt·GTP
S or Gt
·GDP
(10 µg) were incubated with hRGSr or its mutants (50 µg)
immobilized on glutathione-agarose in 100 µl of 20 mM
Tris-HCl buffer (pH 8.0), containing 100 mM NaCl, 2 mM MgSO4, 6 mM 2-mercaptoethanol,
and 5% glycerol (buffer A). Where indicated, the buffer contained 30 µM AlCl3 and 10 mM NaF. After incubation for 20 min at 25 °C, the agarose beads were spun and washed twice with 1 ml of buffer A, and the bound proteins were eluted
with a sample buffer for SDS-polyacrylamide gel electrophoresis.
Single Turnover GTPase Assay--
Single turnover GTPase
activity measurements were carried out in suspensions of dROS membranes
containing 5 µM rhodopsin and 0.4 µM
transducin essentially as described in Refs. 22 and 23. Transducin
concentration of 0.4 µM was determined using the
[35S]GTPS binding assay as described previously (22).
Bleached dROS membranes were mixed with different concentrations of the tested peptides, hRGSr or hRGSr mutants, and preincubated for 5 min at
25 °C. The GTPase reaction was initiated by addition of 100 nM [
-32P]GTP (~5 × 104
dpm/pmol) in a total volume of 20 µl. At 5, 10, 20, 40, and 60 s
aliquots of the reaction mixture were withdrawn and quenched with 7%
perchloric acid. Nucleotides were then precipitated using activated
Norit A charcoal (10% w/v) in 50 mM sodium phosphate buffer (pH 7.5), and 32Pi formation was
measured by liquid scintillation counting. The GTPase rate constants
were calculated by fitting the experimental data to an exponential
function: % GTP hydrolyzed = 100 (1
e
kt), where k is a rate
constant for GTP hydrolysis.
Peptide Synthesis--
A peptide, CSEAPKEVNIDHETRELT,
corresponding to residues 123-140 of hRGSr was custom made by Genosys
Biotechnologies Inc. The N and C termini of the peptide were acetylated
and amidated, respectively. The peptide was purified by reverse-phase
high pressure liquid chromatography on a preparative Dynamax-300A
column (Rainin). The purity and chemical formula of the peptide were
confirmed by fast atom bombardment-mass spectrometry and analytical
high pressure liquid chromatography. Preparation of synthetic peptides corresponding to residues 21-31, 461-491, 492-516, and 517-541 of
rod PDE -subunit was described previously (24).
Miscellaneous-- Protein concentrations were determined by the method of Bradford (25) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (26) in 12% acrylamide gels. Rhodopsin concentrations were measured using the difference in absorbance at 500 nm between "dark" and bleached ROS preparations. Fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prizm (version 2) software. The results are expressed as the mean ± S.E. of triplicate measurements.
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RESULTS |
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Effects of Synthetic Peptide hRGSr-(123-140)--
The
Gi binding region of RGS4 containing Asn128
resides in the loop
5-
6 of RGS4 and contains 7 amino acid
residues making contacts with all three switch regions of G-protein
(17). The number of interactions is sufficient to ensure a relatively
high affinity between the corresponding synthetic peptide and G
,
provided that the peptide is able to adopt a functional conformation.
For the preliminary testing of the catalytic role of Asn131
of hRGSr, the hRGSr-(123-140)-peptide was synthesized. The length of
the peptide was chosen to allow the G
contact residues to be flanked
by at least 3 terminal residues. hRGSr-(123-140) was first examined
for its ability to stimulate GTPase activity of transducin in
suspensions of dROS membranes containing 5 µM rhodopsin and 0.4 µM transducin. dROS membranes lacked intrinsic
catalytic PDE
and inhibitory PDE
subunits. Use of such ROS
avoided interference of PDE
effects with effects of RGS protein or
RGS peptide (22, 27, 28). The peptide at concentrations of up to 2 mM had no effect on GTPase activity of transducin (not
shown). To determine if hRGSr-(123-140) is capable of binding to
transducin, we investigated effects of the hRGSr peptide on the
stimulation of GTPase activity of transducin by hRGSr. Fig.
