(Received for publication, February 6, 1996, and in revised form, August 19, 1996)
From the Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
The subunit of the retinal cGMP
phosphodiesterase (
PDE) acts as an inhibitor of
phosphodiesterase (PDE) catalytic activity and mediates enzyme
regulation by the
subunit of the GTP-binding protein transducin
(
T). In order to characterize conformational changes in
the 87-amino acid
PDE subunit that may accompany the activation of the holoenzyme,
PDE was labeled with the
fluorescent probes 5-iodoacetamidofluorescein and
eosin-5-isothiocyanate for use in resonance energy transfer
measurements. 5-Iodoacetamidofluorescein specifically labeled a
cysteine residue at position 68 and served as a resonance energy
transfer donor. The site of modification of eosin-5-isothiocyanate,
which served as the resonance energy transfer acceptor, was determined
to be within the first seven residues of the amino terminus of
PDE. Energy transfer between the labeled sites on free,
unbound
PDE indicated that they were separated by a
distance of 63 Å, consistent with a random conformation. Upon binding
the catalytic
subunits of the PDE, the distance between the two
probes on
PDE increased to 77 Å. Binding of the labeled
PDE by
T·guanosine
5
-3-O-(thio)triphosphate did not affect the distance
between the probes under conditions where the PDE was activated. These
data are consistent with the view that the binding of activated
T to
PDE, which is essential for the
stimulation of PDE activity, does not impart significant alterations in
the tertiary structure of the
PDE molecule. They also
support a model for PDE activation that places active
T
in a complex with the holoenzyme.
The vertebrate phototransduction system has served as a paradigm
for understanding how receptors containing seven transmembrane helices
couple to heterotrimeric GTP-binding proteins (G proteins) and how
activated G proteins regulate the activities of their biological
effectors. The receptor in this system, rhodopsin (made up of the
protein backbone opsin and the chromophore retinal), initiates the
signaling pathway following the absorption of light. This leads to the
formation of a complex between rhodopsin and the G protein transducin
(which consists of a 39-kDa subunit, designated
T, a
35-kDa
subunit, and an ~8-kDa
subunit, designated
T). Within this complex, rhodopsin stimulates the
exchange of GDP for GTP, which in turn causes the dissociation of
transducin into an
T·GTP species and intact
·
T complex. The
T·GTP species then
stimulates the biological effector, the cyclic GMP phosphodiesterase (PDE),1 a tetrameric enzyme consisting of
two larger subunits (designated
PDE and
PDE, molecular mass ~85 kDa) and two identical smaller subunits designated
PDE (~14 kDa). The
PDE subunits serve as the binding sites for the
GTP-bound
T subunit, although the specific mechanism by
which
T binding to
PDE results in the
stimulation of cyclic GMP hydrolysis by the catalytic core of the
enzyme (i.e. the
PDE and
PDE
subunits) is still not understood. The stimulation of enzyme activity
continues until the bound GTP is hydrolyzed to GDP; thus, the GTPase
activity returns the signaling system to its starting point.
Recently, a significant amount of information has been reported
regarding the tertiary structural features of G protein subunits,
including x-ray crystallographic structures for GDP- and GTP
S-bound
forms of
T (Noel et al., 1993
; Lambright
et al., 1994
) and
i1 (Coleman et
al., 1994
). This structural information has raised a number of
possibilities regarding the identity of the regions and amino acid
residues on the G protein
subunits that are involved in the
regulation of effector activity. However, thus far, no tertiary
structural information is available for a G protein-effector complex,
and consequently, very little is known regarding the specific
mechanisms by which G protein binding is translated into effector
regulation. The phototransduction system would seem to be especially
amenable to such structure-function characterization, given that the
target sites on the effector molecule for the G protein
(i.e. the
PDE subunits) are relatively small.
However, based on NMR structure studies performed in this laboratory,
all indications are that the
PDE subunits do not possess
significant secondary structure, at least when these subunits are free
in solution (i.e. when dissociated both from the larger PDE
subunits and the
T subunit).
