(Received for publication, October 23, 1995; and in revised form, December 4, 1995)
From the
N-methyl-3`-O-anthranoyl (MANT) guanine
nucleotide analogs are useful environmentally sensitive fluorescent
probes for studying G protein mechanisms. Previously, we showed that
MANT fluorescence intensity when bound to G protein was related to the
degree of G protein activation where
MANT-guanosine-5`-O-(3-thiotriphosphate) (mGTPS) had the
highest fluorescence followed by mGTP and mGDP, respectively (Remmers,
A. E., Posner, R., and Neubig, R. R.(1994) J. Biol. Chem. 269,
13771-13778). To directly examine G protein conformations with
nucleotide triphosphates bound, we synthesized several nonhydrolyzable
MANT-labeled guanine nucleotides. The relative maximal fluorescence
levels observed upon binding to recombinant myristoylated G
(myrG
) and myrG
were:
mGTP
S > MANT-5`-guanylyl-imidodiphosphate >
MANT-guanylyl-(
,
-methylene)-diphosphonate > MANT-guanosine
5`-O-2-(thio)diphosphate. Using protection against tryptic
digestion as a measure of the activated conformation, the ability of
the MANT guanine nucleotides to maximally activate
myrG
correlated with maximal fluorescence. Biphasic
dissociation kinetics were observed for all of the MANT guanine
nucleotides. The data were consistent with the following model,
where G protein
activation (G*-GXP) is determined by a conformational equilibrium
between two triphosphate bound states as well as by the balance between
binding and hydrolysis of the nucleotide triphosphate. Compared with
myrG, maximal mGTP fluorescence was only 2-fold
higher for the myrG
Q204L mutant, a mutant with
greatly reduced GTPase activity, and only 24% that of mGTP
S,
indicating that partial activation by mGTP was not just due to
hydrolysis of mGTP. These results extend our previous conclusion that
GTP analogs do not fully activate G protein.
Receptor-mediated activation of heterotrimeric GTP binding
proteins is a common mechanism for signal transduction in biological
systems. Receptors on the cell surface regulate G protein function by
catalyzing the release of GDP from the subunit of the G protein,
allowing GTP to bind and activate the G protein. G protein activation
is thought to be concomitant with
subunit dissociation from
subunits. Activated G protein
and
subunits
interact with effector proteins to modulate intracellular second
messenger metabolism (for review, see (1, 2, 3) ). G protein deactivation is
mediated by
subunit GTPase activity. The deactivation rate, which
may be limited by the rate of phosphate release from
-GDP-P
(4) is catalyzed by membrane-bound GTPase accelerating
proteins(5, 6) . Upon deactivation,
and
subunits reassociate. Perhaps the best understood G protein
mechanism is the rhodopsin-mediated activation of transducin in the
retinal rod outer segment. The rhodopsin conformations can be easily
studied because of the retinal chromophore attached to the protein. In
addition, changes in G protein intrinsic fluorescence (due largely to
changes in tryptophan 207(7) ) can be used to monitor
transducin activation and deactivation (8, 9) and have
been used to model the kinetic mechanism of the rhodopsin-stimulated
activation/deactivation cycle of transducin(10) .
Kinetic
mechanisms of the predominant G proteins purified from brain (G and G
) have also been studied extensively in solution
or when reconstituted into phospholipid vesicles (for review, see (11) ). G
is a G protein abundant in brain that
contains two tryptophans in the
subunit. Upon activation by GTP
and magnesium, an increase in the intrinsic fluorescence of G protein
tryptophan is observed(12) . The effects of
subunits, ions, and activating ligands on G
tryptophan
fluorescence have been well characterized in detergent
solutions(13, 14) . Recent structural data of
transducin bound to GDP and GTP
S (
)indicate differences
in structure in the
2 helix containing Trp
. In the
GTP
S-bound state the
2 helix rotates such that the Trp is in
a more hydrophobic environment, which is presumably the cause of the
increase in its quantum yield. However, the receptor-stimulated G
protein activation/deactivation cycle has not been studied, most likely
due to the high background and relatively small signals available from
intrinsic tryptophan fluorescence. One of our goals has been to study G
protein activation and deactivation kinetics in real time. To that end,
we have synthesized fluorescently labeled G protein
and
subunits (15) and studied the kinetics of their
association(16) . In addition, we have found that N-methyl-3`-O-anthranoyl (MANT) guanine nucleotide
analogs are useful environmentally sensitive fluorescent probes to
study heterotrimeric G proteins(17) . MANT-GTP
S
(mGTP
S) displays a marked increase in fluorescence upon binding to
G
. The fluorescence increase is greater when the MANT
moiety is excited via energy transfer from tryptophan (excitation, 280
nm). The fluorescence increase observed when mGTP is bound is lower.
