From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The visual GTP-binding protein, transducin,
couples light-activated rhodopsin (R*) with the effector enzyme, cGMP
phosphodiesterase in vertebrate photoreceptor cells. The region
corresponding to the Upon transduction of the visual signal in vertebrate photoreceptor
cells, photoexcited rhodopsin
(R*)1 binds the retinal G
protein, transducin (Gt), leading to Gt
activation. The The two central interactions of Gt In light of the importance of the Gt Preparation of ROS Membranes, Gt Ala-scanning Mutagenesis of the
Single substitutions of Gt GTP Fluorescence Assays--
Fluorescence assays of interaction
between Gt PDE Activation Assay--
HoloPDE was extracted from ROS
membranes and purified as described earlier (24). PDE (0.2 nM) was reconstituted with 2 µM Gt Miscellaneous Procedures--
Protein concentrations were
determined by the method of Bradford (25) using IgG as a standard or
using calculated extinction coefficients at 280 nm. 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 (v.2) software. The results are expressed as the
means ± S.E. of triplicate measurements. Examination of the
crystal structure of Gt Expression and Characterization of the Receptor and Effector
Competent Chimeric Gt Expression of Gt
The ability of Gt R*-induced GTP
A substantial loss of the receptor function was observed when
Asp311 was replaced by Ala. The D311A mutant in comparison
with Gt Binding of Gt Activation of Rod PDE by Gt The The critical R*-binding region, Gt Mutations Y298A, I299A, and F303A caused the loss of Gt The key event in photoactivation of PDE is a direct interaction between
the GTP-bound Gt The Ala-scanning mutational analysis performed in this study
demonstrated that none of the The most surprising finding in this work is that none of the mutations
of five Gt4-helix and
4-
6 loop of the transducin
-subunit (Gt
) has been implicated in
interactions with the receptor and the effector. Ala-scanning
mutagenesis of the
4-
6 region has been carried out to elucidate
residues critical for the functions of transducin. The mutational
analysis supports the role of the
4-
6 loop in the
R*-Gt
interface and suggests that the Gt
residues Arg310 and Asp311 are involved in the
interaction with R*. These residues are likely to contribute to the
specificity of the R* recognition. Contrary to the evidence previously
obtained with synthetic peptides of Gt
, our data
indicate that none of the
4-
6 residues directly or significantly
participate in the interaction with and activation of
phosphodiesterase. However, Ile299, Phe303, and
Leu306 form a network of interactions with the
3-helix
of Gt
, which is critical for the ability of
Gt
to undergo an activational conformational change.
Thereby, Ile299, Phe303, and Leu306
play only an indirect role in the effector function of
Gt
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-subunit of Gt (Gt
)
complexed with GTP is then released to stimulate the effector enzyme,
cGMP phosphodiesterase (PDE), by reversing the inhibiton imposed by two
PDE
subunits (P
) on the PDE catalytic dimer (P
). Activated
PDE rapidly hydrolyzes cGMP resulting in closure of cGMP-gated channels
in the photoreceptor plasma membrane (1-3).
with R* and P
during visual excitation have been extensively investigated, and the
Gt
interaction sites have been localized. Evidence
points to the C terminus of Gt
as the major R* contact
site that is critical for Gt
activation (4-9). A second
essential site of Gt
interaction with R* includes the
4/
6 loop (residues 305-315) (6, 10, 11). A peptide,
Gt
-311-328, competed for the Gt-R*
interaction (6). The tryptic cleavage site at Arg310 of
Gt
was protected upon Gt
binding to
R* (10). Several mutants with Ala substitutions of residues from the
4/
6 loop had impaired binding to R* and reduced degrees of
activation (11). Interestingly, this R* binding site overlaps with a
region of Gt
, Gt
-293-314, that has been
implicated in the transducin-effector interaction (12-16). A synthetic
peptide, Gt
-293-314, corresponding to the
4-
6
region was shown to activate PDE in vitro and to bind to
P
(12, 13). Sites of chemical cross-linking of the P
-subunit to
Gt
were localized to within the
4-
6 loop (14, 15).