In studying the protein-protein interactions important in visual signal
transduction, our aim has been to develop fluorescence spectroscopic
approaches to examine different aspects of the GTP-binding/GTPase cycle
of transducin (Phillips and Cerione, 1988; Guy et al., 1990
; Mittal et al., 1994
) and to probe the mechanisms underlying
the activation of the cyclic GMP PDE (Erickson and Cerione, 1989, 1991;
Erickson et al., 1995
). In the present study, we have used resonance energy transfer approaches to determine whether the
PDE subunit adopts a unique tertiary structure when it
is bound to the
PDE and
PDE subunits
(versus when it is free in solution) and when it binds
GTP-bound
T. To do this, we developed procedures for
generating doubly labeled
PDE subunits, with one label
serving as an energy donor and the other as an energy acceptor. Using these labeled
PDE subunits, we are able to show that the
PDE subunit does change its tertiary conformation upon
binding to the
PDE and
PDE subunits; this
change extends the distance between the amino terminus and cysteine 68 of the
PDE molecule. However, the binding of the
T subunit does not appear to perturb the relative juxtaposition of these two sites on
PDE. Thus, these
results suggest that the changes in the
PDE subunit that
accompany the binding of the GTP-bound
T subunit and are
responsible for the stimulation of cyclic GMP hydrolysis by the
PDE and
PDE subunits do not occur between
residue 68 and the amino terminus of the
PDE molecule.
Furthermore, measurements of PDE enzyme activation and inhibition,
along with the characterization of corresponding spectroscopic states
of the doubly labeled
PDE subunit, support a model in
which the activated
T·
PDE complexes
remain associated with the
PDE core of the effector
enzyme during the stimulation of cyclic GMP hydrolysis.
SP-Sepharose, phenyl-Sepharose, and blue Sepharose were obtained from Pharmacia Biotech Inc. Factor Xa was purchased from New England Biolabs (Beverly, MA). 5-Iodoacetamidofluorescein and eosin-5-isothiocyanate were purchased from Molecular Probes, Inc. (Eugene, OR). Dark-adapted bovine retina were purchased from Hormel Meat Packers (Austin, MN). All other chemicals and enzymes were purchased from Sigma. The pLCIIFXSG plasmid was a gift from Dr. Heidi Hamm (University of Illinois College of Medicine, Chicago, IL).
Recombinant
PDE was expressed in Escherichia coli, and
cells were lysed as described by Brown and Stryer (1989)
. Briefly, E. coli strain AR68 containing the pLCIIFXSG plasmid was
grown at 30 °C in a Labline fermentor and induced by temperature
jump to 42 °C at an A600 of 0.5. After the
temperature jump, the cells were grown at 37 °C for an additional
2 h. Cells were harvested by centrifugation at 4000 × g and lysed with lysozyme. cII-
PDE fusion
protein was partially purified based on its insolubility in detergent,
and this particulate fraction was subsequently solubilized in 50 mM Tris, 50 mM NaCl, 1 mM EDTA, 6 M urea, pH 8.0.
The cII-PDE present in the urea-solubilized extract was
further purified by binding to an SP-Sepharose cation exchange
chromatography matrix and eluting the
PDE fusion protein
with a linear gradient of NaCl (50-300 mM), 50 mM Tris (pH 8.0), 1 mM EDTA, 6 M
urea. The cII-
PDE fusion protein was diluted to a final
[NaCl] of 100 mM and bound again to the SP-Sepharose
column. While bound to the column matrix, the buffer was changed to 50 mM Tris, 50 mM NaCl, 1 mM EDTA, pH
8.0, with no urea. The protein was again eluted with the same NaCl
gradient. At this point, the
PDE was >90% pure based
on Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis.
The purified PDE was clipped with factor Xa at
4 °C overnight. Ammonium sulfate was added to the
PDE
to a final concentration of 0.75 M, and the clipped
PDE was then loaded onto a phenyl-Sepharose column. The
PDE eluted from the phenyl-Sepharose column when there
was no (NH4)2SO4 in the column
buffer. This eluted protein was >99% pure as judged by Coomassie
Blue-stained SDS-polyacrylamide gel electrophoresis. This sample was
dialyzed in Spectropore 2,000-Da molecular mass cut-off tubing
versus 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 0.2 mM DTT, pH 7.4, and
stored in aliquots at
80 °C until use.
