From the
Heterotrimeric G-proteins mediate between receptors and
effectors, acting as molecular clocks. G-protein interactions with
activated receptors catalyze the replacement of GDP bound to the
The activation-inactivation cycle of the photoreceptor
G-protein, transducin, begins when photoexcited rhodopsin catalyzes the
exchange of transducin-bound GDP for GTP. Transducin then stimulates
the activity of its target, rod cGMP phosphodiesterase (PDE),
Our previous study with synthetic peptides corresponding to
different segments of PDE
G
The data were
analyzed after subtraction of background signal (blank injections) with
the BIAevaluation software (Pharmacia Biosensor). The kinetic
parameters of the PDE
On-line formulae not verified for accuracy where R is the
PDE
Alanine scanning mutagenesis (Gibbs and Zoller, 1991) was
used to determine the residues on PDE
The second approach to
analysis of transducin-PDE
The W70A mutation, although
decreasing PDE
Here we report the identification of amino acid
residues within the Asp
We thank Dr. M. I. Simon for many helpful discussions
and Dr. R. Swanson for help with the BIAcore instrument.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunit with GTP.
-Subunits then modulate the activity of
downstream effectors until the bound GTP is hydrolyzed. In several
signal transduction pathways, including the cGMP cascade of
photoreceptor cells, the relatively slow GTPase activity of
heterotrimeric G-proteins can be significantly accelerated when they
are complexed with corresponding effectors. In the phototransduction
cascade the GTPase activity of photoreceptor G-protein, transducin, is
substantially accelerated in a complex with its effector, cGMP
phosphodiesterase. Here we characterize the stimulation of transducin
GTPase by a set of 23 mutant phosphodiesterase
-subunits
(PDE
) containing single alanine substitutions within a
stretch of the 25 C-terminal amino acid residues known to be primarily
responsible for the GTPase regulation. The substitution of tryptophan
at position 70 completely abolished the acceleration of GTP hydrolysis
by transducin in a complex with this mutant. This mutation also
resulted in a reduction of PDE
affinity for transducin,
but did not affect PDE
interactions with the
phosphodiesterase catalytic subunits. Single substitutions of 7 other
hydrophobic amino acids resulted in a 50-70% reduction in the
ability of PDE
to stimulate transducin GTPase, while
substitutions of charged and polar amino acids had little or no effect.
These observations suggest that the role of PDE
in
activation of the transducin GTPase rate may be based on multiple
hydrophobic interactions between these molecules.
(
)until bound GTP is hydrolyzed (reviewed in
Stryer(1986), Chabre and Deterre(1989), and Hurley (1992)). It has
remained as a paradox for a number of years that the rate of intrinsic
transducin GTPase activity measured in vitro was substantially
slower that the duration of the photoresponse (reviewed in Chabre and
Deterre(1989) and Arshavsky et al.(1991)). Recent studies have
shown that, under more physiological conditions, for example in
concentrated suspensions of disrupted ROS, the rates of transducin
GTPase are high enough to cause the termination of PDE activation on
the time scale of the photoresponse (Dratz et al., 1987;
Wagner et al., 1988; Arshavsky et al., 1989; Angleson
and Wensel, 1993). Data from several laboratories now show that
transducin's interaction with PDE (Arshavsky and Bownds, 1992;
Pagès et al., 1992, 1993; Otto-Bruc et al.,
1994), and more specifically with PDE
(Arshavsky and
Bownds, 1992; Angleson and Wensel, 1994; Arshavsky et al.,
1994), results in an acceleration of transducin GTPase that can exceed
20-fold. The effect of PDE requires the presence of a membrane-bound
factor whose nature has not yet been identified (Angleson and Wensel,
1994; Arshavsky et al., 1994; Otto-Bruc et al.,
1994).
has shown that the epitope
responsible for transducin GTPase activation is located within a
stretch of 25 C-terminal amino acid residues of PDE
(Arshavsky et al., 1994). Here we report the
identification of amino acid residues in this region that are directly
involved in the GTPase regulation. We have found that the alanine
substitution of Trp
results in a complete abolishment of
the GTPase activation and seven other substitutions of hydrophobic
residues result in the reduction of the GTPase stimulation by
50-70%. These data indicate that the transducin GTPase activation
by an effector may be a result of hydrophobic interaction between
relatively long stretches of these proteins.
