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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Known RGS proteins stimulate GTPase activity of
Gi and Gq family members, but do not
interact with Gs and G12
. To determine the role of specific G
residues for RGS protein recognition, six RGS
contact residues of chimeric transducin
-subunit (Gt
) corresponding to the residues that differ between Gi
and
Gs
have been replaced by Gs
residues. The
ability of human retinal RGS (hRGSr) to bind mutant Gt
subunits and accelerate their GTPase activity was investigated.
Substitutions Thr178
Ser, Ile181
Phe,
and Lys205
Arg of Gt
did not alter its
interaction with hRGSr. The Lys176
Leu mutant had the
same affinity for hRGSr as Gt
, but the maximal GTPase
stimulation by hRGSr was reduced by ~2.5-fold. The substitution
His209
Gln led to a 3-fold decrease in the affinity of
hRGSr for the Gt
mutant without significantly affecting
the maximal GTPase enhancement. The Ser202
Asp mutation
abolished Gt
recognition by hRGSr. A counteracting replacement of Glu129 by Ala in hRGSr did not restore the
interaction of hRGSr with the Gt
Ser202
Asp mutant. Our data suggest that the Ser residue at position 202 of
Gt
is critical for the specificity of RGS proteins
toward Gi and Gq families of G-proteins.
Consequently, the corresponding residue, Asp229 of
Gs
, is likely responsible for the inability of RGS
proteins to interact with Gs
.
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INTRODUCTION |
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Heterotrimeric GTP-binding proteins (G-proteins) are components of
many major signaling systems that are used by cells to transduce a
variety of signals from specific cell surface receptors to
intracellular effector proteins. Regulation of G-protein GTPase activity represents an important mechanism for establishing proper signal duration. A novel class of proteins called
RGS1 for
regulators of G-protein signaling
has been identified (1-5). Evidence has been accumulated that members
of this family negatively regulate signaling via Gi and
Gq-like G-proteins by stimulating their GTPase activity
(6-10). Identification of RGS proteins has helped to solve a long
standing discrepancy between the fast signal termination in
vivo and relatively slow intrinsic GTPase rates typically observed
under in vitro conditions (6, 11). However, no RGS protein
or other GTPase-activating protein (GAP) specific toward
Gs has been described to date (9, 10). The recently solved crystal structure of RGS4 bound to
Gi
1·AlF4
provides the first structural insights into the mechanism of RGS
protein GAP function and offers a starting point for studying the
structural basis of the specificity of known RGS proteins (12). RGS4
interacts with the switch regions of Gi
1
that are likely to have a similar general conformation with the
corresponding regions of Gs
(12). The incompetence of
RGS proteins to bind and stimulate the GTPase activity of
Gs
therefore originates from the differences between
amino acid residues of Gi
1 contacting RGS4
and corresponding residues of Gs
.
In this study we investigate molecular determinants of the specificity
of RGS/G-protein interaction using transducin -subunit (Gt
) as a prototypical member of the Gi
family and a human homologue (hRGSr) of mouse retinal mRGSr (13, 14).
We have carried out mutational analysis of specific amino acid residues
of chimeric Gt
corresponding to the RGS contact residues
that are different between Gi
and Gs
to
determine their specific role for RGS protein recognition.
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EXPERIMENTAL PROCEDURES |
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Materials--
GTP was a product of Boehringer Mannheim.
[-32P]GTP (>5000 Ci/mmol) was purchased from Amersham
Corp. All other chemicals were acquired from Sigma.
Preparation of Rod Outer Segment (ROS) Membranes,
Gt and hRGSr--
Bovine ROS membranes were prepared
as described previously (15). Urea-washed ROS membranes (uROS) were
prepared according to protocol in Ref. 16. Gt
was
prepared by the procedure described in Ref. 17. GST-hRGSr and hRGSr
were prepared and purified as described previously (14). The purified
proteins were stored in 40% glycerol at
20 °C or without glycerol
at
80 °C.
