(Received for publication, March 25, 1997, and in revised form, May 5, 1997)
From the Division of Biology, California Institute of Technology, Pasadena, California 91125
Several GTP binding proteins, including EF-Tu,
Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been
reported to bind xanthine nucleotides when the conserved aspartate
residue in the NKXD motif was changed to asparagine.
However, the corresponding single Go mutant protein (D273N) did not
bind either xanthine nucleotides or guanine nucleotides. Interestingly,
the introduction of a second mutation to generate the Go
subunit
D273N/Q205L switched nucleotide binding specificity to xanthine
nucleotide. The double mutant protein Go
D273N/Q205L (Go
X) bound
xanthine triphosphate, but not guanine triphosphate. Recombinant Go
X
(Go
D273N/Q205L) formed heterotrimers with
complexes only in
the presence of xanthine diphosphate (XDP),
and the binding to
was inhibited by xanthine triphosphate (XTP).
Furthermore, as a result of binding to XTP, the Go
X protein
underwent a conformational change similar to that of the activated
wild-type Go
. In transfected COS-7 cells, we demonstrate that the
interaction between Go
X and
occurred only when cell membranes
were permeabilized to allow the uptake of xanthine diphosphate. This is
the first example of a switch in nucleotide binding specificity from
guanine to xanthine nucleotides in a heterotrimeric G protein
subunit.
G proteins transduce receptor-generated signals across the plasma
membranes of eukaryotic cells. They are heterotrimeric complexes composed of ,
, and
subunits. Each of the subunits belongs to
a multigene protein family, containing at least 18 distinct
, 5
,
and 11
subunits. Hundreds of seven-transmembrane receptors activated by a great variety of hormones, neuromediators, and growth
factors are coupled to G proteins. Receptor-induced activation of a G
protein leads to exchange of GDP for GTP bound to the
subunit. The
GTP-bound
subunit is released from the
trimeric complex,
and both free
and
dimers are capable of modulating activities of effector enzymes and ion channels (1-3). G
protein-mediated signaling is complicated; a single receptor can
activate more than one kind of heterotrimer, and both the activated
and the
subunits can interact with multiple effectors. For
example, the thrombin receptor is known to couple to G12,
Gi, and Gq family members (4), and physiological responses may be the
result of contributions by both
and
subunits. Furthermore,
cross-talk between these different G protein-regulated pathways makes
the networks even more complex.
One way to analyze this complex network is to specifically activate a
particular G in vivo to discern its function without interference from other G proteins. As a first step toward this goal,
we used site-specific mutagenesis to switch the nucleotide specificity
of G
from guanine to xanthine nucleotides. In cells, xanothine
monophosphate is an intermediate in the biosynthesis of GMP; however,
the steady-state concentrations of XDP1 and XTP are relatively low (5).
Thus, by subsequent introduction of XTP, we should be able to
specifically activate the mutant protein. The
subunits of
heterotrimeric G proteins belong to the GTPase superfamily that also
includes factors involved in ribosomal protein synthesis, such as
EF-Tu, and a large number of Ras-like small guanine nucleotide binding
proteins (6, 7). Crystal structures of the
subunits of transducin
and Gi have been recently solved (8-11). Both G
structures had
nearly identical binding pockets for the guanine nucleotide, which was
similar to the guanine nucleotide binding pocket revealed in the
crystal structures of Ras (12) and EF-Tu (13, 14). One of the conserved features was the interaction between a specific G
amino acid residue
and the guanine nucleotide ring, i.e. a hydrogen bond from
the side chain of a conserved aspartic acid (Asp-268 in transducin) to
the N-1 nitrogen and the N2 amine of the guanine ring (see Fig.
1a). Asp-268 of transducin belongs to a conserved motif
(NKXD) found in the GTPase superfamily. It has been shown
that the characteristic hydrogen bond formed with the aspartic acid
residue determines the specificity of guanine nucleotide binding in
other GTP-binding proteins, such as EF-Tu and Ras (15, 16). A mutation
of aspartate to asparagine at this position in several GTP binding
proteins, including EF-Tu (17, 18), Ypt1 (19), rab-5 (20, 21), and FtsY
(22) and adenylosuccinate synthetase (23), leads to active proteins
regulated by xanthine nucleotides instead of guanine nucleotides. In
this report, we studied the effect of the similar D273N mutation on
nucleotide binding specificity of Go
.
Myristoylated
recombinant mouse GoA was expressed in Escherichia coli.