PDE was
reacted with 1 mM IAF at pH 7.4 for 1 h. The reaction
was quenched with the addition of 30 mM DTT, and the
labeled protein was separated from free probe by SDS-polyacrylamide gel electrophoresis. Labeled protein was visualized in the gel using UV
trans-illumination and excised (Wensel and Stryer, 1990
; Erickson and
Cerione, 1991
). Gel purification of the labeled
PDE
subunit provides a means to obtain only
PDE that
contains the IAF (fluorescence donor) molecule. This is due to the fact
that the
PDE subunit possesses a single reactive residue
at cysteine 68 and that the labeled
PDE subunit
undergoes an apparent shift in molecular mass from ~11 to ~15 kDa
after IAF labeling. Thus, pure IAF-
PDE subunit can be
isolated using a preparative gel; amino acid analysis together with
fluorescein absorption measurements at 495 nm (
max = 75,000 M
1 cm
1) (Carraway
et al. 1989
) indicate a stoichiometry of IAF incorporation of 1 ± 0.1 mol of IAF/mol of
PDE. The gel slice
containing the labeled
PDE was incubated in 4 volumes of
distilled water at 4 °C overnight, and the gel eluate was dialyzed
against a 100 × volume of 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 0.2 mM DTT, pH 7.4. The fluorescein-labeled
PDE
was then reacted in this buffer with 1 mM EITC for 3 h. The reaction was quenched with 15 mM Tris, pH 6.8, and
the protein was run on SDS-polyacrylamide gel electrophoresis and again
eluted from a gel slice.
The ratio of eosin to fluorescein in the doubly labeled
PDE was determined to be 1:1.08 ± 0.04 (S.E.,
n = 3) by absorbance spectroscopy using a
Hewlett-Packard 8451A spectrophotometer. The fluorescein absorbance was
measured at 495 nm and corrected for any contribution from EITC by
deconvolution (this represented 20% of the total absorbance). The
concentration of EITC in the doubly labeled protein was determined by
eosin absorbance at 522 nm, using a molar extinction coefficient of
83,000 M
1 cm
1 for EITC (Cherry
et al., 1976
). There was no detectable contribution of
fluorescein to this absorbance.
Purification of
transducin and holo-PDE from bovine retina were performed as described
previously (Kroll et al., 1989). Rod outer segments were
purified as described by Gierschik et al. (1984)
and washed
several times with isotonic buffer (10 mM HEPES, 5 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 100 mM NaCl, 0.3 mM
phenylmethylsulfonyl fluoride, pH 7.5). The rod outer cell membranes
were then resuspended in hypotonic buffer (10 mM HEPES, 1 mM DTT, 0.1 mM EDTA, 0.3 mM phenylmethylsulfonyl fluoride, pH 7.5) to release PDE from the membranes and centrifuged at 39,000 × g. The
supernatant from the hypotonic wash (containing the holo-PDE) was
concentrated using an Amicon 30,000-Da molecular mass cut-off
membrane.
Transducin was recovered from the hypotonically washed pellet by
resuspending the membranes in hypotonic buffer supplemented with 100 mM GTP or GTPS (for inactive GDP-bound
T
or active GTP
S-bound
T, respectively). The membranes
were washed several times with nucleotide-containing buffer, and the
supernatants were pooled. The pooled extract containing crude
transducin was purified by blue Sepharose chromatography as described
by Pines et al. (1985)
.
Trypsin-activated PDE
(tPDE) was prepared by limited tryptic digest of purified PDE (Kroll
et al., 1989). Trypsin at 65 µg/ml was added to 1 µM PDE and incubated at room temperature for 2 min. The
reaction was quenched by the addition of 260 µg/ml soybean trypsin
inhibitor. PDE activity was determined using a pH microelectrode as
described by Yee and Liebman, 1978
. Activity was measured in 5 mM HEPES, 100 mM NaCl, 2 mM
MgCl2, 5 mM cGMP, pH 7.5, at room temperature.
Proton release from cGMP hydrolysis due to PDE activity was recorded in
mV at one determination per second.
Phospholipid vesicles were prepared by sonication
of 17 mg of lecithin in 1.0 ml of deionized and distilled water. PDE
purified from ROS was incubated with the phospholipid vesicles to allow binding, and the vesicles were pelleted by centrifugation in a Beckman
Airfuge (10 min, 30 p.s.i.). Membrane-bound PDE was activated as
described by Brown (1992). Briefly, PDE was digested with ArgC protease
at a concentration of 0.1 units/µl for 6 h at room temperature. The PDE that remained in the membrane was then purified by
recentrifugation in the Beckman Airfuge. Approximately 15-20% of the
total PDE was recovered after binding and ArgC activation, and the
ArgC-proteolyzed PDE had an activity of greater than 75% of that
achieved by trypsinization. The PDE was inhibited by the addition of
labeled recombinant
PDE or unlabeled, purified
recombinant
PDE while activity was monitored to ensure
that the minimum amount of
PDE required for full
inhibition of PDE activity was added. The reconstituted, lipid
vesicle-bound PDE was stimulated by the addition of the
T·GTP
S subunit.