Site-directed Mutagenesis of Chimeric
Gt--
Mutagenesis of Gt
residues was
performed using the vector for expression of His6-tagged
Gt
/Gi
1 chimera 8 (Chi8) as a
template for PCR amplifications (18). The Gt
Lys176
Leu and Thr178
Ser substitutions
were introduced using 5'-primer 1 and 3'-primers 2 and 3, respectively,
for PCR amplification (see below). The PCR products were digested with
BsmBI and subcloned into the BsmBI-digested pHis6Chi8. Primer 3 also contained silent mutations
creating the unique XbaI site that was used to make the
Ile181
Phe mutant. The 5'-primer 4 and 3'-primer 5 were
used to obtain the PCR product carrying the Ile181
Phe
mutation. The product was cut with XbaI and
HindIII and subcloned into the
XbaI/HindIII-digested pHis6Chi8
Thr178
Ser. The Ser202
Asp and
Lys205
Arg substitutions were introduced by
PCR-directed mutagenesis using 5'-primer 6 and 3'-primers 7 and 8, respectively, followed by insertion of the
NcoI/BamHI-digested PCR products into
pHis6Chi8. Mutation His209
Gln was
generated using 5'-primer 9 and 3'-primer 5 and subcloning of the PCR
product into the BamHI and HindIII sites of
pHis6Chi8. The sequences of all mutants were verified by
automated DNA sequencing at the University of Iowa DNA Core Facility.
Chi8 and all mutants were expressed and purified as described
previously (18). The purified proteins were tested in the trypsin
protection assay as described (19). The following primers were used to
generate mutant Chi8 (the restriction sites are underlined and mutated codons are in bold): Primer 1, TGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGG; Primer 2, GAACTG
CGTCTC AAT GAT ACC CGT GGT CAG GAC ACG GG;
Primer 3, GAACTG CGTCTC AAT GAT ACC CGA GGT CTT
GAC TCT AGA GCG C; Primer 4, GCGC TCT AGA GTC
AAG ACC ACG GGT ATC TTT GAG; Primer 5, TCGTCTTCAAGAATCGATAAGCTT; Primer 6, ATC ACG CC ATG
GGG GCT GGG GCC AGC; Primer 7, A GCA GTG GAT CCA CTT
CTT GCG CTC ATC GCG CTG CC; Primer 8, A GCA GTG GAT
CCA CTT GCG GCG CTC TGA GC; Primer 9, AAG TGG
ATC CAG TGC TTT GAA GGC.
Site-directed Mutagenesis of hRGSr--
A substitution
Glu129 Ala of hRGSr was performed using PCR
amplifications from the pGEX-KG-hRGSr template (14) similarly as
described (20). GST-hRGSr and the mutant were expressed in DH5
Escherichia coli cells, and the GST portion was removed as described earlier (14).
Binding of Chimeric Gt and Its Mutants to
GST-hRGSr--
Chi8 or its mutants (1 µM final
concentration) were mixed with glutathione-agarose retaining ~10 µg
of GST-hRGSr in 200 µl of 20 mM HEPES buffer (pH 7.6)
containing 100 mM NaCl, 2 mM MgCl2, 30 µM AlCl3, and 10 mM NaF
(buffer A). After incubation for 20 min at 25 °C, the agarose beads
were spun down, washed three times 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 uROS membranes
(5 µM rhodopsin) reconstituted with chimeric
Gt or its mutants (2 µM) and
Gt
(1 µM) essentially as described in
Refs. 14 and 21. Bleached uROS membranes were mixed with different
concentrations of hRGSr or hRGSrGlu129
Ala and
preincubated for 5 min at 25 °C. The GTPase reaction was initiated
by addition of 100 nM [
-32P]GTP (~4 × 105 dpm/pmol). 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.
Miscellaneous-- Protein concentrations were determined by the method of Bradford (22) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (23) 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 hRGSr on GTPase Activity of Gt
Mutants--
Six residues directly interacting with RGS4 are different
in Gi
1 and Gs
(12). These
residues correspond to Lys176, Thr178,
Ile181, Ser202, Lys205, and
His209 of Gt
. Except for a conservative
substitution, Gt
Ile181/Gi
1 Val185,
these residues are identical in Gt
and
Gi
1. To analyze functional consequences of
the replacement of these Gt
residues by corresponding Gs
residues we took advantage of the efficient
expression of functional
Gt
/Gi
1 chimeras in E. coli (18). All the Gt
mutants were made based on
Chi8 that contains 80% of Gt
amino acid sequence, including all three Gt
switch regions (18). Analysis of
Chi8 GTPase activity showed properties similar to native
Gt
. The GTP hydrolysis by Chi8 alone or in the presence
of uROS was negligible (not shown). In the presence of both, uROS and
Gt
, the basal GTPase activity of Chi8 was 0.016 ± 0.002 s
1 (Fig. 1). A
similar rate of GTP hydrolysis (0.019 s
1) was observed
earlier for holotransducin, Gt
, reconstituted with
uROS under similar conditions (14). This suggests that despite a lack
of myristoylation and the His6-tag attached to the N
terminus, Chi8 was competent to interact with Gt
and
light-activated rhodopsin. The GTPase activity of Chi8 was
substantially enhanced in the presence of hRGSr. Addition of 1 µM hRGSr led to acceleration of the GTPase activity by
almost 8-fold (k = 0.126 ± 0.018 s
1) (Fig. 1). Stimulation of GTPase activity of
transducin by hRGSr under similar conditions was ~10-fold (14).
