(Received for publication, March 18, 1997, and in revised form, July 1, 1997)
From the Department of Pharmacology, Veterinary Medical Center, Cornell University, Ithaca, New York 14853-6401
Comparisons of the tertiary structures of the
GDP-bound and guanosine 5-O-(thiotriphosphate)
(GTP
S)-bound forms of the
subunit of transducin
(
T) indicate that there are three regions that undergo
changes in conformation upon
T activation. Two of these
regions, Switch I and Switch II, were originally identified in Ras,
while Switch III appears to be unique to trimeric GTP-binding proteins
(G proteins). We find that replacement of the Switch III region
(aspartic acid 227 through asparagine 237) with a single alanine
residue yields an
T subunit that fully binds and
hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP
phosphodiesterase (PDE), the physiological target for transducin. We
also show that changing glutamic acid 232 of
T to a
leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange
nor on the GTP hydrolytic activity of
T. However, the
GTP
S-bound form of the
TE232L mutant was unable to
stimulate the activity of the cyclic GMP PDE. The lack of stimulation
was not due to an inability of the
TE232L mutant to bind
to the target. Taken together, these results indicate that glutamic
acid 232 mediates a conformational coupling between Switch II and
Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.
GTP-binding proteins (G proteins) serve as molecular switches in a
wide variety of biological response systems. Two large families, the
Ras-related small G proteins and the trimeric G proteins, have
received a great deal of attention because of their central role in
signal transduction. The trimeric G proteins serve as intermediate
signal transducers for seven-transmembrane-spanning (also called
heptahelical or serpentine) receptors that are involved in responses to
sensory, hormonal and neurotransmitter signals (1, 2). These G proteins
consist of two functional units, the guanine nucleotide-binding subunit (G
) and the
complex (G
). The molecular switch
capability of a trimeric G protein is mediated through the G
subunit, which cycles between the GDP-bound (inactive) and GTP-bound
(active) states. A signal received from a receptor promotes the
activation of a trimeric G protein by catalyzing the exchange of GTP
for GDP on G
. This results in the dissociation of the GTP-bound G
from the G
complex, thereby enabling these subunits to regulate
the activities of downstream target/effectors. GTP hydrolysis on the
G
subunit promotes its re-association with G
, thus terminating
the signal.
The vertebrate phototransduction system represents one of the best
characterized G protein-coupled signaling cascades. In this system, the
photoreceptor rhodopsin activates a trimeric G protein, transducin,
generating a GTP-bound subunit (
T-GTP), which
stimulates the target/effector enzyme, the cyclic GMP phosphodiesterase (PDE).1 The three-dimensional
structures for
T in different guanine nucleotide-bound
states, and more recently for the
T-
T
holotransducin complex, have been solved by x-ray crystallography
(3-6), as have the corresponding structures for the inhibitory
GTP-binding protein of the adenylyl cyclase system, Gi1
(7-9). This structural information now provides the foundation for
understanding the molecular basis of many aspects of G protein-mediated
signaling.
Three distinct regions on trimeric G protein subunits have been
shown to undergo conformational changes in response to GTP/GDP exchange
(4, 5). Two of these regions, designated Switch I (Ser173
to Thr183 in
T) and Switch II
(Phe195 to Thr215), are structurally analogous
to the two conformationally-sensitive regions found in Ras (10) and
EF-Tu (11), whereas the third region, designated Switch III
(Arg227 to Arg238 in
T), is
unique to the
subunits of trimeric G proteins. The various
available x-ray crystallographic structures of G proteins show that the
conformational changes in Switch I and II are the direct result of GTP
binding to residues within these regions. Specifically, the structural
changes in Switch I are induced by the interaction of the
-phosphate
of GTP with Thr177, while the changes in Switch II result
from a hydrogen bond between Gly199 and the
-phosphate
(4). However, Switch III does not directly contact GTP. Rather, it was
shown to respond to Switch II through a series of polar interactions
that were mediated and/or promoted by GTP-induced conformational
changes in Switch II (4). At present, the functional role of the Switch
III domain or the importance of its conformational coupling to Switch
II is not known. The fact that the residues proposed to be responsible
for this conformational coupling are conserved in all trimeric G
protein
subunits (4, 7) supports a critical role for Switch
II-Switch III communication in some event associated with G protein
activation, such as the GTP-mediated dissociation of rhodopsin and/or
T from
T or the GTP-dependent interaction of
T with the
cyclic GMP PDE. In the present work, we have examined the importance of
Switch III in G protein function and find that a conserved glutamic
acid residue within the Switch III domain of
T is
essential for the regulation of target/effector activity.
