(Received for publication, March 10, 1995; and in revised form, February 1, 1996)
From the
The stimulatory guanine nucleotide binding protein
(G) is heterotrimeric (
), and mediates
activation of adenylyl cyclase by a ligand-receptor complex. The
subunit of G
(G
) has a guanine nucleotide
binding site, and activation occurs when tightly bound GDP is displaced
by GTP. Together, GDP and fluoroaluminate
(AlF
) form a transition state analog of
GTP that activates G
. The work of other investigators
suggests that AlF
causes subunit
dissociation when it activates G
. We have observed that in
solution AlF
did not cause G
subunits to dissociate unless NaCl was also present. The effect
of NaCl was concentration dependent (10-200 mM).
Omitting F
, Al
, or Mg
prevented the NaCl-induced dissociation of G
subunits. Na
SO
could not substitute for
NaCl in causing subunit dissociation, but KCl could, suggesting that
the anion was responsible for the effect. G
subunit
reassociation occurred when the concentration of Cl
was reduced even though the concentrations of
AlF
and Mg
were
maintained. The absence of Cl
did not prevent
AlF
binding to G
. We
have concluded that AlF
, a ligand which
is capable of activating G proteins, can bind to G
in
solution without causing subunit dissociation.
It has been more than 35 years since F was
first identified as being an activator of adenylyl cyclase (Rall and
Sutherland, 1958). In the interim the heterotrimeric (
)
stimulatory G protein (G
) (
)was identified as
mediating activation of the enzyme by hormone-receptor complexes, by
GTP and its analogs, and by F
(Howlett et
al., 1979). In order for G
to activate adenylyl
cyclase, GDP which is tightly bound to the guanine nucleotide binding
site of the
subunit (G
) must be displaced by GTP
or a nonhydrolyzable GTP analog such as GTP
S or Gpp(NH)p. This
process is facilitated by the hormone-receptor complex which explains
its role in the activation of G
. The intrinsic GTPase
activity of G
provided a means for terminating
activation of the adenylyl cyclase (Gilman, 1987; Birnbaumer, 1990;
Simon et al., 1991; Clapham and Neer, 1993). The mechanism by
which F
activated G
remained a mystery
until it was recognized that Al
or Be
was also required for the activation (Sternweis and Gilman,
1982). It is believed that together Al
and
F
form fluoroaluminate
(AlF
), a phosphate analog, that binds to
the guanine nucleotide binding site of G
and together
with bound GDP mimics the effects of GTP (Chabre, 1990). Following the
activation of G
by GTP analogs or
AlF
it is thought that G
dissociates from the G protein
subunit complex
(G
). This dissociation is believed to be a critical part of
the activation process. Contributing to the subunit dissociation
hypothesis are data suggesting that activation of G
by
AlF
is accompanied by G
subunit dissociation (Sternweis et al., 1981; Northup et al., 1983; Kahn and Gilman, 1984). However, we have
recently reported that AlF
does not cause
G
subunits to dissociate (Toyoshige et al., 1994).
In an attempt to reconcile our findings with those of other
investigators we have discovered that Cl
is required
for AlF
-induced G
subunit
dissociation. Here we report the experimental results of our
investigation.
For
immunoprecipitation the samples were diluted to 100 µl and treated
as described previously (Toyoshige et al., 1994). During
immunoprecipitation the salt concentrations were maintained, decreased
or increased as indicated in the figures. Immunoprecipitates were
assayed for G subunits by SDS-polyacrylamide gel
electrophoresis and immunoblotting (Toyoshige et al., 1994).
In addition to the
and
subunits of G
, the
immunoblots show a protein with slower electrophoretic mobility than
G
. This is the heavy chain of the RM/1 antibody. When
percent dissociation of G
is reported, it is based on zero
percent being defined as the amount of G
present when G
was immunoprecipitated following incubation in solution A
containing 2 mM MgSO
and 0.1% Lubrol PX.