A study using substituted peptides identified five nonconserved effector residues within this region (16). Despite the large body of
evidence, the significance of the Gt
4-
6 region in the effector interaction remains unclear. An insertion of the Gt
-295-314 segment into Gi
1
only marginally improved the latter's ability to bind P
(17). This
finding suggests that if the
4-
6 region is important for the
interaction with PDE, then likely the conserved residues within
4-
6 are essential for the function of the effector.
Alternatively, even small differences in Gt
and
Gi
folding may interfere with the ability of
Gt
-293-314 to assume the proper effector-binding
conformation in the context of Gi
. More importantly, the
apparent ability of peptide Gt
-293-314 to potently
stimulate PDE (12, 16) is inconsistent with the mutational analysis of
Gt
(18, 19). The latter indicates the requirement of the
switch II and
3 regions for effector activation (18, 19).
4-
6 region
for the Gt-R* interaction and substantial but conflicting
evidence on its role for PDE activation, we carried out Ala-scanning
mutational analysis of the
4-helix (residues 293-304) and the
4-
6 loop of Gt
. Our analysis of mutant
Gt
interactions with R* and PDE has underscored the role
of the
4-
6 loop for the receptor function but revealed only
indirect involvement of the
4-helix in the Gt
effector function via requirement of the
4/
3 coupling for the
activational conformational change.
EXPERIMENTAL PROCEDURES
GDP,
Gt
, and P
BC--
Bovine ROS membranes were
prepared as described previously (20). Urea-washed ROS membranes (uROS)
were prepared according to protocol described by Yamanaka et
al. (21). Gt
was purified according to Kleuss
et al. (22). P
labeled with the fluorescent probe,
3-(bromoacetyl)-7-diethyl aminocoumarin (P
BC), was obtained and
purified as described previously (23).
4-
6 Region of
Gt
--
Substitutions of Gt
residues by
Ala were introduced into
Gt
/Gi
1 chimeric protein,
Gt
*, which contains only 16 residues from
Gi
. Gt
* was made based on another
Gt
/Gi
1 chimeric protein, Chi8, which is competent to interact with R* and Gt
(17, 19). To generate Gt
*, all the Gi
residues in the
3-helix and the
3-
5 loop of Chi8, except for
Met247 (corresponding to Leu243 of
Gt
) were replaced by Gt
residues. The
following Gt
residues were introduced into Chi8:
His244, Asn247, His252,
Arg253, Tyr254, Ala256, and
Thr257. The PCR-directed mutagenesis was carried out
essentially as described in Natochin et al. (19).
residues at positions 293, 294, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 311, 312, 313, and 314 were introduced by PCR-directed mutagenesis. In
the PCR reactions, forward mutant primers were paired with a reverse
primer carrying a HindIII site and corresponding to a
sequence 50 base pairs downstream of the stop codon. The
pHis6-Gt
* plasmid was used as a template. The PCR
products (~200 base pairs) were purified on agarose gel and used for
a second round PCR amplification as reverse primers combined with a
forward primer containing the unique Gt
BamHI
site. The 500-base pair PCR products were digested with
BamHI and HindIII and ligated into
pHis6-Gt
* cut with the same enzymes. The sequences of
all mutants were verified by automated DNA sequencing at the University
of Iowa DNA Core Facility. Gt
* and all mutants were
expressed and purified as described previously (19).
S Binding Assay--
Gt
* or mutants (0.4 µM each) were premixed with Gt
(2 µM) in 0.5 ml of 20 mM HEPES buffer (pH 8.0)
containing 100 mM NaCl and 8 mM
MgSO4. The binding of GTP
S to Gt
* or
mutants was initiated by addition of 5 µM
[35S]GTP
S (0.2 µCi) and uROS membranes (100 nM rhodopsin). Aliquots (100 µl) were withdrawn at the
indicated times, passed through the Whatman cellulose nitrate filters
(0.45 µm), washed three times with an ice-cold binding buffer and
counted. The kapp values for the binding
reactions were calculated by fitting the data to the equation, GTP
S
bound (%) = 100(1
e
kt).