Preparation of ROS, Test Membranes, and
Proteins
ROS were purified from frozen retinas (TA & WL
Lowson Co., Lincoln, NE) under infrared illumination by double step
sucrose flotation (Smith et al., 1975). Rhodopsin
concentration was determined spectrophotometrically according to Bownds et al.(1971). Test membranes used for the measurements of
transducin GTPase activity were obtained as described by Arshavsky et al.(1994). Briefly, ROS were bleached on ice to achieve
tight binding of transducin with rhodopsin and homogenized in a
glass-to-glass homogenizer. The membranes were washed once by an
isotonic buffer containing 100 mM KCl, 2 mM
MgCl, 1 mM dithiothreitol, and 10 mM Tris-HCl (pH 7.5) and three times by a hypotonic buffer containing
5 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1
mM dithiothreitol. Analysis of these membranes by SDS-gel
electrophoresis shows that they retain >80% of their transducin and
are depleted of >98% of their endogenous PDE. Before being used,
test membranes were incubated for 5 h at room temperature to achieve
practically irreversible binding of GTP upon transducin activation (see
Arshavsky et al.(1994) for a detailed explanation).
GTP
S was eluted from the test membranes by 20
µM GTP
S, and then purified to at least 95%
homogeneity by gel filtration on a Superose-12 column (Pharmacia
Biotech Inc.). PDE was extracted from ROS as described by Baehr et
al.(1979). Soluble
PDE
dimer
lacking the isoprenylated and carboxymethylated C termini was prepared
by tryptic proteolysis (Catty and Deterre, 1991). PDE extract
containing
1 mg/ml PDE was incubated with 40 µg/ml trypsin for
90 min at 20 °C which resulted in a complete enzyme activation. The
proteolysis was terminated by an addition of soybean trypsin inhibitor
at a final concentration of 400 µg/ml.
PDE
was then purified to >95%
purity by gel filtration of a Superose-6 column (Pharmacia Biotech
Inc.). Protein concentration was determined by the Bradford(1976) assay
using bovine serum albumin as the standard.
Preparation of PDE
To obtain PDE and Its
Mutants
and its mutants the coding
sequence of the PDE
from the synthetic gene for the
fusion protein (Brown and Stryer, 1989) was subcloned into an
expression vector pET-11a (Novagen) under control of the isopropyl
-D-thiogalactoside-sensitive promoter T7. The alanine
substitutions were introduced by a ``cassette mutagenesis''
strategy. For each mutation two complementary oligonucleotides
containing desired mutations and protruding ends matching appropriate
restriction sites were annealed and ligated with the vector replacing
the wild type sequence. The vectors were transfected into Escherichia coli BL21-DE3 strain. Protein expression was
induced by isopropyl
-D-thiogalactoside. PDE
or its mutants were then purified by a combination of
cation-exchange and reverse-phase chromatography (Brown and Stryer,
1989). The purity of PDE
was estimated to be >95%;
the PDE
concentration was determined
spectrophotometrically at 280 nm using a molar extinction coefficient
of 7,100. The concentration of the W70A mutant whose absorbance at 280
nm is small was determined either based on the results of a complete
amino acid analysis of this protein or by the Bradford(1976) method
using the wild type PDE
as the standard. Both methods
yielded identical results. The preparation of PDE
LY and
the W70A mutant labeled by lucifer yellow vinyl sulfone was performed
as described by Artemyev et al.(1992).
GTPase Measurements
Transducin GTPase activity was
determined by a single-turnover technique described in detail by
Arshavsky et al.(1991). All the measurements were conducted at
room temperature (22-25 °C) in a buffer containing 10 mM Tris-HCl (pH 7.8), 100 mM NaCl, and 8 mM
MgCl. The reaction was started by mixing 20 µl of the
test membranes (20 µM final rhodopsin concentration) with
20 µl of [
-
P]GTP (
4
10
dpm/pmol, 0.2 µM final concentration)
supplemented by various concentrations of PDE
or its
mutants. The reaction was stopped by the addition of 100 µl of 6%
perchloric acid.