In most cases, relative
fluorescence was measured on an SLM 8000C spectrofluorimeter using a
1 × 0.3-cm quartz cuvette. Samples were diluted into 5 mM HEPES, 100 mM NaCl, 2 mM
MgCl2, pH 7.5, and were stirred continuously. Excitation
from a xenon lamp passed through a monochrometer set at 465 nm, and
orthogonal emission was monitored continuously at 520 nm. Emission was
corrected for changes in the lamp intensity by recording in ratio mode.
The amplitude of the fluorescence changes (for example, following the
addition of PDE and
PDE to labeled
PDE) were measured by subtracting the peak height of the
fluorescence change (i.e. after it has leveled off,
typically after 800 s) from the fluorescence that is measured
after unlabeled
PDE is added (i.e. conditions where the effect has been reversed by competition). Contributions to
the fluorescence signals due to intrinsic protein fluorescence and
light scattering were negligible (<1%) in all experiments presented
here.
The distance between donor and acceptor fluorophores was calculated
according to Förster energy transfer theory (Lakowicz, 1983).
When making resonance energy transfer measurements for the doubly
labeled
PDE, we measured the change in IAF (donor) fluorescence that occurred after trypsin treatment (since this treatment effectively separates the donor (IAF) and acceptor (EITC) probes and eliminates energy transfer) and subtracted any changes caused by trypsin treatment of singly labeled IAF-
PDE.
The efficiency of energy transfer (E) was calculated as
follows,
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Fluorescence emission anisotropy was measured on an SLM 8000C fluorimeter. Emission in the horizontal and vertical orientations was measured simultaneously using a monochrometer set at 520 nm and a band pass filter of 520 nm. The G factor was set to 1.0 by adjusting the photomultiplier gain prior to beginning acquisition. All measurements were made at 20 °C.
Data Analysis forActivity data in Fig. 2 were fit to the binding equation,
![]() |
(Eq. 3) |
Anisotropy data in Fig. 2 were fit to the binding equation,
![]() |
(Eq. 4) |
For the comparison of the kinetics of relative fluorescence and
fluorescence anisotropy changes (e.g. Fig. 5B),
relative fluorescence was determined using the mathematical
relationship between fluorescence intensity and anisotropy (Lakowicz,
1983),
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
![]() |
(Eq. 7) |
The primary aim of these studies was to generate a
PDE subunit that was labeled at two distinct sites so
that resonance energy transfer approaches could be used to monitor
changes in the juxtaposition of these sites caused by the binding of
other signaling molecules to the
PDE. In particular, we
were interested in determining if the structure of
PDE
changes upon its binding to the core of the PDE molecule and/or upon
its binding to an activated
T subunit.
We used the following strategy to generate a doubly
labeled PDE subunit. One site of labeling (with IAF) was
the single cysteine residue at position 68, both because this site is
located near one end of the
PDE molecule and because the
conditions for its selective and stoichiometric modification have been
well established (e.g. Erickson et al. (1995)
).
We then set out to label the primary amino group of the amino-terminal
methionine residue, because previous studies have demonstrated that the
N-terminal primary amino group of a protein can be selectively labeled
when performing the modification at pH values below 7.5 (Carraway
et al., 1990
).
However, given that the PDE subunit contains a number of
lysine residues, we first examined whether the labeling with EITC was
in fact occurring at the amino-terminal residue rather than at one or
more of the lysine residues on
PDE. When mass
spectrometry was performed on the full-length recombinant
PDE labeled with both EITC and IAF, a single predominant
peak was determined with a size of 10,880 (Fig.
1A). The position of this peak is within the
experimental error of the calculated size for a full-length
PDE molecule containing just one IAF moiety and one EITC
moiety (10,762 Da).