Previous modeling of the mGTP fluorescence kinetics indicated that the
low peak fluorescence with mGTP was not just due to slow binding and
rapid hydrolysis. We concluded that mGTP does not fully activate G
protein even when bound as triphosphate. It is possible that the
natural nucleotide, GTP, also does not fully activate the G protein and
is a partial agonist regarding G protein activation. However, the
intrinsic GTPase activity of G protein makes it difficult to discern
whether a conformational change resulting in partial activation limits
GTP effects in addition to the well known role of GTP hydrolysis. For
example, GTP
S and Gpp(NH)p inhibited cyc
S49 cell adenylyl cyclase activity more than GTP(18) .
However, GTP
S and GTP activated cGMP phosphodiesterase to the same
extent, suggesting that GTP can fully activate transducin in rod outer
segment membranes (19) .
To test the hypothesis that partial
activation of G by MANT-GTP was not just due to the balance
between binding and hydrolysis and to explore the G protein
activation/deactivation reactions, we synthesized several
nonhydrolyzable MANT-labeled guanine nucleotides. The MANT-labeled
nonhydrolyzable analogs displayed differing levels of maximal
fluorescence, which correlated with their ability to activate G
protein. In addition, the MANT guanine nucleotides with slower
dissociation rates were able to more fully activate G protein, which is
consistent with predictions of a two-step activation model. Partial
activators showed biphasic dissociation kinetics, indicating that we
are able to observe MANT-nucleotide fluorescence signals from both the
inactive and active G protein states. We also compared mGTP
fluorescence when bound to a hydrolysis-deficient G protein,
G
Q204L, to that of wild type protein. While mGTP
fluorescence was greater when bound to the Q204L mutant than when bound
to wild type protein, it was still only 24% of the fluorescence of
mGTP
S. Taken together, these results further support the use of
MANT guanine nucleotide fluorescence as a measure of G protein
activation and a probe of G protein conformational states. This also
represents the first demonstration that nonhydrolyzable triphosphate
nucleotides can produce partial activation of heterotrimeric G
proteins. Regulation of this activating conformational change could be
a mechanism of receptor or GTPase accelerating protein effects on G
proteins.
Recombinant myristoylated G proteins were further purified by chromatography on Source 15Q (7.8 ml, Pharmacia), hydroxyapatite (Bio-Rad), and Mono Q HR5/5 (Pharmacia) columns using a BioCAD SPRINT System (PerSeptive Biosystems, Inc., Framingham, MA). The Source 15Q column was equilibrated with 50 mM Tris/bis-Tris propane, 1 mM dithiothreitol, pH 8.0, at 10 ml/min. Before loading onto the column the concentrated sample was filtered through a 0.45-µm millipore filter. Following sample loading, the column was washed with 2 column volumes of equilibration buffer followed by a 0-225 mM NaCl gradient over 20 column volumes. Hydroxyapatite column chromatography was performed as described previously(21) . An additional final chromatography on the Mono Q HR5/5 column removed significant amounts of inactive G protein from the preparation.
Figure 1:
Equilibrium fluorescence of MANT
guanine nucleotides bound to r-myrG and
r-myrG
. A, recombinant myristoylated
G
(100 nM) was incubated with 0.03-7
µM MANT guanine nucleotides for 20 min at 20 °C in
HEDNML buffer. Fluorescence was measured at 440 nm for mGTP
S
(
), mGpp(NH)p (
), mGpp(CH
)p (
), and
mGDP
S (
) as described under ``Experimental
Procedures.'' The fluorescence of MANT guanine nucleotide in
buffer was subtracted to determine specific fluorescence (counts per
second
10
(cps
10
)). B, recombinant
myristoylated G
(50 nM) or G
(100 nM) was incubated with MANT guanine nucleotides for
20 min at 30 °C for G
and 20 °C for
G
in HEDNML buffer, and fluorescence levels were
measured as described above. Maximal fluorescence and apparent K
values were determined by fitting the
data to a one site binding hyperbola as described under
``Experimental Procedures.'' Maximal fluorescence values and
S.E. (relative to mGTP
S) are shown here for recombinant
myristoylated G
(left) and G
(right). Maximal fluorescence and K
values are listed in Table 1.