Conditions for growth, induction, and lysis of the Go
-expressing
cells were described previously (24). The D273N mutation was introduced
in both wild-type Go
and the activated mutant Go
Q205L by
oligonucleotide-directed mutagenesis. The oligonucleotide TTTCTAAACAAGAAAAATTTATTTGGCGAGAAGATTAAGAAGTC was annealed to
uracil-containing single-stranded DNA from the plasmids pGo
and
pGo
Q205L. The resulting vectors were designated as pGo
D273N and
pGo
X.
We subcloned wild type and mutant Go cDNAs into the
E. coli expression vector pET-15b (Novagen), which added a
peptide of 20 amino acids MGSS(H6)SSGLVPRGSH containing
the His6 tag and a thrombin site upstream of the amino
terminus of Go
. These clones were used to transform the E. coli strain BL21(DE3), and proteins were expressed. After
harvesting the culture, cell extracts were resuspended in the binding
buffer (5 mM imidazole, 0.5 M NaCl, 160 mM Tris-HCl, pH 7.9, 1 mM
Me). Binding to
the Ni2+-NTA resin was according to the protocol provided
by Novagen. The His6-tagged protein was eluted with a
gradient of imidazole concentration (5-500 mM). The Go
and various mutant proteins eluted at about 250 mM
imidazole. Proteins were then transferred to TED buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT) with 0.1 mM MgCl2 and 0.1 mM nucleotide diphosphate (GDP or XDP as appropriate) by
gel filtration. Purified proteins were stored in 50% glycerol at
70 °C.
XTPS was synthesized from XDP and
ATP
S with nucleotide diphosphate kinase (NDK) as described
previously (25). To produce 35S-labeled XTP
S, the
reaction contained 10 µM XDP, 1 µM
[35S]ATP
S, and 10 units NDK (Sigma) in 100 µl of NDK
buffer (1 mM MgCl2, 5 mM DTT, 20 mM Tris-HCl, pH 8.0). The mixture was incubated at room
temperature for 2 h. The resulting concentration of
[35S]XTP
S was about 1 µM (1 µCi/pmol).
The radiochemical purity of XTP
S was monitored by thin layer
chromatography on Avicel/DEAE plates (Analtech) in 0.07 N
HCl.
Binding of [35S]GTPS
and [35S]XTP
S to the recombinant Go
and the mutant
proteins was performed as described (24). The binding reaction
contained 0.5 µg of purified protein or 200 µg of crude E. coli protein in TED buffer with 0.1 mM
MgCl2, 1 µM ATP, and 0.1 µM
GTP
S or XTP
S (20,000 cpm/pmol). For the time course experiments, 20-µl aliquots were withdrawn from a 200-µl reaction, diluted 10-fold with ice-cold TED buffer containing 0.1 mM
MgCl2, filtered through a 0.45-µm nitrocellulose filter,
washed, and dried. The amount of bound radioactivity was determined by
scintillation counting.
Approximately 0.1 µg of purified
recombinant Go was preincubated with nucleotide at room temperature
for 30 min in the TED buffer. 10 ng of trypsin was then added to the
mixture, and the reaction was terminated after 10 min by addition of an
equal volume of 2 × SDS-PAGE sample buffer and heating for 3 min
at 100 °C. The proteolytic pattern was subsequently analyzed by
Western blot using antibodies against Go
.
Pertussis
toxin-catalyzed ADP-ribosylation was performed as described (24).
Briefly, 0.1 µg of recombinant Go was mixed with 0.1 µg of
purified retinal
subunit complex in the presence of the
appropriate nucleotide and incubated for 10 min at room temperature
before addition of the reaction mixture (final concentration of 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.5 µM [32P]NAD (20,000 cpm/pmol), and 10 µg/ml pertussis toxin (List Biologicals)). Reactions were incubated
for 30 min at room temperature and terminated by the addition of 5 × SDS-PAGE sample buffer. Samples were resolved on SDS-PAGE. Gels were
stained with Coomassie Blue, dried, and exposed to x-ray film.
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
1 × 105 cells/well were seeded in 12-well plates 1 day before transfection. All transfection assays contained a total
amount of 1 µg of DNA; the plasmid pCIS encoding -galactosidase
was used to maintain a constant amount of DNA. To each well, 1 µg of DNA was mixed with 5 µl of lipofectamine (Life Technologies,
Inc.) in 0.5 ml of Opti-MEM (Life Technologies, Inc.), and five h
later, 0.5 ml of 20% fetal calf serum in Dulbecco's modified Eagle's
medium was added to the cells. After 48 h, cells were assayed for
inositol phosphate levels as described previously (26, 27).