For
zonal sedimentation samples of bovine brain G incubated as
described above were diluted to 100 µl so that, with the exception
of G
, the concentration of components in the solution were
unchanged. The samples were then transferred onto the top of sucrose
density gradients for zonal sedimentation experiments. G
from S49 membranes was also used for some zonal sedimentation
experiments. Samples containing 250 µg of membrane protein were
diluted with one volume of solution A, and centrifuged at 43,000
g for 30 min at 4 °C to collect the membranes. The
membrane pellets were suspended in 100 µl of solution A containing
0.1% Lubrol PX and the combination of salts indicated in the figure
legend. The samples were incubated for 30 min at 30 °C, and
centrifuged at 43,000
g for 30 min at 4 °C to
remove insoluble material. The supernatant was then transferred onto
the top of a sucrose density gradient for zonal sedimentation
experiments as follows.
Sample volumes of 30 µl from each fraction were then
used to reconstitute adenylyl cyclase in the membranes of G deficient S49 cyc
cells. Just prior to use the
S49 cyc
membranes were thawed and diluted with a
large volume of solution A containing 2 mM MgSO
.
The membranes were recovered by centrifugation and suspended at a
membrane protein concentration of 2.5 mg/ml in solution A containing 2
mM MgSO
. Ten µl of the suspended S49
cyc
membranes were added to each sample, and after
reconstitution of adenylyl cyclase, effector stimulated enzyme activity
was assayed as described previously (Toyoshige et al., 1994).
Effectors included 10 mM AlF
for
samples originally treated with AlF
, and
30 µM GTP
S plus 20 µM isoproterenol for
samples of G
that were not activated before zonal
sedimentation. No effector was added when samples of G
were
activated with GTP
S before zonal sedimentation.
RM/1 antiserum raised against a synthetic decapeptide
corresponding to the carboxyl-terminal of G has been
used by us (Toyoshige et al., 1994) and others (Simonds et
al., 1989; Morris et al., 1990) to immunoprecipitate
G
. The amount of antiserum used for immunoprecipitation is
of consequence since either too little or too much will decrease the
efficacy of precipitation (Morris et al., 1990). It is
reported that RM/1 will immunoprecipitate 30 to >90% of the G
present in detergent extracts of cell membranes (Simonds et
al., 1989; Morris et al., 1990). Using conditions
described previously (Toyoshige et al., 1994) we found that
RM/1 precipitated 62% of the detected G
from
preparations of bovine brain G
(Fig. 1). In the
absence of RM/1 there was no detectable precipitation of
G
. Sample to sample variation in the amount of
G
precipitated was ± 12% (standard deviation
for n = 10 from a representative experiment), and there
was no significant difference (
)between the amount of
G
precipitated from samples of dissociated and
undissociated G
(see for example Fig. 2through 6).
Similar results have been reported by other investigators (Morris et al., 1990). By using RM/1 to immunoprecipitate
G
, we found that in the absence of NaCl,
AlF
was unable to cause G
subunit dissociation (Fig. 2). NaCl caused a concentration
dependent dissociation of G
subunits in the presence of 2
mM MgSO
and AlF
.
Dissociation was easily detectable with 10 mM NaCl, and was
nearly complete when it was 200 mM. In the presence of 2
mM MgSO
and AlF
, 150
mM NaCl caused 76 ± 2% dissociation when compared with
samples of G
incubated in the absence of NaCl. In order to
better understand what was required for G
subunit
dissociation we varied the ion composition of the solutions during
incubation and immunoprecipitation. Omitting AlCl
(Fig. 3A) or NaF (Fig. 3B) during
the incubation and subsequent immunoprecipitation prevented G
subunit dissociation. G
subunit dissociation was also
prevented if, at the time of immunoprecipitation, the NaF concentration
was reduced by dilution to 200 µM even though the
concentration of AlCl
was maintained and 150 mM NaCl was added (Fig. 3B, last lane).
Although not all combinations were tested, it appeared that the
presence of NaF, AlCl
, and NaCl was required only during
the immunoprecipitation to cause G
subunit dissociation (Fig. 3, A and B). MgSO
was also
required since its omission prevented G
subunit
dissociation in the presence of NaCl and AlF
(Fig. 4). G
subunit dissociation occurred when
KCl was substituted for NaCl but not when Na
SO
was substituted (Fig. 5). Reducing the NaCl concentration
was sufficient to allow dissociated G
subunits to
reassociate even though the concentrations of MgSO
and
AlF
were maintained, and the G
subunit concentration was reduced by 50-fold as a consequence of
dilution (Fig. 6).