We performed an additional experiment where the full-length
PDE, labeled with IAF and EITC, was trypsin-treated and
then subjected to reverse-phase HPLC in order to resolve the resulting peptides. This was followed by mass spectrometry of the eosin-labeled species. The trypsin treatment resulted in the generation of only three
peaks showing (EITC) absorbance at 525 nm. Two of these peaks showed
lower relative levels of 525 nm absorbance and were subsequently found
not to contain peptides as revealed by mass spectral analysis. It seems
most likely that these peaks represented fluorescent dye aggregates,
and we therefore concentrated on the third peak of 525-nm absorbance
for further analysis. When this peak was subjected to mass
spectrometry, two observable mass spectrum peaks were obtained (Fig.
1B). One peak of 1555 Da corresponds to the first seven
amino acids from the amino terminus (ending with lysine residue 7) plus
a single eosin moiety. The second peak of 2025 Da represents the first
11 amino acids (ending with arginine residue 11). Comparison of these
experimentally determined labeled peptide masses revealed no other
close matches when compared with hypothetical tryptic fragments in the
PDE sequence (not shown). These results indicate that
the EITC label was attached either at the amino terminus as originally
intended or at the lysine located seven residues from the terminus.
Because the modification of lysine residues with isothiocyanate
reagents typically requires several hours of incubation at pH > 8.5 (compared with the 3-h modification at pH 7.4 used here), we feel
it is most likely that the EITC is attached to the amino-terminal
methionine of
PDE. However, even if the EITC were placed
at position 7, this would not affect the general aim of the study,
which was to determine whether the position of the amino terminus of
PDE relative to cysteine 68 changed when binding to the
larger subunits of the PDE and/or to the
T subunit of
transducin.
It has been
well documented that trypsin treatment of the cyclic GMP PDE
selectively degrades the PDE subunits, resulting in a
constitutively active PDE complex (composed of the
PDE
and
PDE subunits) (Wensel and Stryer, 1990
). Readdition
of
PDE to the trypsin-treated enzyme reverses the
constitutive activation and returns the enzyme to its inactive state.
Fig. 2A shows that changes in the
fluorescence anisotropy of IAF-
PDE can be used to
monitor the interaction between the IAF- and EITC-labeled
PDE molecule and the
PDE and
PDE subunits. This anisotropy change directly reflects
the association of the IAF-labeled
PDE subunit with the
significantly larger core subunits (
PDE,
PDE) of the effector enzyme. The resultant titration
profile is consistent with a single class of binding sites between the
labeled
PDE and the trypsin-treated PDE and yields an
apparent KD value of 6 nM. Fig.
2B shows the results of an experiment where the ability of
the doubly labeled
PDE subunit to inhibit the trypsin-activated PDE was assayed. The data again yield a titration profile that can be fit by a single class of sites with an apparent KI value of 7 nM. The close agreement
between these two observed KD values argues that the
doubly labeled
PDE is fully functional in terms of its
ability to bind to the core of the effector enzyme and inhibit cyclic
GMP hydrolysis.
We next determined whether the position of the amino-terminal
domain (labeled with EITC) relative to cysteine 68 (labeled with IAF)
changed upon the binding of the labeled PDE to the trypsin-treated PDE. The rationale here was that any change in the
proximity of these sites on the
PDE molecule would be
reflected by a change in resonance energy transfer between the donor
IAF and the acceptor EITC moiety, as monitored by a change in (donor) IAF fluorescence. The results of this experiment are shown in Fig.
3A. The lower trace represents a
control where
PDE subunits, labeled at just a single
site (cysteine 68) with IAF, were added to the trypsin-treated PDE. A
slight increase (
5%) in the IAF fluorescence was detected,
presumably reflecting a change in the microenvironment of cysteine 68. This small change can be reversed by the addition of excess, unlabeled
PDE. The subsequent addition of trypsin also resulted in
a minor increase in the singly IAF-labeled
PDE
fluorescence. This suggests that the microenvironment of cysteine 68 within the full-length
PDE molecule is altered
relative to the environment of cysteine 68 within the trypsinized
fragment that contains this residue. A parallel set of
controls was performed with a singly labeled eosin-
PDE
species binding to trypsinized
PDE. No detectable
change in either eosin emission (at 545 nm) or absorbance (at 525) was
observed when eosin-
PDE bound to the PDE enzyme core
(data not shown).