Purification of Retinal ProteinsRod outer segments
(ROS) were prepared as described previously (12, 13). Transducin and the cyclic GMP PDE were purified by exposing ROS to room light and
repeated washings with 10 mM Hepes, pH 7.5, 6 mM MgCl2, 1 mM dithiothreitol
(DTT), and 100 mM NaCl (isotonic buffer) and then with 10 mM Hepes, pH 7.5, 6 mM MgCl2, and 1 mM DTT (hypotonic buffer). The cyclic GMP PDE is released
into the hypotonic wash and is further purified by hydroxyapatite
chromatography (13). Transducin is released from ROS by washing with
hypotonic buffer in the presence of 0.1 mM GTP or 0.1 mM GTP
S. The
T and
T subunit complexes are then resolved by Blue Sepharose chromatography (14).
Dark-adapted ROS membranes
containing hydroxyapatite-purified cyclic GMP PDE were assayed for
cyclic GMP hydrolysis by measuring proton release, as originally
described by Yee and Liebman (15). The assays are performed at room
temperature in a buffer containing 10 mM Hepes, pH 8.0, 60 mM KCl, 30 mM NaCl, 1 mM DTT, and 5 mM MgCl2, together with the protein components
described in the legend to Fig. 3. The assays were initiated by the
addition of cyclic GMP (5 mM), with the pH being recorded
for 1-2 min at one determination/s. The PDE activity (nanomole/s) was
calculated as the ratio of the slope of the pH change (millivolts) and
the buffering capacity of the medium (millivolt/nmol) (16).
Expression of Recombinant
The
coding region of the bovine T was amplified by the
polymerase chain reaction using primers that create a 5
-end
NdeI site and a 3
-end BamHI site. The polymerase
chain reaction product was digested with NdeI and
BamHI and ligated into pET15b (Novagen). The resultant
vector was digested with NcoI and PstI to release the coding region of
T with its 5
-end fused in-frame to
the hexa-His tag present in the vector pET15b. This released fragment was then blunt-ended using T4 DNA polymerase and ligated into pVL1393.
To generate the
T
I2 deletion mutant
(Asp227 through Asn237 replaced by a single
Ala), and the
TE232L point mutant (Glu232
replaced by Leu), we employed a single-stranded DNA-based mutagenesis strategy (17), using synthetic oligonucleotide primers containing the
indicated deletion or point mutation
(
T
I2:
5
-CAGGCTCTCGTGCATTCGAGCGTAGGCGCTCAG-3
; and
TE232L:
5
-CACTTCGTCCTCGAGCACCAGC-3
). The pVL1393 vector carrying either the
wild type,
T
I2, or
TE232L
gene was introduced into Sf9 insect cells using the Baculogold
transfection kit (PharMingen). The recombinant extracellular virus
(rECV) was purified by a limiting dilution procedure (18). For
production of the recombinant proteins, Sf9 insect cells were infected
at 80% confluence with the purified rECVs at a multiplicity of
infection of 5 and harvested typically 60 h post-infection. The
His-tagged
T proteins were purified through
Ni2+-nitrilotriacetic acid affinity chromatography
following a protocol provided by Qiagen. The purified proteins were
finally dialyzed against HMDN buffer (20 mM Hepes, pH 7.4, 5 mM MgCl2, 150 mM NaCl, and 1 mM DTT) containing 40% of glycerol and stored at
20 °C.
The original finding that a third region on heterotrimeric G
protein subunits undergoes structural changes upon GTP-GDP exchange
(i.e. in addition to the Switch I and Switch II regions originally identified in Ras and EF-Tu (10, 11)) suggests that it may
play a critical role in a GTP-dependent G protein function.