Figure 2:
[H]GDP equilibrium
binding in the presence of MANT guanine nucleotides. Recombinant
myrG
was preincubated at 20 °C with MANT guanine
nucleotides followed by incubation with [
H]GDP as
described under ``Experimental Procedures.'' The K
values for mGTP
S (
),
mGpp(NH)p (
), mGpp(CH
)p (
), and mGDP
S
(
) are (mean ± S.E., n = 4) 3 ± 1
nM, 160 ± 90 nM, 1.6 ± 0.5
µM, and 250 ± 200 µM,
respectively.
Figure 3:
Activation of r-myrG by
MANT guanine nucleotides. A, one microgram of recombinant
myristoylated G
was preincubated with the indicated
concentrations of mGTP
S, mGpp(NH)p, mGpp(CH
)p, and
mGDP
S followed by incubation with trypsin and 11%
SDS-polyacrylamide electrophoresis as described under
``Experimental Procedures.'' In the lanes marked
with 0 and *, recombinant myristoylated G
was
preincubated with no additional guanine nucleotide or 100 µM GTP
S, respectively. Shown is a representative experiment that
was performed 4 times. B, following trypsin digestion in the
presence of GTP
S or the MANT guanine nucleotide analogs, the
amount of 37-kDa r-myrG
was quantitated for mGTP
S
(
), mGpp(NH)p (
), mGpp(CH
)p (
), and
mGDP
S (
) as described under ``Experimental
Procedures,'' and the results from four experiments were averaged
(±S.E.). Background and maximal protection from trypsin
digestion was defined using no added guanine nucleotide and 100
µM GTP
S, respectively.
Figure 4:
G protein activation by and maximal
fluorescence of MANT guanine nucleotides. Plotted on the abscissa is the maximal MANT guanine nucleotide fluorescence when bound to
recombinant myristoylated G relative to mGTP
S (Table 1). On the ordinate, the ability of 100
µM MANT guanine nucleotides to protect against trypsin
digestion is plotted where protection by 100 µM GTP
S
is 1.00 (Fig. 3B).
We examined the rates of
fluorescence decrease of prebound fluorescent guanine nucleotide,
following the addition of competing ligand, GTPS. The rates of
fluorescence decrease varied greatly among the MANT guanine nucleotide
analogs (Fig. 5A). MANT-GTP
S displayed very slow
kinetics, followed by mGpp(NH)p and mGpp(CH
)p with
intermediate rates and mGDP
S with the fastest kinetics. In
addition, all four nucleotides showed biphasic kinetics with results of
nonlinear least square fits summarized in Table 2. We propose
that the two kinetic components reflect the disappearance of the G-mGXP
and G*-mGXP states. The fraction of fluorescence that decreased rapidly
was dependent on the guanine nucleotide where mGpp(CH
)p
(10.3 ± 0.2%) had a larger fast component than mGpp(NH)p (4.1
± 0.2%) (Fig. 5B and Table 2). The
half-times for the fast and slow processes were 3.6 and 33 s for
mGpp(CH
)p and 16 and 330 s for mGpp(NH)p. The fluorescence
decrease for mGTP
S never reached a plateau, so the rapidly
decreasing fraction was estimated to be very small (
0.4%; see Fig. 5legend for details), and GDP
S had the greatest fast
component (63%). If there was an equilibrium between nucleotide-bound
and activated G protein, guanine nucleotide analogs that more
completely activate the G protein would have a smaller amount of fast
component, which reflects direct dissociation of mGXP from the
nonactivated G-mGXP state.