The upper trace in Fig. 3A shows the results
obtained when PDE labeled with both IAF and EITC was
added to the
PDE and
PDE subunits,
generated by trypsin treatment of the PDE. There was a significant
increase in the IAF fluorescence of the doubly labeled
PDE upon binding to
PDE and
PDE. We have found that this increase, which ranged from
20 to 40% in different experiments (34 ± 5%, S.E.,
n = 4), could be completely eliminated by adding excess
PDE, consistent with the interaction between
PDE and the
PDE and
PDE
subunits being fully reversible. Significantly, the addition of trypsin
to the free, unbound doubly labeled
PDE caused a
significant enhancement in the IAF fluorescence. After correction for
the small changes caused by the addition of trypsin to the control IAF-labeled
PDE subunit (shown in the lower
trace of Fig. 3A), the average value for
trypsin-induced enhancement of the free IAF-
PDE
fluorescence was 36 ± 2% (S.E., n = 8). In
control experiments, the magnitude of the enhancement was not
influenced by the presence of other nonfluorescent proteins
(e.g. bovine serum albumin; data not shown). This level of
IAF fluorescence represents the donor fluorescence in the absence of
resonance energy transfer, since tryptic digestion of the
PDE molecule results in the IAF moiety being attached to
a (tryptic) peptide that is distinct from the peptide containing the
EITC moiety. Thus, when this level of IAF fluorescence is compared with
the IAF fluorescence for the doubly labeled
PDE molecule
when it is free in solution, this yields an efficiency of energy
transfer of 36%. When that value is used to calculate the distance
separating the IAF and EITC moieties, using an
R0 value of 56.8 Å (see "Experimental
Procedures"), an apparent distance between the IAF and EITC moieties
of 62.7 ± 1.05 Å (S.E., n = 8) is obtained. The
same measurement made for the doubly labeled
PDE subunit
when it is bound to the
PDE and
PDE
subunits, generated by treatment of the PDE with trypsin or with ArgC
(see below), yielded an efficiency of energy transfer of 15 ± 3%
(S.E., n = 4) and an effective distance of 77.3 ± 3.3 Å (S.E., n = 4). Thus, these results indicate that
the
PDE molecule becomes significantly more extended
when it binds to the
PDE and
PDE
subunits.
The results shown in Fig. 3B show the corresponding real
time fluorescence assay for doubly labeled PDE
interactions with the
PDE and
PDE using
the changes in fluorescence anisotropy for the IAF moiety bound to
PDE. The fluorescence anisotropy change that occurs upon
the addition of trypsin-treated PDE to the doubly labeled
PDE subunit can be rapidly reversed by the addition of
excess
PDE (as the change in IAF fluorescence can also
be rapidly reversed; Fig. 3A). The addition of trypsin to this sample then results in an immediate decrease in the anisotropy for
IAF due to the degradation of the labeled
PDE subunit
and the dissociation of labeled
PDE peptide fragments.
The raw anisotropy data shown in Fig. 4B can
be further analyzed taking into account the disproportionate
contribution of the
PDE-bound state of the doubly
labeled
PDE to the overall 520 nm emission (see
"Experimental Procedures"). When the corrected data for the
decrease in the fluorescence anisotropy, upon the addition of unlabeled
PDE, and the corresponding decrease in the fluorescence
enhancement, are fit to a single exponential decay, the rate of the
decay in the IAF fluorescence (0.066 s
1) is similar to
the decrease in doubly labeled
PDE anisotropy (0.042 s
1).
Fluorescence Read-out for the Binding of
We next examined
whether the position of cysteine 68 relative to the amino terminus of
PDE changed upon binding to an activated
T subunit. This was done employing the same fluorescence
read-outs as those used to examine changes in
PDE that
accompanied its binding to the
PDE and
PDE subunits. Fig. 4A shows the results from
a control experiment where the
T·GTP
S complex was
added to an IAF-labeled
PDE subunit. We found that the
T·GTP
S/IAF-
PDE interaction resulted
in an ~10% enhancement in the IAF fluorescence, presumably
reflecting an
T-induced change in the microenvironment of cysteine 68, as previously reported (Erickson et al.,
1995
). This fluorescence change was immediately reversed upon the
addition of excess (unlabeled)
PDE, due to its
competition with the IAF-labeled
PDE for the activated
T subunit. As shown earlier (Fig. 3A), trypsin treatment of the IAF-
PDE then results in a minor
increase in the IAF fluorescence.