Figure 1:
Efficacy of immunoprecipitation of
G with RM/1 antiserum. A sample of bovine brain G
was immunoprecipitated with RM/1 antiserum as described under
``Experimental Procedures.'' The supernatant remaining after
immunoprecipitation was concentrated with a Centricon 30
ultrafiltration unit that was treated with bovine serum albumin to
block nonspecific binding of G
. The solute and Lubrol PX
concentrations of the immunoprecipitated sample were adjusted to make
them the same as that in the supernatant sample. As a control preimmune
serum (PI) was substituted for RM/1 antiserum. All of the
samples received
-mercaptoethanol and sodium dodecyl sulfate, and
the proteins in the immunoprecipitate and supernatant were separated on
10% polyacrylamide gels. Detection of G
was
accomplished as described under ``Experimental Procedures.''
The preparation of bovine brain G
used for these studies
contained both the short (G
) and long (G
) forms of
G
, but the former predominated. The other bands in the
immunoprecipitate and supernatant are the heavy chain of IgG and bovine
serum albumin, respectively.
Figure 2:
Dose-dependent effects of NaCl on G subunit dissociation in the presence of
AlF
. Bovine brain G
was
incubated as described under ``Experimental Procedures'' with
the indicated concentrations of NaCl in the presence of 10 mM AlF
and 2 mM MgSO
. The concentrations of NaCl and fluoroaluminate
were maintained during the immunoprecipitation of G
with RM/1 antiserum. The amount of precipitated G
and
G
was determined as described under ``Experimental
Procedures.''
Figure 3:
G subunit dissociation
requires the simultaneous presence of NaCl, NaF, and AlCl
at the time of immunoprecipitation. During the incubation step
bovine brain G
was treated with 2 mM MgSO
as described under ``Experimental Procedures'' in the
presence or absence of 150 mM NaCl, 10 mM NaF, or 10
µM AlCl
as indicated in the figure. Samples
were subsequently diluted at which time the concentration of these
salts were either maintained, increased by addition, or decreased by
dilution as indicated for immunoprecipitation. The concentration of
MgSO
was maintained at 2 mM during the
immunoprecipitation step.
At the time of
immunoprecipitation the concentration of NaF was 200
µM.
Figure 4:
G subunit dissociation in the
presence of AlF
and NaCl requires
magnesium ion. Bovine brain G
was incubated and
immunoprecipitated as described under ``Experimental
Procedures'' in the presence of 10 mM AlF
, 150 mM NaCl, and/or 2
mM MgSO
as indicated in the figure. The
concentration of each salt was maintained during the incubation and
immunoprecipitation.
Figure 5:
Cation and anion effects on G
subunit dissociation. Bovine brain G
was incubated and
immunoprecipitated as described under ``Experimental
Procedures.'' Solutions for both the incubation and
immunoprecipitation contained 2 mM MgSO
as well as
10 mM AlF
plus either 150 mM NaCl, KCl, or Na
SO
as
indicated.
Figure 6:
G subunit reassociation can
occur in the presence of AlF
. Bovine
brain G
was incubated with 2 mM MgSO
as described under ``Experimental Procedures.'' The
incubations were done in the absence or presence of 10 mM AlF
and 150 mM NaCl as
indicated in the figure. Subsequently, the samples were diluted for
immunoprecipitation, but the addition of 1 µl of RM/1 antiserum was
postponed for 1 h while the samples were incubated at 30 °C. The
dilutions were made in such a way that the MgSO
and
AlF
concentrations were not changed, and
the NaCl concentration was either maintained or reduced to 3 mM (150/3) by dilution as indicated. After antiserum was
added the immunoprecipitation was completed as described under
``Experimental Procedures.''