Transfected COS-7
cells were washed twice with phosphate-buffered saline and incubated in
200 µl of permeabilization solution consisting of 115 mM
KCl, 15 mM NaCl, 0.5 mM MgCl2, 20 mM Hepes-NaOH, pH 7, 1 mM EGTA, 100 µM ATP, 0.37 mM CaCl2 (to give a
free Ca2+ concentration of 100 nM), and 200 units/ml -toxin with or without 0.1 mM XDP for 10 min at
37 °C. Then 2 µl of 1 M LiCl was added before the
inositol phosphate assay.
To change the binding specificity of Go from guanine
nucleotides to xanthine nucleotides, we replaced Asp-273 by an
asparagine residue, which was expected on the basis of structural
analysis to coordinate with xanthine instead of guanine (Fig.
1b). This mutation was introduced into both
the wild-type Go
subunit and the GTPase-deficient Go
mutant
(Q205L). We chose Go
because myristoylated Go
can be expressed in
E. coli, and it has been shown that many of the
characteristics of the recombinant Go
protein are similar to those
of the protein isolated from brain. To further characterize the
function of XTP-bound Go
mutants, we purified these proteins in the
form of non-myristoylated His6-tagged Go
by affinity
chromatography on a Ni2+-NTA column. It has been shown that
the non-myristoylated form of Go
has identical nucleotide binding
properties compared with the myristoylated form, and it also forms
trimeric complexes with
subunits although the affinity to
is much less than the myristoylated form (44).
The nucleotide binding of
Go, Go
D273N, and Go
X (Go
D273N/Q205L) was assayed with
[35S]GTP
S and [35S]XTP
S. In E. coli crude extracts, Go
reached maximum binding of GTP
S in
about 30 min (Fig. 2a). As expected, Go
showed no affinity for XTP
S. However, Go
X revealed a switch in
nucleotide specificity. As shown in Fig. 2b, Go
X had high
affinity for XTP
S but not for GTP
S. Interestingly, only the
double mutant was active while Go
D273N did not bind either GTP
S
or XTP
S (data not shown). Go
binds GTP
S very tightly in the
presence of 1 µM Mg2+ (28, 29). Both Go
(Fig. 2c) and Go
X (Fig. 2d) did not exchange bound [35S]NTP
S when excess non-radioactive
nucleotides were subsequently added.
The purified His6-tagged proteins in general retained the
properties of the untagged myristoylated subunits. However, we detected some differences in nucleotide binding.
His6-tagged Go
or Go
X bound GTP
S or XTP
S,
respectively, but the binding was less stable than with the untagged
myristoylated protein. In the case of His6-tagged Go
,
the bound GTP
S could be exchanged after excess non-radioactive
GTP
S was added (Fig. 2c). Similar behavior was observed
in the XTP
S binding of pure His6-tagged Go
X, which also showed distinct nucleotide exchange after non-radioactive XDP or
XTP were added to the binding reaction (Fig. 2d). The
decrease in nucleotide affinity was apparently the result of the
presence of the His6-tag. Although the nucleotide binding
of His6-tagged proteins was less stable, the specificity of
binding was clearly maintained, and the mutant bound the xanthine
nucleotides rather than the guanine nucleotides. As expected, the
purified single mutant Go
D273N did not show any nucleotide binding
activity (data not shown).
Guanine nucleotides protect G protein subunits,
including Go
, from complete proteolytic degradation (30-32). The
pattern of fragments derived from partial tryptic digestion can be used as an indicator of the conformation of the protein. In the presence of
GDP, Go
is hydrolyzed by trypsin resulting in two products, a stable
25-kDa and an unstable 17-kDa peptide. Binding of non-hydrolyzable analogs of GTP can induce an active conformation of the Go
subunit, which is resistant to proteolytic degradation, and protects a stable
37-kDa polypeptide from further degradation. In the case of the
activated mutant Go
Q205L, GTP can also protect the remaining 37-kDa
polypeptide from complete proteolytic digestion by trypsin because
Go
Q205L lacks GTPase activity. Fig. 3a
shows that XTP protects Go
X from proteolysis by trypsin (lanes
4 and 5), whereas in the control experiment, GTP
S
protected wild-type Go
(lane 8). This experiment
indicates that Go
X binds XTP without hydrolyzing it. After binding
to XTP, Go
X must have assumed a conformation similar to that of
GTP
S-bound wild-type Go
. In this experiment, wild-type Go
needed only 1 µM GTP
S to prevent complete proteolysis. Similarly, Go
X was sufficiently protected in the presence of 1 µM XTP. It is noteworthy that GTP
S, but not GTP, was
also able to protect Go
X from complete tryptic digestion although
this protection required GTP
S concentrations above 100 µM (lanes 1, 2, and 3). Thus,
Go
X has a much lower affinity for GTP
S than for XTP. We did not
detect any of GTP
S binding activity of Go
X in our nucleotide
binding assay because the highest concentrations of
[35S]GTP
S used in the reaction were micromolar.