Fig. 4B shows the results obtained when the same type of
experiment was performed using the doubly labeled PDE.
Essentially, the same results were obtained as those seen with the
singly (IAF-) labeled
PDE. Specifically, there was an
immediate enhancement (~10%) in the IAF fluorescence of the doubly
labeled
PDE molecule upon the addition of
T·GTP
S, and the
T-induced
enhancement was rapidly reversed by the addition of excess unlabeled
PDE. However, in this case, trypsin treatment of the IAF
and EITC-labeled
PDE subunit resulted in a significant
enhancement of the IAF fluorescence (as shown earlier in Fig.
3B), because the IAF and EITC labels end up on different
tryptic fragments, and thus the resonance energy transfer between these
labels is eliminated. Consequently, an increase in IAF fluorescence
occurs. Thus, although
PDE binding to the
PDE and
PDE subunits effectively results in an extension in the distance between the amino terminus and cysteine
68 of the
PDE molecule, the results in Fig. 4 argue that
there is no such change in the juxtaposition of these sites when
PDE binds to the activated
T subunit.
An important question was whether any change in the PDE
structure occurred when an activated
T subunit bound to
an intact PDE molecule (i.e. an
PDE·
PDE·
PDE complex).
The results shown in Fig. 5 suggest that this is not the
case. In this experiment, the IAF fluorescence of an IAF and
EITC-labeled
PDE subunit was monitored after protease
treatment of the holo-PDE molecule. The protease ArgC was used because
it has been shown to have some selectivity in degrading the
PDE subunits of the holo-PDE complex, leaving intact the
ability of an activated
T subunit to stimulate cyclic
GMP hydrolysis following the addition of this G protein subunit
together with fluorescently labeled
PDE subunits (Brown, 1992
). The addition of the doubly labeled
PDE subunits
to holo-PDE molecules that were pretreated with ArgC resulted in an
enhancement in IAF fluorescence, consistent with our previous findings
that a reduction in energy transfer occurs between the IAF and EITC labels when the doubly labeled
PDE subunit binds to the
PDE and
PDE subunits. Subsequently, no
additional change in the IAF fluorescence was observed upon the
addition of an
T·GTP
S complex under conditions
where we found that the addition of the G protein to this mixture
caused a significant stimulation of cyclic GMP hydrolysis (Fig. 5,
inset). Thus, under conditions where the activated
T subunits were clearly binding to the IAF and
EITC-labeled
PDE subunits (which in turn are part of a
holo-PDE complex), there was no detectable change in
PDE
structure as read-out by changes in resonance energy transfer between
the two labels.
In the present study, we set out to use fluorescence spectroscopic
approaches to determine whether structural changes occur within the
PDE molecule when it binds to the larger
PDE and
PDE subunits of the effector
enzyme. We also were interested in determining whether the tertiary
structure of
PDE is affected by the binding of an
activated
T subunit, because such information could
provide insight into the molecular mechanism by which the retinal G
protein stimulates PDE activity (i.e. cyclic GMP
hydrolysis). Our general strategy for examining tertiary structural
changes within
PDE was to attach a fluorescence donor
moiety at cysteine 68 (IAF) and an acceptor chromophore (EITC) at the
amino-terminal end of the
PDE molecule and then to use
resonance energy transfer to monitor the juxtaposition of these labels
under different experimental conditions. By using this approach, we
found that the distance between the fluorescent probes (i.e.
the distance between the EITC-labeled amino group and the IAF-labeled
cysteine) was increased from 63 Å when
PDE was free in
solution to 77 Å when it was bound to the larger subunits of the PDE
molecule. However, there was no significant change in the proximity of
these two sites on the
PDE molecule upon the binding of
an activated
T subunit.