P
formation was measured
according to a modified method of Godchaux and Zimmerman(1979)
described by Arshavsky et al.(1991). The GTPase rate constant
was determined by the exponential fit of the time course of P
formation.
Determination of G
Fluorescent measurements were performed as described
earlier (Artemyev et al., 1992) on a Perkin Elmer LS5B
spectrofluorometer in a buffer containing 10 mM HEPES, 100
mM NaCl, and 1 mM MgClGTP
S Affinity to
PDE
and Its Mutants by Fluorescent
Assay
. The excitation
wavelength was 430 nm, and the emission was measured at 520 nm.
Fluorescence of 25 nM PDE
LY in the presence of
50 nM G
GTP
S was measured before and
after additions of increasing concentrations of PDE
or
its mutants. PDE
or mutants cause a decrease in the
fluorescence due to their competition with PDE
LY for
binding to G
GTP
S. The K
values in all cases were calculated from the competition
curves considering 36 nM as the K
value for the
PDE
LY
G
GTP
S complex
(Artemyev et al., 1992).
Determination of G
PDEGTP
S and
PDE
Binding with PDE
and Its Mutants on the BIAcore Sensor Chip
was covalently attached to the surface of the BIAcore sensor chip
(Pharmacia Biosensor) via primary amines following the activation of
the carboxymethyl groups of dextran on the chip. Briefly, the CM5 chip
was activated at the flow rate of 5 µl/min with 30 µl of 0.2 MN-(3-dimethylaminopropyl)-N-ethylcarbodiimide and 0.4 M of N-hydroxysuccinimide, and then 15-45
µl of 0.5 µM PDE
in 100 mM NaCl with 10 mM sodium formate (pH 4.3) were flown
through the activated surface. Unbound groups were blocked by 30 µl
of 1 M ethanolamine (pH 8.5). The noncovalently bound
PDE
was then removed by a 5-µl pulse of 6 M guanidine, 100 mM NaCl, 1 mM dithiothreitol, and
10 mM Tris-HCl (pH 8.0). For kinetics studies 35 µl of
varying concentrations of G
GTP
S or
PDE
were injected at a flow rate of 5
µl/min in a buffer containing 120 mM NaCl, 8 mM
MgCl
, 1 mM dithiothreitol, 0.05 mg/ml bovine serum
albumin, and 10 mM HEPES-KOH (pH 7.5). Each injection was
followed by a buffer flow for
7 min to monitor the dissociation of
the complex. For regeneration the cycle was concluded by a 5-µl
pulse of 6 M guanidine, 100 mM NaCl, 1 mM
dithiothreitol, and 10 mM Tris-HCl (pH 8.0).
G
GTP
S
interaction were determined by fitting the data to the general rate
equation:
G
GTP
S complex
concentration (it is proportional to the amplitude of the SPR signal), t is time, T is G
GTP
S
concentration (which remains constant during each injection due to the
constant flow of fresh solution through the reaction cell), P is the total amount of immobilized PDE
, k
and k
are the
association and dissociation rate constants. As seen from the
rearranged equation, the change in the response
(dR/dt) is linearly related to the response amplitude (R) with the slope proportional to transducin concentration.
The values of the slopes for the lines dR/dt versus R obtained at different transducin concentrations were then
replotted as a function of transducin concentration. The k
for PDE
and each of the
mutants could now be determined as slopes of these lines, while k
is the ordinate intercept. Alternatively the
values of k
were determined from the
exponential analysis of the SPR signal decay after replacement of the
G
GTP
S solution by the buffer. The k
values determined by these two methods did
not differ more than 3-fold. The second method, however, provided more
reliable data, so it was used for the calculations of the K
values presented in this study. The
same analysis was performed in the case of
PDE
.