* and P
BC were performed on a F-2000
fluorescence spectrophotometer (Hitachi) in 1 ml of 20 mM
HEPES buffer (pH 7.6), 100 mM NaCl, 5 mM
dithiothreitol, and 4 mM MgCl2 essentially as
described in (19, 23). Where indicated, the buffer contained 30 µM AlCl3 and 10 mM NaF.
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. The
AlF4
-induced increases in the
tryptophan fluorescence of Gt
*GDP and its mutants were
recorded on an AB2 fluorescence spectrophotometer (Spectronic
Instruments) in a stirred 1-ml cuvette with excitation at 280 nm and
emission at 340 nm as described previously (19).
*GDP or the GDP-bound Gt
* mutants and 2 µM Gt
in suspensions of uROS membranes
containing 10 µM rhodopsin. GTP
S (10 µM)
was added to the reaction mixture, and PDE activity was measured using [3H]cGMP similarly as described previously (19).
was performed using RasMol
(v.2.6) software.
RESULTS
*--
We have previously found
that residue Leu243 of Gt
is mainly
responsible for the low level expression of
Gt
/Gi
chimeras containing the
Gt
-237-270 (
3-
5) segment (19). Gt
*
was obtained based on the Gt
/Gi
chimera,
Chi8, which contains the
3-
5 region of Gi
(17).
The nonconserved Gi
residues from the
3-helix and the
3-
5 loop of Chi8 were replaced by the corresponding Gt
residues (except for Leu243). Two of the
introduced Gt
residues, His244 and
Asn247, are important for the Gt
-PDE
interaction (19). The resulting chimeric Gt
was not only
efficiently expressed in Escherichia coli (yields of soluble
protein of 3-5 mg/liter culture) but was also fully competent for
interaction with Gt
and R* and capable of high
affinity effector binding. The ability of Gt
* to
interact with R* in the presence of Gt
was evaluated
using the GTP
S binding assay. The very slow GTP
S binding rate to
Gt
* (kapp = ~0.004
s
1), which is limited by the rate of GDP dissociation
(26), was significantly accelerated in the presence of R* and
Gt
(kapp = ~0.079
s
1) (Fig. 1). A
fluorescence read-out assay was utilized to monitor the interaction
between Gt
and the P
subunit (23). Using this assay,
Gt
*GDP bound fluorescently labeled P
, P
BC, with a
Kd value of 28 nM (Fig.
2A and Table
I). When Gt
*GDP was
activated in the presence of AlF4
it
bound to P
BC with an almost 6-fold higher affinity
(Kd of 5.1 nM) (Fig. 2B and
Table I). Thus, Gt
*, which contains only 16 Gi
residues, represents a well suited tool for
mutational analysis to identify residues that are essential for both
receptor and effector interactions of transducin.
View larger version (12K):
[in a new window]
Fig. 1.
The time course of
GTP S binding to
Gt
*. The binding of GTP
S
to Gt
* (0.4 µM) was initiated by addition
of 5 µM of [35S]GTP
S (0.2 µCi) (
).
When the effect of R* was measured, Gt
* was premixed
with Gt
(2 µM), and the binding was
initiated by addition of 5 µM of
[35S]GTP
S and uROS (100 nM rhodopsin)
(
). Aliquots were withdrawn at the indicated times, and GTP
S
bound to Gt
* was determined using the filter binding
assay. The calculated kapp values are 0.004 s
1 (
) and 0.079 s
1 (
).
View larger version (10K):
[in a new window]
Fig. 2.