Consistent with the results of the nucleotide binding experiments, the
single mutant Go
D273N was not protected by any nucleotides including GTP, GTP
S, and XTP up to millimolar concentrations (data not shown).
Pertussis Toxin-induced ADP-ribosylation
The interaction of
Go with the
complex can be assayed by ADP-ribosylation of the
subunit induced by pertussis toxin (PTX) because ADP-ribosylation
requires the formation of the heterotrimeric complex (33, 34).
Modification (by ADP-ribosylation) of recombinant Go
catalyzed by
PTX is the same in the presence of GTP or GDP because of the GTPase
activity of Go
. However, GTP
S strongly inhibits the modification
since Go
cannot hydrolyze GTP
S. GTP
S binding thus promotes the
dissociation of the trimeric
complex and prevents the
ADP-ribosylation of the Go
subunit. The activated Go
Q205L mutant
lacks GTPase activity, and the effect of GTP on ADP-ribosylation is
similar to that of GTP
S on the wild-type Go
. Therefore, PTX
labeling can be used not only to examine
binding but also GTPase
activity. Fig. 3b shows that purified Go
was
ADP-ribosylated by pertussis toxin (lane 7), and the
labeling was strongly inhibited by GTP
S (lane 6). In
contrast, Go
X was modified by pertussis toxin only in the presence
of XDP (lane 4) but not with GDP (lane 5), and as
expected, the reaction was strongly inhibited by XTP (lane
2), whereas GTP had no effect (lane 3). Therefore, only
XDP-bound Go
X can form trimeric complexes with
, and binding
of XTP induces dissociation of the trimeric complex. As a control, we
did not detect any ADP-ribosylation of Go
X when GTP
S, GTP, or XTP
alone was present (data not shown). Consistent with the results of
trypsin digestion, this experiment indicated that XTP was not
hydrolyzed by Go
X. The quantitation of [32P]ADP-ribose
incorporation revealed that the labeling of Go
X was proportional to
the amount of
used and reached a maximum at a Go
X:
ratio of 1:1, similar to wild-type Go
(data not shown).
Interestingly, high concentrations (over 100 µM) of
GTP
S also inhibited the ADP-ribosylation of Go
X (Fig.
3b, lane 1), offering further evidence that
Go
X was able to bind GTP
S with low affinity. As expected,
Go
D273N did not interact with
and was not modified by
pertussis toxin in the presence of either GDP or XDP (data not
shown).
In transfected COS-7 cells,
1
2 is able to activate
PLC
2, and the activation of PLC
2 can be
inhibited by cotransfection with Go
because of competition for
(35). We cotransfected COS-7 cells with PLC
2,
1,
2, and
Go
D273N or Go
X and found that both Go
mutants did not inhibit
PLC
2 activity, whereas wild-type Go
did. This
experiment indicates that both mutants do not bind
in COS-7
cells and is consistent with the in vitro experiments on
PTX-induced ADP-ribosylation. Go
X bound
only in the presence
of XDP, and because XDP concentration is negligible inside the cell,
the interaction did not occur. To deliver XDP into cells, we tried to
permeabilize COS-7 cells by several methods including digitonin
treatment, electroporation, and
-toxin (36). We found that only
-toxin gave us consistent results and had no effect on the PLC
2
activities stimulated by
. After incubating cells with
-toxin
in the presence of XDP, we found that Go
X inhibited PLC
2
activity, whereas Go
D273N was not affected by XDP (Fig.
4). In the control experiments, we found that adding GDP
or GTP to the permeabilization buffer had no effect on the PLC
2
activity of cells transfected with the Go
mutants (data not shown).
This experiment shows that the Go
mutants behave similarly in
vitro and in cultured cells; Go
X binds
only when exogenous XDP is available.