which are
responsible for the stimulation of GTPase activity of the rod
G-protein, transducin. A previous study (Arshavsky et al.,
1994) had shown that the segment comprised of the 25 C-terminal amino
acid residues of PDE
,
DITVICPWEAFNHLELHELAQYGII
, is able to
stimulate transducin GTPase practically to the same extent as
full-length PDE
, and thus the mutagenesis was limited
to this area. Since two alanine residues are present in this segment,
the total number of mutants was 23. Their ability to stimulate
transducin GTPase was compared with that of the wild type PDE
in the test system containing photoreceptor membranes with most
of their transducin, but depleted of endogenous PDE. A single turnover
approach described in detail in our previous publications (Arshavsky et al., 1989, 1991, 1994) was used to monitor the rate of
transducin GTPase. Briefly, the GTPase reaction was initiated by the
addition of [
-
P]GTP in the amount less than
transducin. GTP was quickly bound to transducin due to a relatively
high concentration of transducin in this assay, so the subsequent
formation of the
P
reflected a single
synchronized turnover of transducin GTPase. This approach is
illustrated in Fig. 1showing a family of the GTP hydrolysis
curves obtained with increasing concentrations of PDE
.
Figure 1:
Activation
of transducin GTPase by increasing concentrations of
PDE. The GTPase reaction was started by mixing equal
volumes (20 µl) of the test membranes (40 µM rhodopsin) with 0.4 µM [
-
P]GTP, and the time course of the
P
formation was determined after quenching
samples by perchloric acid. In all experiments, besides the control,
GTP solution contained PDE
concentrations
indicated in the figure. The rate constants of transducin GTPase were
determined by single exponential fit of the data. The data are taken
from one of three similar experiments.
Based on
their ability to stimulate transducin GTPase the mutants can be
separated into three groups (Fig. 2A). The first group
of 15 mutants has GTPase activating ability similar to that of wild
type PDE. The seven members of the second group, V66A,
F73A, L76A, L78A, L81A, I86A, and I87A, retain only 35-50% of
their GAP activity. The third group includes only one mutant, W70A, in
which the ability to activate transducin GTPase is abolished.
Interestingly, all the mutations leading to a change of phenotype have
substitutions of hydrophobic rather than charged or polar amino acid
residues.
Figure 2:
PDE mutants: acceleration
of transducin GTPase (A) and binding affinities for transducin (B). The GTPase measurements in the test membranes and the
determinations of binding affinities with the fluorescent assay were
performed as described under ``Experimental Procedures.'' A, saturating, 2 µM concentrations of
PDE
and its mutants were used routinely. No GTPase
acceleration was observed with up to 30 µM W70A mutant.
The bars represent mean ± S.D. for at least two
independent determinations. The extent of GTPase regulation by the wild
type PDE
was taken as 100%. B, the bars represent mean ± S.D. for three independent determinations.
For the wild type PDE
the K was 10 ± 2
nM.
In principle, the reduction of the PDE mutants' ability to stimulate transducin GTPase might be
caused by two mechanisms. It might be the result of lower efficiency of
formation of the complex between transducin and PDE
or
inability of the mutant PDE
to accelerate GTP
hydrolysis after forming a complex with transducin. To decide which of
these is correct, we studied transducin association with PDE
mutants by two complementary techniques. The first is based on
the ability of transducin's
-subunit complex with GTP
S
(G
GTP
S) to enhance the fluorescence of
PDE
labeled with lucifer yellow vinyl sulfone (Artemyev et al., 1992). The K
for
PDE
LY binding with G
GTP
S was
determined from the measurements of fluorescence changes, and then the K
values for the wild type PDE
and all the mutants were calculated from the analysis of their
competition with PDE
LY for binding to
G
GTP
S. Fig. 2B shows that the only
mutation causing a substantial (4-5-fold) reduction of the
PDE
affinity for transducin was W70A. Direct
measurements of G
GTP
S binding to the W70A mutant
labeled with lucifer yellow vinyl sulfone revealed a similar,
10-fold loss in affinity (data not shown). This is in general
agreement with an earlier observation that a W70F substitution results
in
100-fold reduction of PDE
affinity for
transducin (Otto-Bruc et al., 1993).