To obtain experimental support for this suggestion, we examined the
properties of an
T deletion mutant in which the entire
Switch III domain (residues Asp227 through
Asn237) was replaced by a single alanine residue. The
deletion mutant, designated
T
I2, was
expressed in Spodoptera frugiperda (Sf9) cells as a
hexahistidine (His)-tagged fusion protein and purified by
Ni2+ affinity chromatography. This results in a rapid and
highly effective purification of the recombinant
T
subunit, as shown in Fig. 1. The first
three lanes in A show the Coomassie Blue-stained profiles for the
T subunit purified from bovine retina, the
recombinant wild type His-tagged
T purified from Sf9
cells (which has a slightly slower mobility on SDS gels because of the
His-tag), and the His-tagged
T
I2 mutant
purified from Sf9 cells. B shows the corresponding Western
blots that were obtained using a specific antibody raised against the
carboxyl-terminal 10 amino acids of
T (16).
We first examined whether the deletion of the Switch III domain from
T affected rhodopsin- and
T-promoted
[35S]GTP
S/GDP exchange. Fig.
2A shows that as has been
documented previously (19, 20), when
T purified from
bovine retina was added to urea-stripped ROS containing light-activated
rhodopsin, there was a marked increase in [35S]GTP
S
binding that was strongly stimulated by the addition of purified
retinal
T. Virtually identical results were obtained with the Sf9-expressed, His-tagged wild type
T and the
His-tagged
T
I2 deletion mutant. Likewise,
the
T
I2 mutant was able to fully
hydrolyze [
-32P]GTP (Fig. 2B). Taken
together, the results presented in Fig. 2, A and
B, indicated that the deletion of the Switch III domain did
not impair the ability of
T to interact with rhodopsin
nor with the
T subunit complex and that removal of
Switch III did not interfere with the GTP-binding/GTP hydrolytic cycle
of the G protein.
We then examined the ability of the T
I2
mutant to functionally couple to the cyclic GMP PDE, by first loading
the
T
I2 mutant with GTP
S (by incubation with ROS
and bovine retinal
T) and then assaying cyclic GMP
PDE activity, by measuring the H+ release that accompanies
cyclic GMP hydrolysis. The results presented in Fig.
3A illustrate that the bovine
retinal
T subunit and the recombinant wild type
T were essentially equivalent in their abilities to
stimulate cyclic GMP hydrolysis. However, the
T
I2 deletion mutant was unable to stimulate PDE activity (relative to the
basal activity measured in the absence of added
T).
Thus, these results suggested that the integrity of the Switch III
domain was essential for
T-mediated regulation of its
target/effector enzyme.
An interesting possibility that was originally proposed following an
examination of the x-ray crystallographic structure of the
T·GTP
S complex (3) was that the acidic amino acid
residues, Asp233, Asp234, and
Glu235 formed a potential binding site for a basic stretch
of amino acids on the
PDE subunit. If this were the
case, it would then explain why the deletion of the Switch III domain
yields an
T subunit that is unable to stimulate effector
activity. However, we have expressed and purified an
T
mutant from Sf9 cells in which the three acidic amino acids were
replaced by alanine residues and found that this triple mutant was
fully active, not only in its ability to bind and hydrolyze GTP, but
also in its ability to stimulate cyclic GMP PDE activity (data not
shown).
This then led us to examine another possibility, namely that a
conserved glutamic acid residue in the Switch III region,
Glu232, is responsible for mediating the conformational
communication between the Switch II and Switch III domains (4). To test
this, we generated a mutant of T in which a leucine
residue was substituted for Glu232 (
TE232L)
and expressed it in S. frugiperda (Sf9) insect cells as a
hexahistidine (His)-tagged protein (see lane 4 in Fig. 1, A and B). Like the
T
I2 deletion
mutant, we found that the
TE232L mutant was able to
functionally couple to rhodopsin and/or
T, as
read-out by its ability to undergo [35S]GTP
S/GDP
exchange and GTP hydrolysis in a rhodopsin- and
T-dependent manner (Fig. 2, A
and B). Moreover, just as was the case for the Switch III
deletion mutant, the
TE232L mutant was unable to
stimulate target/effector (PDE) activity (Fig. 3A), even
when using amounts of the mutant that were in 10-fold excess relative
to the retinal or recombinant wild type
T proteins (data
not shown). Thus, the mutation of the single conserved
Glu232 residue appeared to fully mimick the effects
obtained upon the removal of the entire Switch III domain.