We engineered a mutant of Go that switched nucleotide
binding activity from guanine nucleotides to xanthine nucleotides. The
mutation (D273N) was at a conserved residue of the NKXD
motif that appears in all GTPase superfamily proteins. Crystal
structures of transducin and Gi showed that this aspartic acid residue
participated in hydrogen bonding to the guanine ring (Fig.
1a). The proposed interaction between the mutagenized Asn
and the xanthine ring is shown in Fig. 1b in which the
hydrogen bond is "flipped" when compared with wild-type G
.
Similar single Asp
Asn mutations have been made in other GTP binding
proteins, including EF-Tu (17, 18), Ypt1 (19), rab-5 (20, 21), and FtsY
(22), and E. coli adenylosuccinate synthetase (23),
resulting in active proteins regulated by xanthine nucleotides instead
of guanine nucleotides. However, the similar D119N mutant of H-Ras
induced transformation of NIH-3T3 cells with efficiency
indistinguishable from wild-type H-Ras (16, 37). Although the mutant
D119N Ras exhibited decreased affinity for GTP and increased affinity
for XTP (by 2 to 3 orders of magnitude), the high intracellular
concentration of GTP (millimolar) probably ensures that the protein is
still bound to the guanine nucleotides in the cell. Interestingly, we
found the corresponding D273N mutation in Go
did not result in
binding of either GTP
S or XTP
S, whereas the D273N/Q205L double
mutant, Go
X, switched nucleotide binding ability. When examining the
crystal structure of transducin, it is not clear why the Gln
Leu
mutation (position 200 in transducin
), which is at the opposite
side of the nucleotide binding pocket from the Asp
Asn mutation
(position 268 in transducin
), rescued the xanthine nucleotide
binding of Go
D273N. It is interesting to note that Go
X binds
GTP
S at concentrations higher than 100 µM. In our
nucleotide binding experiments, we could not observe this binding
because the affinity was weak, requiring concentrations higher than 1 µM [35S]GTP
S, which was the highest
concentration that we could use. The P-S bond of the
phosphate in
GTP
S is longer than the P-O bond in GTP, which not only prevents
nucleotide hydrolysis when binding to G protein
subunits, it also
results in qualitatively different interactions and different
affinities.
In vitro experiments using limited trypsin digestion and
PTX-induced ADP-ribosylation showed that GoX retained the
characteristic properties of wild-type Go
in the presence of XDP or
XTP. In addition, our data confirm the assumption that diphosphate
nucleotides are required for the interaction of G protein
subunits
with
subunits. XTP-bound Go
X assumed a trypsin-resistant
conformation similar to that of the activated wild-type Go
and
stimulated
dissociation from the trimeric complex, suggesting
that Go
X can be activated by XTP. In transfected COS-7 cells,
PLC
2 is activated by G protein
subunits, and the activity is
inhibited when cotransfecting with Go
because of the competition for
. To study
binding of the mutant Go
X in
vivo, we looked for inhibition of PLC
2 activity as an
indication of
binding. We found that Go
X did not affect
-stimulated PLC
2 activity because of the absence of XDP. To
turn on
binding, we used
-toxin to make cell membranes
permeable to XDP, and indeed under these conditions, Go
X attenuated
PLC
2 activity. G protein-derived
subunits are shown to be
able to bind many proteins other than G
, and may be involved in many
signal transduction pathways. We demonstrated that XDP can be delivered
into cells and Go
X may be used as a
quencher that can make
the cellular
pool unavailable to other
effectors. The
ability to turn on and off
in vivo could be useful to
better understand the physiological function of
.
Go is one of the G protein
subunits whose functions are not well
understood although there is some evidence supporting a role in the
regulation of calcium channels (38-42). Since
subunits are also
proposed as regulators of calcium channels (43), it is difficult to
differentiate the activities of Go
and
in some situations
when activated receptors release both Go
and
subunits. This
is one of the problems that the Go
X mutant might be used to address.
The channel may be activated directly by adding XTP without releasing
free
in cells that have been transfected with cDNA
expressing the mutant protein. Cross-talk between the different G
protein-mediated signaling pathways has been well demonstrated.
Activating Go
X directly and instantly by XTP would avoid the
interference of other pathways and help us to differentiate individual
pathways. Introducing this mutation into other G protein
subunits
may be used to study their functions as well.
We thank members of the Simon laboratory for helpful discussions and Drs. Lorna Brundage and Tau-Mu Yi for comments on the manuscript.