Furthermore, the dose dependence of the stimulation of Chi8 GTPase
activity by hRGSr yielded an EC50 value of 109 ± 15 nM (Fig. 2A),
which correlates well with the EC50 value of 85 nM for the effect of hRGSr on transducin (24). Effects of
hRGSr on the GTPase activity of Chi8 suggest that this chimeric
G-protein was an appropriate target for the site-directed
mutagenesis.
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Binding of Gt Mutants to GST-hRGSr--
Binding
between the Gt
mutants and hRGSr was examined using
precipitation of mutants by glutathione-agarose beads containing immobilized GST-hRGSr. hRGSr, as many other RGS proteins, binds with
high affinity to the AlF4
conformation
of G-protein
-subunits (7, 9, 14). The binding assay demonstrated
that GST-hRGSr in the presence of AlF4
was able to precipitate nearly stoichiometric amounts of Chi8, and all
of the Gt
mutants, except Gt
Ser202
Asp (Fig.
3A). The competence of hRGSr
to efficiently precipitate the mutant Lys176
Leu is
consistent with the EC50 value of 129 nM for
the stimulation of its GTPase activity, even though the maximal GTPase
enhancement by hRGSr for this mutant was substantially decreased.
Gt
Ser202
Asp failed to bind GST-hRGSr
using this assay (Fig. 3A). The failure of Gt
Ser202
Asp to bind GST-hRGSr is not caused by its
inability to bind AlF4
and assume an
active conformation. Chi8 and the Ser202
Asp mutant
demonstrated equivalent degrees of protection of their switch II region
from tryptic cleavage upon binding of
AlF4
(Fig. 3B). The binding
data indicate correlation between the stimulatory effects of hRGSr on
the Gt
mutants in the GTPase assay and ability of hRGRr
to bind these mutants. The deficiency of hRGSr to stimulate GTPase
activity of Gt
Ser202
Asp has resulted
from the loss of the affinity of this interaction. However, the
Lys176
Leu substitution appeared to produce a different
result. The reduction in the maximal GTPase acceleration of
Gt
Lys176
Leu occurred without a
concurrent decrease in affinity of the G-protein/RGS interaction.
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Effects of the hRGSr Mutant Glu129 Ala on GTPase
Activity of Chimeric Gt
and Its Ser202
Asp Mutant--
Based on the crystal structure of RGS4 bound to
Gi
1·AlF4
(12), a residue Ser202 makes a contact with hRGSr residue
Glu129. We have tested the possibility that a complementary
replacement of hRGSr residue Glu129 by Ala would restore
the ability of hRGSr to interact and stimulate GTPase activity of
Gt
Ser202
Asp. hRGSr Glu129
Ala was fully active toward Chi8 and five of its mutants, but deficient of any GAP activity toward Gt
Ser202
Asp (not shown). Similarly, to the wild type
hRGSr, the Glu129
Ala mutant failed to bind
Gt
Ser202
Asp, whereas its binding to
Chi8 was intact (Fig. 3C).
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DISCUSSION |
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Since its recent discovery, the family of RGS proteins has been
rapidly growing. Those RGS proteins that have already been extensively
characterized share a common specificity pattern. These RGS proteins
interact with G-protein -subunits from Gi and
Gq families but have no activity toward Gs
(6-8, 10) and G12 (9). Both possibilities remain open: a
member(s) of the RGS family capable of interaction with
Gs
(G12
) has not been yet identified or
characterized, or none of the RGS proteins would be a GAP for
Gs
(G12
). The answer to this question
lies in understanding the structural details and requirements for
RGS/G-protein interaction.