Binding of
Gt * to
P
BC. The relative increase in
fluorescence (F/Fo) of P
BC (10 nM)
(excitation at 445 nm; emission at 495 nm) was determined after
addition of increasing concentrations of Gt
*GDP in the
absence (A) or in the presence (B) of
AlF4
.
Interaction of Gt* mutants with P
BC and activation by R*
* Mutants with Ala Substitutions of
the
4-
6 Residues--
Residues at positions 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 308, 309, 310, 311, 312, 313, and 314 within the
4-helix and the
4-
6 loop of Gt
are
surface exposed (27) and were substituted with Ala residues. In
addition to a modestly solvent-exposed Leu306, two buried
residues, Ile299 and Phe303, are involved in
coupling the
4-helix with the
3-helix (27). This linkage might be
important for stabilization of the receptor and/or effector-competent
conformations of Gt
. Substitutions of Ile299
and Phe303 were made to test this possibility. Expression
of all but three of the Gt
* mutants in E. coli have yielded similar amounts of soluble proteins (~3-5
mg/liter of culture). Mutants Y298A, I299A, and F303A had notably
reduced expression levels (~0.5-1 mg/liter). The crystal structure
of Gt
GTP
S shows that the Tyr298 side
chain makes contact with Tyr286 from the
G-
4 loop,
whereas Ile299 and Phe303 interact with the
3-helix (27). Perhaps, the reduction in mutant expression reflects
lower rates of proper protein folding due to the lack of stabilizing
contacts between
4 and the
G-
4 loop or the
3-helix.
mutants to undergo a conformational
change upon addition of AlF4
was
analyzed by measuring their intrinsic tryptophan fluorescence (18).
Mutants Y298A, I299A, and F303A failed to display an increase in
tryptophan fluorescence upon addition of
AlF4
, whereas the fluorescence change
for L306A was intermediate to that for Gt
* (not shown).
S Binding to Gt
* Mutants--
The
ability of R* to interact with Gt
* mutants and cause
them to release GDP was examined by measuring the rates of GTP
S binding to these mutants in the presence of R* and
Gt
. The release of GDP is a rate-limiting step in
activation of G protein
subunits, and thus it controls the rate of
GTP
S binding (26). Three Gt
* mutants, Y298A, I299A,
and F303A, did not appreciably bind GTP
S. A correlation between the
low expression levels of these mutants and the lack of GTP
S binding
indicates that defects in the overall folding might be responsible for
the loss of the R*-dependent activation. However, the
finding that these mutants were able to specifically interact with the
effector (see below) rules out gross misfolding. Alternatively, the
4-
3 coupling could represent an important element in maintaining
proper conformation of the R*-binding regions, or it is essential for
the ability of Gt
to undergo a conformational change
upon binding of GTP
S. The latter possibility is supported by the
lack of the tryptophan fluorescence enhancement with addition of
AlF4
to Y298A, I299A, and F303A. The
GTP
S binding properties of the L306A mutant, in which another
residue that contacts
3 was substituted, were seriously compromised
but not abolished. Fitting of the GTP
S binding data for L306A
yielded a value for maximal binding at ~35% of that for
Gt
* with an ~4-fold lower rate
(kapp = ~0.019 s
1) (Fig.
3 and Table I). Gt
*L306A
was expressed in E. coli comparably to Gt
*
but showed diminished ability for the conformational change in the
presence of AlF4
. This suggests that
L306A has a similar but more mildly expressed phenotype than mutants
Y298A, I299A, and F303A.
View larger version (16K):
[in a new window]
Fig. 3.
GTP S binding to
Gt
* mutants. The binding of
GTP
S to Gt
* mutants (0.4 µM mutant
Gt
*; 2 µM Gt
) was
initiated by addition of 5 µM [35S]GTP
S
(0.2 µCi) and uROS membranes (100 nM rhodopsin).
Protein-bound GTP
S was determined using the filter binding assay.