To investigate AlF binding to G
we prepared
[
S]methionine-G
by in vitro transcription and translation of the cDNA for rat olfactory
G
. This technique produced the G
with
a molecular mass of 52 kDa as well as two shorter products with
molecular masses of 40 and 36 kDa (Fig. 7). The latter two
proteins resulted respectively from initiation of translation of this
cDNA at the codons for methionines 60 and 110. Only traces of the in vitro translation proteins were able to survive tryptic
digestion in the absence of AlF
. In the
presence of AlF
, a 37-kDa fragment was
protected from proteolysis. The ability of AlF
to protect this fragment was improved by the presence of NaCl
although there was significant protection in the absence of this salt
suggesting that AlF
binding to
G
did not require NaCl.
Figure 7:
Protection of in vitro translated
G from tryptic digestion by
AlF
in the presence and absence of NaCl.
[
S]Methionine-G
was prepared by in vitro translation, and incubated in the presence or absence
of G
as described under ``Experimental
Procedures.'' Subsequently, a Centricon 30 ultrafiltration unit
was used to exchange the solution containing the
[
S]methionine-G
for one without
NaCl and having 2 mM MgSO
. Then, 10 mM AlF
and/or 100 mM NaCl was
added as indicated, and the samples were incubated for 30 min at 30
°C before digestion with trypsin as described under
``Experimental Procedures.'' The proteolytic products were
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and autoradiography. The acute arrow indicates the core 37-kDa
fragment of G
that is protected from proteolysis by
AlF
.
Since sedimentation on
sucrose density gradients has been used to show that
AlF causes G
subunit
dissociation, similar experiments were performed for these studies.
G
was incubated with AlF
in
the presence or absence of NaCl and subjected to zonal sedimentation.
AlF
was present throughout the sucrose
gradients, and NaCl was included or omitted so as to be consistent with
the way G
was treated before application to the sucrose
gradients. In the absence of NaCl, G
from S49 cell
membranes (Fig. 8A), and from bovine brain (Fig. 8B) sedimented as a heterotrimer despite the
presence of AlF
. For experiments with
G
from bovine brain, it was necessary to substitute
Na
SO
for NaCl when the latter was omitted from
the gradients. This prevented the purified G
from
aggregating, and sedimenting to the bottom of the centrifuge tube.
However, the addition of Na
SO
to gradients
reduced the sedimentation rate of heterotrimeric G
when
compared with sedimentation through gradients that did not contain NaCl
or Na
SO
(compare the sedimentation of
heterotrimeric G
in Panels A and B of Fig. 8). The reduced rate of sedimentation was due, either in
part or entirely, to increased density caused by adding
Na
SO
to the solutions used for making
gradients.
Figure 8:
Zonal sedimentation of G through sucrose density gradients in the presence and absence of
NaCl. G
from wild type S49 membranes (Panel A) or
bovine brain (Panel B) was incubated as described under
``Experimental Procedures.'' Samples were incubated with
AlF
in the presence (
,
) or
absence (
) of NaCl and either 2 mM MgSO
(
,
) or 10 mM MgCl
(
). When
samples of bovine brain G
(Panel B) were incubated
in the absence of NaCl (
), 50 mM Na
SO
was substituted for the NaCl. Controls were as follows: 1)
heterotrimeric G
was prepared by incubating G
with 2 mM MgSO
in the absence (Panels
A) or presence (Panel B) of 50 mM Na
SO
and 2) the free
G
-GTP
S subunit was prepared by incubating G
with 100 µM GTP
S, 120 mM MgSO
, and 100 mM NaCl. Before layering
experimental and control samples of bovine brain G
onto the
gradients, their volume was increased to 100 µl in such a way that
the concentration of components in the solutions, except for
G
, were not changed. The volume for experimental and
control samples of G
from S49 membranes was 100 µl and
therefore did not need adjusting before layering them onto gradients.
The sucrose density gradients were made up in solution B and contained
the same concentration of salts and effectors present in the samples
except for the gradients used for the G
-GTP
S
controls. These gradients were made without GTP
S and they
contained 2 mM MgSO
and 100 mM NaCl.
After centrifugation, the gradients were divided into fractions and
aliquants were assayed for their ability to reconstitute adenylyl
cyclase in S49 cyc
membranes as described under
``Experimental Procedures.'' The peak of reconstituted
activity for G
-GTP
S and heterotrimeric G
are indicated by the labeled arrows. The first fraction
represents the top of the gradient.