We used the TE232L mutant to further examine the
importance of Switch II domain-Switch III domain coupling in the
stimulation of target/effector activity. We found that the inability of
the
TE232L mutant to stimulate PDE activity cannot be
attributed to its inability to bind to its PDE target. This was
determined through competition experiments. Fig. 3B shows
that like the GDP-bound wild type
T subunit (open
bar in column 2), the GDP-bound form of the
TE232L mutant (open bars in columns
3 and 4) did not competitively inhibit the PDE
stimulatory activity of the GTP
S-bound retinal
T
subunit (shown as the solid bar in column 1).
This was as expected, since the GDP-bound form of
T has
only a weak affinity for the
PDE subunit. However, the
GTP
S-bound form of
TE232L showed a
dose-dependent inhibition (hatched bars in
columns 3 and 4 in Fig. 3B), thus
indicating that the
TE232L mutant can bind to
PDE in a GTP
S-dependent manner. The fact
that the GTP
S-bound wild type
T subunit did not
competitively inhibit the stimulatory activity of the retinal
GTP
S-bound
T (column 2, hatched bar, in
Fig. 3B) illustrates that the activated wild type
T subunit can fully substitute for the activated retinal
T. Moreover, these results demonstrate that the
inhibitory effects are specific for the GTP
S-bound
TE232L mutant, such that the
TE232L
molecule can act as a dominant-negative mutant.
Thus, GTPS can both bind and induce the appropriate conformational
changes within the
TE232L mutant that enable it to
specifically interact with the target/effector molecule. This is
further indicated by the results of limited trypsin treatment (Fig.
4). It has been well documented that
trypsin treatment of the retinal
T subunit gives rise to
defined proteolytic patterns that are absolutely dependent on the
guanine nucleotide-bound state of
T (21, 22). Trypsin
treatment of the GDP-bound wild type
T yields two stable fragments, an ~23-kDa fragment (shown in lane 2 under
Twt in Fig. 4) and an ~9-kDa fragment (not shown),
whereas trypsin treatment of the GTP
S-bound wild type
T yields a stable 32-kDa (precursor) fragment
(lane 3 under
Twt in Fig. 4). Based on
the information provided from the tertiary structures for the different nucleotide forms of
T (4, 5), it is now clear that the protection afforded by GTP
S directly reflects a
GTP
S-dependent conformational change that occurs within
the Switch II domain and effectively moves the trypsin-sensitive
Arg204 residue from a solvent-exposed environment to a less
accessible position (by virtue of its interaction with
Glu241). Thus, the protection against trypsin proteolysis
afforded by GTP
S serves as a highly sensitive read-out for
GTP
S-induced conformational changes within the Switch II domain and
has frequently been used as a monitor for
T activation
(23). The results presented in Fig. 4 (lanes 2 and
3 under
TE232L) show that GTP
S binding to the
TE232L mutant provides a similar
protection against trypsin proteolysis, as observed with the wild type
T subunit. Therefore, the mutation of Glu232
neither perturbs GTP
S binding nor the GTP
S-induced conformational alteration of Switch II. However, mutation of Glu232, while
preserving the GTP-dependent binding of the
T subunit to the cyclic GMP PDE, completely uncouples
this binding from target/effector stimulation.
The location of Glu232 in the loop connecting the
4 strand and
3 helix of
T
places it in a prime position to couple conformational transitions
between Switch II and Switch III. In particular, x-ray crystallographic
analysis shows that upon GTP
S binding, Glu232 is engaged
in direct interactions with Arg201 and Arg204
of Switch II and in a water-mediated interaction with
Gly199 of Switch II (5). Given our findings, we conclude
that the conformational coupling between Switch II and Switch III is
responsible for converting a primary binding interaction between
activated
T and its target (
PDE), perhaps
involving residues in Switch II (24) or in other regions of
T (25-27), into a secondary stimulatory interaction
between the
PDE subunit and the
4-
6 residues 305-314 of
T
(28). Moreover, these results indicate that Glu232 plays an
essential role in mediating this conformational coupling, thereby
translating
T-target (PDE) interactions into a specific regulatory event. The fact that this glutamic acid residue is conserved
in all trimeric G
subunits further suggests that it plays a
fundamental role in converting target binding into target/effector regulation in a wide variety of G protein-coupled signaling
pathways.
We acknowledge Al Berger, Jon Erickson, and Rohit Mittal for helpful discussions and other assistance during the course of these studies. We also thank Cindy Westmiller for her expert technical assistance.