The V301A mutant (
) is representative of Gt
* mutants
with intact kinetics of GTP
S binding. The L306A (
), R310A (
)
and D311A (
) mutants had impaired GTP
S binding
characteristics.
* maximally bound only ~50% GTP
S with a
reduced rate of 0.023 s
1 (Fig. 3 and Table I). A
relatively mild alteration in R* activation was found for the R310A
mutant. Gt
*R310A had a saturating level of GTP
S
binding similar to that of Gt
*, but the rate of binding was decreased by ~2-fold (Fig. 3 and Table I). Previously,
Arg309, Val312, and Lys313 were
implicated in the Gt
-R* interaction using an assay of
Gt
activation in microsomes of COS7 cells expressing
rhodopsin and mutant Gt (11). We observed no significant
changes in the kinetics of Gt
* activation caused
by these three or other remaining mutations under our
experimental conditions (Table I).
* Mutants to P
BC--
To delineate
potential effector residues within the
4-
6 region, the
Gt
* mutants in the GDP-bound or active
AlF4
-induced conformations were tested
for binding to P
BC. Interestingly, mutants Y298A, I299A, and F303A,
which had low expression levels and lacked R*-induced GTP
S binding,
in the GDP-bound conformations displayed affinities for P
BC
comparable with Gt
*GDP (Table I). This result indicates
that in the inactive conformation their effector interface is not
significantly affected. Predictably, these three Gt
*
mutants had significant defects in binding to P
in the presence of
AlF4
. Addition of
AlF4
produced no enhancement in the
mutant interaction with P
BC, evidently due to the inability of these
mutants to assume an active conformation. In addition, the interaction
of the L306A mutant with P
BC was less sensitive than that of
Gt
* to AlF4
. In the
presence of AlF4
, L306A bound to
P
BC with a Kd only 2-fold lower than when
AlF4
was absent (Table I). This is
consistent with the limited competency of L306A to assume an active
conformation. The Gt
* mutants, V301A and K313A, had mild
defects in effector binding. These mutants retained a high affinity for
P
BC in the AlF4
-bound conformations
but revealed a somewhat reduced interaction with the effector in
the absence of AlF4
(Table I). All
other Gt
* mutants demonstrated affinities for P
BC
comparable with that of Gt
(Table I).
* Mutants--
The
ability of Gt
* mutants to stimulate activity of holoPDE
(P
2) was tested in the reconstituted system with
additions of uROS membranes and purified Gt
in the
presence of GTP
S. Gt
* as well as the majority of its
mutants activated holoPDE under these conditions by
~12-18-fold. Not surprisingly, mutants Y298A, I299A, and
F303A were incapable of stimulating PDE (not shown). Mutants L306A and
D311A were notably less effective in the PDE activation assay (Fig.
4). This reduction in the effector function seems to correlate well with the decreased capacity of these
mutants to bind GTP
S in the presence of R*. Therefore, residues
Leu306 and Asp311 are unlikely to be directly
involved in interaction with and activation of PDE.
View larger version (28K):
[in a new window]
Fig. 4.
Activation of PDE by
Gt * mutants. The cGMP
hydrolytic activity of rod holoPDE (0.2 nM) was measured in
suspensions of uROS membranes (10 µM rhodopsin)
reconstituted with 2 µM Gt
and 10 µM GTP
S in the presence of 2 µM
Gt
* mutants. The PDE activation is expressed as a
percentage of that elicited by Gt
* (the basal and
Gt
*-stimulated PDE activities were 14 and 210 mol cGMP/s
mol PDE, respectively).