interaction was direct
monitoring of complex formation by surface plasmon resonance on
Pharmacia Biosensor's BIAcore instrument (Jonsson et
al., 1991; Schuster et al., 1993). PDE
(wild type and one representative of each group of mutants, P69A,
F78A, and W70A) was covalently attached on the dextran layer of the
sensor chip, and different concentrations of
G
GTP
S were applied. The binding of
G
GTP
S with immobilized PDE
or
PDE
mutants was monitored as an increase of the SPR
signal (Fig. 3, upper panels). After 7 min the flow of
transducin solution was exchanged for a flow of buffer, initiating
G
GTP
S dissociation from the chip. The kinetic
parameters of the PDE
G
GTP
S
interaction were determined as described under ``Experimental
Procedures.'' The data obtained with the SPR measurements are in a
good agreement with the determinations of PDE
LY
fluorescence changes. The only mutant showing a substantial,
25-fold, reduction of the affinity to transducin was W70A. This
reduction is due to the decrease of the association rate; the
dissociation rate for this mutant is not affected. The K
values for PDE
and its
mutants obtained with BIAcore were higher than those with the lucifer
yellow method most likely reflecting differences in the properties of
immobilized PDE
and free PDE
in
solution.
Figure 3:
GGTP
S binding with
PDE
(WT) and three mutants (P69A, F78A, and W70A) on the BIAcore sensor chip. The upper panel shows groups of the binding-dissociation curves
obtained with increasing concentrations of G
GTP
S.
The x axis is time, the y axis is the SPR response in
resonance units. In the lower panel the data are replotted for
the determination of rate constants. The x axis is the
G
GTP
S concentration, the y axis is the
value of the slopes of the lines dR/dt versus R (see
``Experimental Procedures'') determined for corresponding
G
GTP
S concentration. The data are taken from one
of four (W70A mutant), three (wild type PDE
), or two
(P69A and F78A mutants) similar experiments. The K values for
the PDE
G
GTP
S complex were
37 ± 8 nM (mean ± S.D.) for the wild type
PDE
, 46 ± 2 nM for the P69A mutant, 94
± 4 nM for the F78A mutant, and 980 ± 450 nM for the W70A mutant.
An important conclusion from the analysis of PDE binding to transducin is that it was completely saturated at the
mutant concentrations (30 µM for W70A and 2 µm for all
other mutants) used in the GTPase assays. Therefore, a reduction of
GTPase stimulation by all the mutants from the second and the third
groups shown in Fig. 2A does not simply reflect lower
efficiency of the transducin-PDE
complex formation but
results from an altered ability of these mutants to activate GTP
hydrolysis in a complex with transducin.
interaction with transducin, does not
alter PDE
interaction with PDE catalytic subunits. The
ability of this mutant to inhibit the activity of
PDE
was identical to that of the wild
type PDE
(not shown). The kinetics of the W70A mutant
interactions with the PDE catalytic subunits was measured with the
BIAcore instrument (Fig. 4). In contrast to transducin, the rate
of the W70A mutant association with
PDE
was identical to that for the PDE
wild type. The
dissociation of
PDE
from the sensor
chip appears to be the same for both mutant and wild type
PDE
. It is slower than the resolution limit of the
instrument, 0.0005 s
, so the value of the K
could be only estimated to be less than
3 nM.
Figure 4:
trPDE binding with
PDE
(A) and W70A mutant (B) on the
BIAcore sensor chip. Panels A and B show groups of
the binding-dissociation curves obtained with increasing concentrations
of
PDE
. In Panel C the data
are replotted for determinations of k
(see
``Experimental Procedures''). The data are taken from one of
two similar experiments. Closed symbols represent
PDE
; open symbols, W70A mutant. The k
values were 1.80 ± 0.12
10
M
s
for PDE
and 1.75 ± 0.18
10
M
s
for
the mutant.
The Functional Topography of
PDE
PDE regulates the activity
of two central components of the phototransduction cascade. First, it
inhibits the catalytic activity of the nonactivated PDE. This
inhibition is released upon PDE activation by the GTP-bound form of
transducin's
-subunit. The second function of PDE
is to stimulate the rate of transducin-bound GTP hydrolysis, thus
regulating the lifetime of PDE activation. This function is most likely
to be a result of coordinated action of PDE
and another
membrane-associated factor whose nature remains unidentified (see
below). Two domains on PDE
are shown to be involved in
both of these interactions. The first domain is located within the
C-terminal third of the molecule. The site of PDE
inhibition resides mainly within the C-terminal sequence
Gly
-Ile
-Ile
(Lipkin et
al., 1988; Brown, 1992; Skiba et al., 1995), while the
residues between Asp
and Leu
(Skiba et
al., 1995) participate in binding to transducin. Our previous
study (Arshavsky et al., 1994) showed that a peptide
corresponding to the sequence between Asp
and Ile
is capable of stimulating transducin GTPase to the same extent as
PDE
. Another part of PDE
which
participates in the binding with both PDE
and
transducin is the lysine-rich area between residues Arg
and Gly
(Morrison et al., 1987, 1989;
Lipkin et al., 1988; Artemyev and Hamm, 1992; Takemoto et
al., 1992). The most likely role of this segment is to increase
the affinity of PDE
to both PDE
and transducin by providing an additional binding site for these
interactions.