The crystal structure of the complex of RGS4 with
Gi1·AlF4
has revealed a structural basis for the inability of RGS4 to interact with Gs
. Six amino acid residues from the RGS/G-protein
interface are different between Gi
and Gs
(12). Three of these residues, corresponding to Thr178,
Ser202, and His209 in Gt
are
conserved among the Gi
, Gt
,
Gq
, and Gz
subunits that are known to
interact with RGS. Another two Gt
residues, Ile181 and Lys205, have homologous
substitutions. Ile181 is substituted by Val in
Gi
and Gz
, and Lys205 is
replaced by Arg in Gq
. To identify the residue(s)
critical for the failure of Gs
to interact with RGS
proteins, we replaced the RGS contact residues in Gt
by
corresponding residues in Gs
and examined the ability
(EC50 and Vmax) of hRGSr to
stimulate GTPase activity of these mutants. hRGSr is a human homologue
(hRGSr) of mouse retinal mRGS, which was originally thought to be a
retina-specific RGS protein, but later it was found in other tissues as
well (13, 25). Like other characterized RGS proteins, hRGSr interacts with Gi- and Gq-like
-subunits, but does not
bind Gs
(24). Substitutions Thr178
Ser,
Ile181
Phe, and Lys205
Arg did not
significantly alter the activity of hRGSr toward these mutants. While
this was not unexpected for the conservative replacement
Lys205
Arg, it was rather surprising for the
Thr178
Ser mutant. The corresponding
Gi
1 Thr182 residue interacts
with seven invariant or highly conserved residues of RGS4 and, thus,
even homologous substitution by Ser could have had a major impact on
the G
/RGS interaction (12). It appears that Ser may substitute
Thr178 suitably in most of the RGS contacts. Another
substitution that did not interfere with the affinity of
Gt
binding to hRGSr is Lys176
Leu. This
is consistent with the lack of conservation at this position between
Gt
, Gq
, and Gz
.
Interestingly, however, this mutation led to a substantial reduction in
the GTPase Vmax value elicited by hRGSr. Perhaps
the lower stimulated GTPase activity of the Lys176
Leu
mutant reflects an intrinsic partial impairment of the catalytic site
not evident from the basal GTPase activity. The adjacent
Gt
Thr177 residue is intimately involved in
the GTP hydrolysis (26) and may not be fully stabilized in the
RGS/Gt
Lys176
Leu complex. The
Lys176
Leu mutation highlights the possibility that
Gs
may have a limited ability for stimulation by RGS
proteins assuming there is one that binds Gs
. A modest
decrease in the affinity for hRGSr without significantly affecting the
maximal degree of the GTPase rate acceleration was observed for
Gt
His209
Gln. The most severe outcome
for the Gt
/hRGSr interaction was caused by the
Ser202
Asp mutation. This mutation resulted in the loss
of hRGSr binding. The crystal structure of
Gi
1 with RGS4 provides a rationale for such
an outcome (12). A negative charge introduced by the Asp residue might
be repelled by the negative charge of the counteracting Glu129 residue of hRGSr, which corresponds to the
Glu126 residue of RGS4. However, the Glu residue is not
absolutely conserved in RGS proteins. A number of RGS proteins, RGS1,
RGS6, and RGS7, have residues other than Glu at this position. Small
uncharged residues such as the Ala residue in RGS7 might be the most
accommodating residue for Asp. We found that the Glu129
Ala substitution in hRGSr cannot rescue the ability of hRGSr to
interact with Gt
Ser202
Asp. Perhaps,
additional residue(s) such as Asn131 of hRGSr
(Asn128 of RGS4) also interferes with the Asp side chain.
RGS4 Asn128 makes a contact with
Gi
1 Ser206 (Ser202
of Gt
). The RGS Asn residue is critical for the RGS/G
interaction (12), and may only be substituted by Ser, though with a
notable loss of the RGS affinity for Gt
(20). Quite
possibly, an interference of the G
Asp residue with the network of
interactions involving the hRGSr Asn131 residue is also
responsible for the lack of interaction between hRGSr and
Gt
Ser202
Asp.
The degree of impairment of the RGS/G interaction in the
Ser202
Asp mutant allows us to speculate that the
corresponding Asp229 of Gs
is mainly
responsible for the inability of Gs
to interact with
characterized RGS proteins. Other differences in RGS contact residues
between Gs
and the Gi-like
-subunits
could be more easily accommodated by limited variability of different
RGS domains. Our results do not support a likelihood that one of the
currently identified RGS proteins may serve as a GAP for
Gs
. Nevertheless, they provide a direction toward
identification of potential candidates for interaction with
Gs
among yet undiscovered RGS proteins.
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ACKNOWLEDGEMENTS |
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We thank R. McEntaffer for technical
assistance and Drs. H. Hamm and N. Skiba for providing us with the
Gt/Gi
expression vector.
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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: RGS proteins,
regulators of G-protein signaling; hRGSr, human retinal RGS protein;
ROS, rod outer segment(s); uROS, urea-washed ROS membranes; GAP,
GTPase-activating protein; Gt, rod G-protein
(transducin)
-subunit; Gi
, Gs
, Gq
, and Gz
,
-subunits of G-proteins;
GST, glutathione S-transferase; Chi8, chimera 8; PCR,
polymerase chain reaction.
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REFERENCES |
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