DISCUSSION
4-
6 region of Gt
is an essential
contributor to the Gt
-rhodopsin interface (6, 11). The
R* binding sites of Gt
, the
4-
6 loop (amino acids
305-315) and Gt
-340-350, are positioned on the same
"receptor" face of Gt
as the N terminus of
Gt
and the C terminus of Gt
(28). The
6-sheet and the
5-helix project inward from the
4-
6 loop
and Gt
-340-350 on the Gt
surface to form
the
6/
5 loop. The latter contains a cluster of residues,
Cys321, Ala322, and Thr323,
intimately involved in binding of the guanine ring (27). Mutations of
the residues within the
6/
5 loop promote dissociation of GDP and
GTP-GDP exchange on several G
subunits (29-31). Thus, the
Gt
activation mechanism is likely to involve interaction of R* with the
4-
6 loop and Gt
-340-350, leading
to conformational changes of the
6/
5 loop and dissociation of GDP.
-340-350, has been
investigated in great detail (6-9, 32). However, the role of the Gt
4-
6 loop and its individual residues in binding
to R* and Gt
activation is not well understood.
Recently, mutants of Gt
with Ala substitutions of
residues in the
4-
6 loop have been translated in vitro
and expressed in COS-7 cells (11). Mutational analysis revealed that
substitutions of four residues in the
4-
6 loop,
Arg309, Asp311, Val312, and
Lys313 impaired Gt
interaction with R* (11).
The Ala-scanning mutagenesis of the Gt
4-
6 region
in the context of Gt
* readily expressed in E. coli has provided us with an opportunity for in depth
investigation of the roles of individual
4-
6 residues in the
receptor interaction. Reconstitution of the purified mutant
Gt
* with Gt
and uROS membranes has
enabled the examination of the effects of mutations on the kinetics of
Gt
* activation by R*. Our results confirm the role of
Asp311 in the R*-dependent activation of
Gt
. A substitution of this residue led to a substantial
decrease in both the rate of and total R*-induced GTP
S binding. A
moderate alteration of the kinetics of R*-induced GTP
S binding was
caused by the substitution of Arg310. The R310A mutant
bound GTP
S with an ~2-fold slower rate. Supporting the involvement
of Gt
Asp311, and probably
Arg310, in the interaction with R* is the fact that the
trypsin cleavage site Arg310-Asp311 is
protected upon binding of Gt
to R* (10). In addition to effective activation of Gt
, R* is capable of activating
Gi
(17) but has no detectable interaction with
Gs
.2
Arg310 and Asp311 of Gt
align
with the Lys-Asp and Ser-Gly pairs in Gi
and
Gs
, respectively. Therefore, Arg310 and
Asp311 along with Gt
-340-350 may contribute
to the specificity of the Gt
-R* interaction.
*
activation by R* or AlF4
, whereas the
L306A mutation resulted in a less severe phenotype. This loss of
function apparently resulted from the inability of the mutants to
undergo the activational conformational change. Residues
Ile299, Phe303, and Leu306 interact
with Met239, Leu243, and Phe246,
respectively (27). This interaction network between the
4 and
3
helices may secure the proper positioning of Glu241,
Leu245, Ile249, and Phe255. The
latter residues, upon activation of Gt
, engage the
switch II residues Arg201, Arg204, and
Trp207 to form another network of interactions, which is
critical for the Gt
progression to the active
conformation (33). Therefore, our results suggest that the coupling
between helices
3 and
4 is critical for the transition of
Gt
to the active state.