Previous resonance energy transfer measurements suggested that upon the
formation of an T·
PDE complex, cysteine
68 of
PDE (which is thought to be close to the
T-binding site (Faurobert et al., 1993
)) is a
significant distance (~40 Å) from the guanine nucleotide binding
site on
T. Various studies have implicated residues
300-310 of
T as being involved in the stimulatory
interaction with the PDE molecule (Rarick et al., 1992
;
Artemyev et al., 1993
); however, the coordinates from the
x-ray crystallographic structure of
T indicate that this
region of the
T subunit is only 20-25 Å away from
lysine 267 at the guanine nucleotide-binding site. Thus, one possible
explanation for the resonance energy transfer measurements that suggest
a longer distance (than 20-25 Å) between the nucleotide binding site
and the
PDE-binding site on
T is that
T makes at least two contacts with
PDE,
one involving residues 300-310 and another involving a site within the
large helical domain of
T (e.g. residues
106-116). If the contact region in the helical domain of
T bound to
PDE in the vicinity of
cysteine 68, this would be consistent with the measured distance of
~40 Å between cysteine 68 of
PDE and lysine 267 of
T. In this view, residues 300-310 of
T
would bind in the vicinity of lysine residues 41, 44, and 45 of
PDE, consistent with the results obtained from chemical
cross-linking studies (Artemyev et al., 1993
). Our present results would suggest that the distance between residues 41-45 and
cysteine 68 (i.e. the two proposed
T-binding
sites on the
PDE molecule) does not change when
PDE binds to an activated
T subunit,
either when
PDE is free in solution or bound to the
PDE and
PDE subunits. We would expect
that even subtle
T-induced conformational changes
occurring upstream from cysteine 68 would have been detected by a
change in the effective distance between cysteine 68 and the
amino-terminal end of the
PDE molecule. However, we
(Erickson et al., 1995
) and others (Faurobert et
al., 1993
) have found that
T binding perturbs the
microenvironment surrounding cysteine 68. Thus, in light of our present
findings, we hypothesize that these
T-induced changes
are communicated to the carboxyl-terminal domain of the
PDE molecule, resulting in the activation of cyclic GMP
hydrolysis by the
PDE and
PDE
subunits.
An important point raised by the energy transfer experiments is that
the T-mediated stimulation of PDE activity occurs while the
T·
PDE complex is still associated
with the core of the effector enzyme (i.e. the
PDE and
PDE subunits). The latter
suggestion has been a subject of controversy over the years, since
various lines of data have argued that
T-mediated
stimulation is accompanied by the dissociation of an
T·
PDE complex from the core of the enzyme (Wensel and Stryer, 1990
), while other lines of evidence have
suggested that
PDE dissociation is not necessary for the stimulation of PDE activity (Erickson and Cerione, 1993
; Catty et
al., 1992
; Clerc and Bennett, 1993). However, the fact that the
changes in the
PDE subunit that are specifically induced by the
PDE and
PDE subunits
(i.e. the increase in the distance between the amino
terminus and cysteine 68 of
PDE) are not reversed upon
the binding of an activated
T subunit (under
conditions where
T causes a stimulation of PDE
activity) argues that the
PDE·
PDE·
PDE complex
remains intact during the
T·GTP
S-mediated stimulation of cyclic GMP hydrolysis (see Fig. 5). The activated
T subunit does not induce similar changes within the
PDE subunit (as those induced by the
PDE
and
PDE subunits) when
PDE is free in
solution (i.e. dissociated from the
PDE and
PDE subunits), and
T appears to bind to
PDE independently of the
PDE and
PDE subunits. Specifically, the KD
values that we have measured for the interaction of
T·GTP
S with
PDE are similar for
PDE that is free in solution (~30 nM;
Erickson et al. (1995)
) or bound to
PDE and
PDE (21 nM; Erickson and Cerione (1989)).
Thus, based on a consideration of microscopic reversibility, these data
argue against the possibility that the activated
T
subunit binds to
PDE and stimulates its dissociation
from the
PDE and
PDE subunits (during PDE
activation) but still maintains the free
PDE subunit in
a specific conformation that was originally induced by the larger PDE
subunits.
Future work will be directed toward further examining both of the
suggestions raised by the present study. In particular, we will set out
to directly demonstrate that T binding to
PDE results in specific conformational changes within
the carboxyl-terminal domain of this subunit that are directly
translated into the stimulation of PDE activity. We also will try to
determine the types of changes that must occur in the orientation of
the
PDE subunits relative to the larger subunits of the
effector enzyme. This remains an important issue, because although our
data argue that the
PDE subunits remain associated with
PDE and
PDE during
T-mediated stimulation of enzyme activity, it has been
well established that the addition of excess free
PDE
molecules will inhibit the
T-stimulated PDE activity
(Wensel and Stryer, 1986). Taken together, these findings imply that a
positional change in the binding interaction of the
PDE
subunits with the
PDE and
PDE subunits
occurs during PDE enzyme activation following the binding of the
activated
T subunit.