1 shows that hRGSr-(123-140) was able to
compete with hRGSr for binding to Gt
resulting in a
dose-dependent (IC50 = 1.6 ± 0.3 mM) decrease of the stimulated GTPase activity of
transducin. hRGSr-(123-140) in the same range of concentrations had no
notable effect on the basal transducin GTPase activity (Fig. 1).
Because the competition experiments were carried out at a concentration
of hRGSr causing half-maximal stimulation of the GTPase activity, the
affinity of hRGSr-(123-140) for Gt
can be estimated as
0.8 mM. In control experiments, four unrelated peptides
(24) corresponding to residues 21-31, 461-553, 492-516, and 517-541
of the rod PDE
-subunit (at concentrations of 8 mM) had
no effect on hRGSr-stimulated GTPase activity of transducin (not
shown).
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Binding of hRGSr Mutants with Substitutions of Asn131
to Different Conformations of Gt--
Recently, we have
shown that similar to other characterized RGS proteins, hRGSr binds
with high affinity to the AlF4
conformations of transducin and very weakly to the GTP
S and GDP-bound conformations (22). We evaluated the interaction between hRGSr mutants with substitutions of Asn131 by Ser, Gln,
Ala, Leu, His, Asp as well as the mutant with deletion of
Asn131 and transducin using precipitation of
Gt
with the GST-hRGSr mutant proteins immobilized on
glutathione-agarose beads. Mutations hRGSr-Ser131 and
hRGSr-Gln131 led to a reduction in affinity of the
corresponding GST fusion proteins for
Gt
·AlF4
(Fig.
2A). Mutants
hRGSr-Leu131, hRGSr-Asp131, and
hRGSr-Ala131 showed a more significant decrease in their
affinity for the Gt
conformation (Fig. 2A).
hRGSr-His131 and hRGSr-
131 failed to
co-precipitate
Gt
·AlF4
. Mutations of
Asn131 could potentially alter hRGSr interaction with
Gt
·GTP
S and Gt
·GDP since the RGS4
Asn residue makes contact with the switch I and II regions of
Gi
1 (17). We have tested this possibility by
preincubating mutant GST-hRGSr containing beads with both conformations of Gt
. None of the seven hRGSr mutants has demonstrated
enhanced affinity for either conformation of Gt
compared
with the native GST-hRGSr (Fig. 2, B and C).
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Stimulation of GTPase Activity of Transducin by Mutant
hRGSr--
Effects of hRGSr mutants with substitutions of
Asn131 were tested in dROS membranes containing 5 µM rhodopsin and 0.4 µM transducin. Under
these conditions, the calculated rate of GTP hydrolysis by transducin
was 0.025 ± 0.004 s1 (Fig.
3). The rates of transducin GTPase
activity were then determined in the presence of increasing
concentrations of hRGSr or individual hRGSr mutants and plotted as a
function of their concentration. Wild-type hRGSr purified after
cleavage of GST-hRGSr with thrombin stimulated GTPase activity of
transducin by ~10-fold to a maximal rate k = 0.27 ± 0.01 s
1 with an EC50 value of
101 ± 14 nM (Fig. 3). All hRGSr mutants had
substantially reduced ability to stimulate the GTPase activity of
transducin. The tested mutants can be arbitrarily separated into three
groups. Two of the mutants, hRGSr-Ser131 and
hRGSr-Gln131, were relatively potent, and saturation of
their GAP effect could be achieved at 10-40 µM
concentration of mutant. hRGSr-Ser131 mutant was the most
effective and stimulated Gt
GTPase activity with an
EC50 value of 1.34 ± 0.17 µM and
Vmax ~80% (k = 0.22 ± 0.01 s
1). The mutant hRGSr-Gln131 was capable
of accelerating the Gt
GTPase activity to
Vmax of 60% (k = 0.16 ± 0.01 s
1) with an EC50 value of 3.9 ± 1.1 µM (Fig. 3). Three mutants, hRGSr-Leu131,
hRGSr-Ala131, and hRGSr-Asp131, began to cause
acceleration of Gt
GTPase activity only at very high
concentrations (>10 µM) (Fig. 3). We were unable to
practically achieve saturation of the GAP activity by these mutants due
to the very high protein concentrations required. Two mutants,
hRGSr-His131 and hRGSr-
131, did not show GAP
activity at the concentration tested (40 µM). Interestingly, the potency of hRGSr mutants in stimulating
Gt
GTPase activity (Fig. 3) appears to correlate well
with their ability to bind and precipitate
Gt
·AlF4
(Fig.