and P
. Both Gt
GTP and
Gt
GDP are capable of binding P
. However,
Gt
GDP binds P
with ~10-30-fold lower affinity and
is incapable of efficient activation of PDE (23, 34). The
Gt
binding sites on P
have been firmly established (13, 35-37). The P
-binding surface on Gt
appears to
be significantly more complex and less understood. Initially, the
putative effector region of Gt
corresponding to the
4-helix and the
4-
6 loop was identified using synthetic
Gt
peptides. A synthetic peptide, Gt
-293-314, potently (Ka of 8 µM) stimulated activity of rod holoPDE (12) via binding
to P
(13). Substitutions within the Gt
-293-314
peptide have been made, and five nonconserved residues,
Asn297, Val301, Glu305,
Met308, and Arg310, were found to contribute to
the activational effects (16). However, evidence contradicting the role
of
4-
6 as a major effector domain of Gt
has
emerged from analysis of chimeric Gt
/Gi
proteins and mutagenesis of Gt
(17-19). Two other
effector-interacting domains of Gt
, the switch II region
and the
3-helix-
3/
5 loop, have been identified (17-19). These
findings led to the apparent discrepancy between the ability of peptide
Gt
-293-314 to activate PDE and the prerequisite of the
switch II and
3-
5 regions of Gt
for the effector
stimulation. Hypothetically, the discrepancy is nonexistent if the role
of switch II and
3-
5 is only to obscure the
4-
6 region in
Gt
GDP. However, such a model is not supported by the
crystal structures of Gt
(27, 33). Moreover, at least three residues, Ile208 (switch II), His244, and
Asn247 (
3) are likely to interact directly with P
in
the GTP-bound Gt
conformation (19).
4-
6 residues appear to participate directly and significantly in the Gt
/P
binding. Even
substitutions of the residues Tyr298, Ile299,
Phe303, and Leu306, which disabled the
activation of Gt
*, had no notable impact on the binding
of the GDP-bound mutants to P
BC. Results on activation of PDE by the
Gt
mutants correlated well with the P
binding experiments. All mutants with unimpaired capacity for R*-induced GTP
S binding were competent to stimulate cGMP hydrolysis by holoPDE. The studies on cross-linking of P
to Gt
attest to a
close proximity of P
to the
4-
6 region in the
Gt
-P
complex (14, 15). Although our analysis seems to
rule out strong major interactions between P
and
Gt
-293-314, a relatively weak van der Waals'
contact(s) at this site cannot be entirely excluded. Rather, the role
of the
4-
6 residues, Ile299, Phe303, and
Leu306, is that they are critical for the activational
conformational change via the interaction with the
3-helix and thus
indirectly are important for the effector function of
Gt
.
residues identified using synthetic peptides (16) meaningfully affected the Gt
*-PDE interaction. A
greater sensitivity of the peptide structure than that of
Gt
* to mutations may explain the different results.
Although the NMR analysis of substituted peptides ruled out gross
misfolding, inactivation of mutant peptides due to a conformational
change remains a possibility (16). However, a more plausible
explanation is that the peptide Gt
-293-314 and
Gt
activate PDE via different mechanisms. This raises a
general concern regarding potential problems with interpretation of
effects that might be observed using synthetic peptides as probes of
protein-protein interactions. The conclusion that
Gt
-293-314 likely represents a major
effector-activating domain of Gt
was reached based on
the ability of the peptide to "mimic" Gt
in PDE
activation (12, 16) and provided the best explanation of the data in
the absence of an alternative approach. Yet, the puzzling mimicking
effect of the Gt
peptide does not appear to reflect the
role of the corresponding region in Gt
.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant EY-10843 and American Heart Association Grant-in-Aid 9750334N. National Institutes of Health Grant DK-25295 supported the services provided by the Diabetes and Endocrinology Research Center of the University of Iowa.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 American Heart Association Iowa Affiliate
Postdoctoral Fellowship.
§ To whom correspondence should be addressed. Tel.: 319-335-7864; Fax: 319-335-7330; E-mail: nikolai-artemyev{at}uiowa.edu.
2 M. Natochin and N. O. Artemyev, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
R*, light-activated
(bleached) rhodopsin;
Gt, rod G-protein (transducin)
-subunit;
PDE, rod outer segment cGMP phosphodiesterase;
P
and
P
,
,
, and
subunits of PDE;
ROS, rod outer segment(s);
uROS, urea-stripped ROS membranes;
P
BC, P
labeled with
3-(bromoacetyl)-7-diethyl aminocoumarin;
GTP
S, guanosine
5'-O-(3-thiotriphosphate);
PCR, polymerase chain
reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|