Figure 5:
Kinetics of MANT guanine nucleotide
fluorescence decrease. A, MANT guanine nucleotides were
prebound to r-myrG, and dissociation was initiated by
the addition of GTP
S as described under ``Experimental
Procedures.'' The data were best fit with a two-phase exponential
decay with floating end point. The fast and slow rate half-times and
the fraction of fluorescence that decreased rapidly are listed in Table 2. B, the same data are replotted showing the
early timepoints for mGpp(NH)p and mGpp(CH
)p. The dashed line represents the slow component of fluorescence
decay to better illustrate the presence and amount of fast
component.
One puzzling aspect of the dissociation
kinetics was why the fast phase of the mGpp(CH)p
fluorescence (which we attribute to dissociation of the nonactivated
G-mGXP) was only 10% when it appeared from the steady state
fluorescence and trypsin protection data that mGpp(CH
)p
left
50% of the G protein inactive. Similarly, the 4% fast
component appeared to underestimate the 30% inactive for mGpp(NH)p.
This paradox is easily understood when we take into account the
relative quantum yields of the two states. If the inactive G-mGXP state
has lower fluorescence than the activated G*-mGXP state, then we would
expect the fast phase of fluorescence decrease to be proportional to
the amount of G-mGXP present at equilibrium but to be quantitatively
less. Based on a comparison of the fraction of MANT guanine nucleotide
fluorescence that decreases rapidly and the fraction inactive measured
by trypsin digest, we estimate that the relative quantum yield of G-GXP
is approximately one-fourth that of G*-GXP. The relative quantum yields
for the G-mGXP state are comparable with the relative maximal
fluorescence observed for mGDP
S bound G
,
indicating that the fluorescence of G-mGXP is similar to that of an
inactive G protein MANT-nucleotide complex. The biphasic kinetics and
the relative amount of fluorescence that decreased rapidly for each
MANT guanine nucleotide suggest that these data can be described by a
two-step G protein activation model ().
Figure 6:
Maximal mGTP fluorescence when bound to
r-myrG and r-myrG
Q204L. Seven
µM mGTP
S or mGTP was added to 100 nM r-myrG
(black line) or
r-myrG
Q204L (gray line), stirring in HEDNML
at 30 °C and monitoring fluorescence as described under
``Experimental Procedures.'' Data were fit to a one-component
exponential rise, and the maximal fluorescence was plotted in the inset. Shown is a representative experiment that was repeated
once with similar results. The relative maximal mGTP fluorescence
compared with that of mGTP
S when bound to r-myrG
and r-myrG
Q204L was 10.2 ± 1.2% and
23.6 ± 0.6%, respectively (mean ± range, n = 2).
In this report we demonstrate that two fluorescent nucleotide
triphosphate analogs, mGpp(NH)p and mGpp(CH)p, only partially activate the G protein as reflected in their maximal
fluorescence when bound to G protein and their ability to induce a
trypsin-resistant state. Rapid measurements of dissociation kinetics
show nucleotide fluorescence decreases from two pools of G protein,
which appear to represent triphosphate-bound but inactive as
well as activated G protein, G-mGXP and G*-mGXP, respectively. Thus,
the MANT guanine nucleotides reveal a novel aspect of G protein
mechanisms and provide a means to dissect the detailed kinetics of the
G protein activation step and study their regulation.
The strongest
support for the G protein activation model lies in the biphasic
dissociation kinetics observed for mGpp(NH)p and mGpp(CH)p,
which we predicted based on their incomplete activation of the
subunits. Although more complicated models can describe G protein
activation, we cannot distinguish among them and thus discuss the data
in the context of a simple two-step equilibrium model. Based on a
two-step reversible reaction where the first (binding) step is fast and
the second (activation) step is slow (), it is possible to
assign molecular rate constants to the observed fast and slow
dissociation. The slow rate of fluorescence decrease is equal to k
* (k
/(k
+ k
)), and the fast rate is equal to k
+ k
(corresponding
to the disappearance of G-mGXP). The MANT derivatives with higher
fluorescence dissociated more slowly, which would be expected because a
small k
would both increase the steady state
activation and decrease the rate of dissociation. The observation that
mGpp(CH
)p had a larger fraction of fluorescence decrease
rapidly compared with mGpp(NH)p is consistent with
mGpp(CH
)p being less effective in forming G*-mGXP than
mGpp(NH)p.