-Ile
segment of
PDE
that are directly involved in the regulation of
transducin GTPase. Eight hydrophobic residues are important for this
function. The alanine substitutions of seven of them, Val
,
Phe
, Leu
, Leu
, Leu
,
Ile
, and Ile
, result in a 2-3-fold
reduction of their ability to stimulate transducin GTPase. The binding
affinity of these mutants to transducin is the same as that of the wild
type PDE
. The alanine substitution of Trp
results in a reduction of the mutant's affinity for
transducin (in agreement with the report of Otto-Bruc et al.,
1993) and also leads to a complete abolishment of the mutant's
ability to stimulate GTP hydrolysis after forming a complex with
transducin. Interestingly, this mutation is crucial only for
PDE
interaction with transducin. No differences in the
interaction between the W70A mutant and PDE
were
revealed in this study.
How Could PDE
Until recently the regulation of transducin GTPase
activity in ROS remained as one of the most controversial aspects of
the phototransduction biochemistry (see Arshavsky et al. (1994) for a more detailed discussion). However, recent data from
three laboratories (Angleson and Wensel, 1994; Arshavsky et
al., 1994; Otto-Bruc et al., 1994) led the authors to a
consensus conclusion that the acceleration of transducin GTPase is a
result of coordinate action of PDE (or PDE Regulate Transducin GTPase
Activity?
) and another
factor, most likely protein, tightly associated with the photoreceptor
membranes. The only minor discrepancy which remains to be resolved is
whether the factor itself is capable of causing some acceleration of
transducin GTPase in the absence of PDE
. This
discrepancy may be apparent and simply reflect different amounts of
residual PDE in the membrane preparations used in these studies. In any
case, it does not appear to be possible to determine the exact role of
PDE
and the membrane factor on the GTP hydrolysis
before the factor is characterized. It may be noted, however, that the
mechanism of the PDE
action is most likely to be
distinct from the intrinsic G-protein GTPase or from the action of
GTPase activating proteins (GAPs) that regulate the small GTP-binding
proteins. Specifically, while a conserved arginine and glutamine are
required for the intrinsic hydrolysis of GTP by the
-subunits of
heterotrimeric G-proteins (Markby et al., 1993; Sondek et
al., 1994; Coleman et al., 1994; Kleuss et al.,
1994), and a functionally similar arginine is presumably supplied by
GAPs (Brownbridge et al., 1993), transducin GTPase
acceleration by PDE
requires the action of eight
hydrophobic amino acid residues. Along with recent observations that
G-protein
-subunits interact with their effectors by multiple
sites not directly involved in GTP-binding (summarized by Artemyev and
Hamm(1994)), our data indicate that PDE
action may be a
result of hydrophobic interactions between long sequences of
PDE
and transducin. The consequences of such
interactions may include an optimal positioning of the residues
directly involved in GTP hydrolysis or better exclusion of bulk water
leading to a decrease of the dielectric content of the catalytic
center. Alternatively, PDE
binding with transducin may
be necessary for a further interaction of the complex with the membrane
factor. These questions will be addressed in future research.
,
the complex of PDE
- and
-subunits;
PDE
, the complex of PDE
-and
-subunits obtained by PDE trypsinization; PDE
, the
-subunit of PDE; PDE
LY, PDE
labeled by lucifer yellow vinyl sulfone; ROS, rod outer segments;
G
, transducin
-subunit; GTP
S, guanosine
5`-(
-thio)triphosphate; GAP, GTPase activating protein; SPR,
surface plasmon resonance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.