2A),
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Competition between hRGSr and hRGSr Mutants in Stimulation of
Gt GTPase Activity--
Experiments in Fig. 2 have
suggested that hRGSr mutants with substitutions of Asn131
have impaired binding to
Gt
·AlF4
. The binding
assay may, however, not be sufficiently sensitive to detect relatively
weak interactions. To determine if the drastically reduced ability of
some RGS mutants to stimulate the GTPase activity of transducin
correlates with the lack of mutant binding to transducin, we carried
out competition experiments. The hRGSr mutants incapable of
accelerating Gt
GTPase activity were examined for their
ability to block stimulation of GTPase activity of transducin by hRGSR. Fig. 4 demonstrates that none of the
tested mutants, hRGSr-Ala131, hRGSr-His131, and
hRGSr-
131, at 5 µM concentration, had any
effect on stimulation of GTPase activity of transducin by 50 nM hRGSr. These data suggest that the hRGSr mutants that
produced no stimulation of Gt
GTPase activity lost their
binding to Gt
.
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DISCUSSION |
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Molecular mechanisms of RGS protein action as GAP for
heterotrimeric GTP-binding proteins are not well understood. Studies on
the Ras-specific p120GAP suggest that the Ras GAP donates
conserved Arg789 residue to the RAS catalytic site (29, 30)
thus providing the catalytic mechanism for p120GAP
activity. The crystal structure of RGS4 bound to
Gi1·AlF4
has suggested two mechanisms for RGS GAP activity toward heterotrimeric G-proteins (17). Interaction of RGS protein with the G-protein switch
regions indicates that the mechanism of the GTPase activation by RGS
may primarily be a reduction in the free energy of the transitional
state via stabilization of G
switch regions and residues directly
involved in GTP hydrolysis (17). An additional putative mechanism for
the RGS GAP activity would be a donation of the catalytic residue to
the active site of G
. The only residue that RGS4 introduces into the
active site of Gi
1 is Asn128.
Although Asn128, in contrast to the Ras GAP
Arg789 or an intrinsic Arg in G
subunits, does not
directly interact with GDP and AlF4
(13, 14, 17, 30), it makes a contact with the side chain of
Gln204 of Gi
1, which stabilizes
and orients the hydrolytic water molecule in the transitional state of
Gi
1. Conceivably, Asn128 is
within hydrogen-bonding distance of the hydrolytic water molecule and
may bind and orient it for nucleophilic attack of the
-phosphate of
GTP (17).
To probe the role of Asn131 of hRGSr for the mechanism of
RGS protein GAP activity, we initially synthesized a peptide of
hRGSr corresponding to the region of interaction between
Gi1·AlF4
and RGS4 containing Asn128. We reasoned that if the
catalytic role of the Asn residue is a major component of RGS GAP
activity, then perhaps a peptide containing the catalytic residue would
alone be capable of eliciting the stimulation of GTPase activity. Our
data demonstrated that hRGSr peptide-(123-140) containing catalytic
Asn131 retained the ability to bind hRGSr but failed to
accelerate the GTPase activity of transducin. This indicates that the
interaction of at least two and likely all three G
binding regions
of RGS protein is required to stimulate G
GTPase activity.
Consistent with this conclusion is the recent finding that even short
deletions within the RGS domain of RGS4 destroyed its GAP activity
(31).