Although only 10% of the mGpp(CH)p
fluorescence decreases rapidly, it appears, based on the steady state
mGpp(CH
)p fluorescence and the trypsin digest experiment,
that approximately 50% of the G protein is activated. Thus, the small
fast signal appears to account for approximately half of the G protein
because G-mGXP most likely has a lower quantum yield than G*-mGXP. This
lower relative quantum yield (20-30% of that of G*-mGXP) is
mostly dependent on the G protein conformational state. Likewise,
although
60% of mGDP
S fluorescence decreases rapidly, most of
the protein is in the bound but not activated state. The ability to
detect conformational transitions with a spectral probe that is more
selective than intrinsic tryptophan fluorescence will enable us to
examine the mechanisms of receptor,
, and effector proteins
in the cycle of G protein activation and deactivation. To evaluate
potential models of G protein activation, ``on kinetics''
with GDP-free G protein will be measured and the molecular rate
constants determined.
MANT moiety renders mGpp(CH)p and
mGpp(NH)p partial agonists-Yamanaka et al.(19) described different affinities of the guanine
nucleotides in interacting with transducin in which GTP
S was most
potent and Gpp(CH
)p had a rather low affinity. In addition
GTP
S is more potent than Gpp(NH)p to compete for the GTP binding
site on G
(28) and to inhibit G
GTPase(29) . Differing affinities of hydrolysis-resistant
GTP analogs were also observed in vivo in cardiac atrial
myocytes. The GTP analogs activated G
(one of the G
subtypes) and opened an inwardly rectifying potassium channel
with the following relative affinities GTP
S > GTP > Gpp(NH)p
> Gpp(CH
)p(30) . However, all produced the same
maximal response. In contrast, Gpp(CH
)p was only able to
partially activate phosphodiesterase in rod outer segment membranes (19) . We observed strikingly different abilities of
MANT-labeled guanine nucleotides to maximally activate r-myr
G
, although affinities are similar to those described
previously for nonfluorescent nucleotides. In the presence of
saturating concentrations of mGpp(NH)p and mGpp(CH
)p, G
protein was only partially activated as indicated by protection against
trypsin digestion. Thus, we conclude that these fluorescent analogs
only partially activate G protein. It is possible that faster
dissociation rate constants for mGpp(NH)p and mGpp(CH
)p
could contribute to the greater trypsin sensitivity compared with
mGTP
S. However, if the nucleotides fully activated G
,
one would expect little nonactivated G
to be present for
tryptic digestion. More direct methods to measure G protein activation
such as use of fluorescent subunit probes (14) or adenylyl
cyclase inhibition by G
(31) will be useful to
definitively test this possibility.
Is partial activation of G
proteins only a property of MANT-nucleotides? Additional data suggest
that GTP may also function as a partial activator. Slepak et al.(32) generated r-myrG Q205L, a GTP
hydrolysis-deficient G protein mutant. This corresponding mutation
reduced the rate of GTP hydrolysis (k
)
approximately 20-fold in Ras (33) and greater than 100-fold in G
(27) . They showed that GTP was
only able to partially protect the G
Q205L
mutant from tryptic digestion as compared to GTP
S. The time course
of the experiment indicated that GTP hydrolysis was not a factor in the
partial protection. However, G
Q204L was protected
from trypsin digest in the presence of GTP to a greater extent than was
G
Q205L (27) .
We pointed out previously (17) that estimates of k from ``single turnover'' experiments (26, 27) may overestimate the true value of the rate
constant of GTP hydrolysis. The actual value determined in such
experiments is k
+ k
.
The fact that the amount of P
released is less than the
amount of GTP
S bound indicates that the dissociation term k
is not negligible as previously assumed. If k
is not as large as previously estimated, then
some other factor (i.e. the activation/deactivation
equilibrium) is required to account for the low degree of steady state
activation of G proteins by GTP.
The proposal that GTP may be a
partial agonist regarding G protein activation suggests a novel role of
agonist-liganded receptor. In addition to stimulating GDP release from
G protein, the receptor may also shift the activation/deactivation
equilibrium toward activated G protein when GTP is bound. These
fluorescent GTP analogs are novel tools to discern guanine nucleotide
binding from activation. In future experiments they will permit us to
provide estimates of the kinetics of these processes and to assess the
role of receptor, , and effector in regulation of the G
protein activation/deactivation conformational equilibrium.