Further analysis of the role of Asn131 of hRGSr was carried
out using mutational substitutions of this residue. The major result from testing all hRGSr mutants is that replacement of
Asn131 with other residues dramatically decreases the
affinity of mutant hRGSr binding to Gt. Substitution of
Asn131 by Ser was intriguing because the Asn residue is not
absolutely conserved in RGS proteins, and some RGS proteins, including
GAIP, have a Ser at this position (5, 17, 31). Serine has proven to be
the best substitution for Asn with respect of retaining the GAP
activity of hRGSr protein. The hRGSr-Ser131 mutant had more
than 10-fold lower affinity for Gt
but can stimulate its
GTPase activity nearly as well as native hRGSr
(Vmax ~80%). Therefore, it is not surprising
that this residue was evolutionary selected instead of Asn for some RGS
proteins. Interestingly, the reported concentrations of the Ser
containing RGS domain of retina-specific RET-RGS1 (1 µM)
and GAIP (5 µM) required for the half-maximal GTPase
stimulation of transducin are comparable to the EC50 value
for hRGSr-Ser131 (28, 32).
An indication that the Asn residue may indeed serve to some
extent as a catalytic residue was provided by the
hRGSr-Gln131 mutant. The effects of this mutant on GTPase
activity of transducin nearly reached a plateau at only ~60%
Vmax of that observed with hRGSr. However,
effects of other hRGSr mutants argue against a catalytic role of the
Asn residue as the key component of the RGS GAP activity. The
hRGSr-Leu131 and hRGSr-Ala131 mutants at very
high concentrations started to have a stimulatory effect on
Gt GTPase activity even though these residues are not expected to form hydrogen bonds which are made by the Asn residue. Our
mutational analysis suggests that although Asn131 of hRGSr
may play a catalytic role in the RGS GAP activity, stabilization of the
switch regions of G-protein and reduction of the energy of the
transition state appear to be the major components of the RGS GAP
function. The Asn residue is absolutely essential for the stabilization
of the transition state for GTP hydrolysis because its replacement or
deletion leads to a drastic reduction in hRGSr affinity for
Gt
.
In addition to their role as GAPs, RGS proteins may act as antagonists
for some G-protein effectors, particularly for phospholipase C. RGS4
has been shown to block activation of phospholipase C
by
Gq
GTP
S (33). In another study, RGS4 inhibited
inositol phosphate synthesis activated by
AlF4
in COS-7 cells overexpressing
Gq (34). Tesmer et al. (17) have suggested that
the RGS proteins lacking the Asn residue may better serve as inhibitors
of effector binding than as GAPs. This would appear to be a likely
scenario if replacements of the Asn residue resulted in a loss of GAP
activity without a concurrent reduction of the RGS protein affinity for
activated G
subunits. The results of this work suggest
that the main consequence of Asn replacement is an impairment of
binding between mutated hRGSr protein and Gt
.
Furthermore, none of the hRGSr mutants have shown enhanced affinity to
the active Gt
·GTP
S conformation which could be
indicative of the potential of such a mutant to serve as an antagonist
for the G-protein effector.
This study only begins to address the questions, introduced by the
first crystal structure between G-protein and RGS protein, about the
mechanism of RGS protein GAP activity (17). Further biochemical
analysis coupled with resolution of other crystal structures between
activated G subunits and RGS proteins would ultimately define a role
of the critical Asn residue.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY-10843. The services provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by National Institutes of Health Grant DK-25295.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 and reprint requests should be addressed:
Dept. of Physiology and Biophysics, University of Iowa College of
Medicine, 5-660 Bowen Science Bldg., Iowa City, IA 52242.. Tel.:
319-335-7864; Fax: 319-335-7330; E-mail:
Nikolai-Artemyev{at}UIOWA.EDU.
1
The abbreviations used are: PDE, cGMP
phosphodiesterase; RGS proteins, regulators of G-protein signaling;
hRGSr, human retinal RGS protein; ROS, rod outer segment(s); dROS,
hypotonically washed ROS membranes; Gt, rod G-protein
(transducin)
-subunit; GTP
S, guanosine
5'-O-(3-thiotriphosphate); PCR, polymerase chain
reaction.
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REFERENCES |
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