In the presence of both AlF and NaCl, G
from S49 cells sedimented at the same
rate as the free G
-GTP
S subunit (Fig. 8A) when the same conditions employed by other
investigators were used (Kahn and Gilman, 1984). These conditions
included the addition of 10 mM MgCl
during
incubation and zonal sedimentation. When MgCl
was replaced
with 2 mM MgSO
, G
from the S49 cell
membranes sedimented at a rate intermediate between the free
G
-GTP
S subunit and heterotrimeric G
(Fig. 8A). The same phenomenon was observed for
G
from bovine brain even in the presence of 10 mM MgCl
(Fig. 8B), and the peak of
G
activity was broad, extending to regions of the gradient
where both the free G
-GTP
S subunit and
heterotrimeric G
sedimented.
Heterotrimeric G proteins are activated when
AlF and GDP form an analog similar to the
transition state created when G
hydrolyzes GTP (Chabre, 1990;
Sondek et al., 1994; Coleman et al., 1994). The
activation is thought to be followed by dissociation of G
from
G
, and dissociation is considered necessary in order for
G
to interact productively with its effector molecule. The
activation of G
by AlF
can be
accompanied by subunit dissociation (Kahn and Gilman, 1984). However,
we have reported that activation of G
by
AlF
/ in the presence of 2 mM MgCl
did not cause G
subunit dissociation
(Toyoshige et al., 1994). We speculated that the difference
between our recent results and the earlier results of other
investigators were a consequence of experimental design. In an effort
to reconcile these differences, and to gain a better understanding of
the effects of AlF
and other ions on
G
subunit interaction we conducted the experiments
described in this article.
We found that dissociation of G subunit in the presence of AlF
required the simultaneous presence of 10 mM NaF, 10
µM AlCl
, 2 mM MgSO
, and
10-200 mM NaCl. Higher concentrations of NaF did not
cause subunit dissociation in the absence of NaCl (data not shown), and
dilution of NaCl to 3 mM allowed the G
subunit to
reassociate even though the concentrations of MgSO
and
AlF
were maintained. The effects of NaCl
are apparently due to Cl
. Other investigations
(Higashijima et al., 1987) indicate that Cl
causes conformational changes in G proteins when they bind
activating ligands, but not when they bind GDP. Recently, we have
investigated the ability of ligands other than
AlF
to cause G
subunit
dissociation. In the presence of low concentrations of Mg
(2 mM or less) neither GTP nor GTP
S caused G
subunit dissociation (Toyoshige et al., 1994, Basi et al., 1996). However, high concentrations of MgCl
(Toyoshige et al., 1994) and MgSO
(data not
shown) do cause G
subunit dissociation both in the absence
and presence of guanine nucleotides. While GTP and GDP were equally
effective in attenuating the Mg
-induced dissociation
of G
subunits (Basi et al., 1996), GTP
S had
little influence (Toyoshige et al., 1994) or augmented the
effect of Mg
(Basi et al., 1996) depending
upon how the experiment was done. We have not observed any effect of
Cl
on the Mg
-induced dissociation
of G
subunits in the presence or absence of GTP
S,
suggesting that Cl
does not influence subunit
dissociation under these circumstances (data not shown).
If
AlF could not bind to G
in
solution in the absence of NaCl it would not be able to cause subunit
dissociation. To investigate this possibility we took advantage of the
fact that ligands which activate G proteins can also protect a core
fragment ranging in molecular mass from 37 to 41 kDa from proteolytic
digestion (Husdon et al., 1981; Fung and Nash, 1983; Hurley et al., 1984). Since the antibodies available to us did not
recognize this core fragment we chose to prepare
[
S]methionine-G
by in vitro transcription and translation. Previously we (Warner et
al., 1996) and others (Journot et al., 1991) have found
that the properties of in vitro translated G
are similar to those of G
prepared from animal tissue.
AlF
was able to protect in vitro translated G
from tryptic proteolysis in the
absence as well as the presence of NaCl indicating that the salt was
not required for AlF
- binding to G
. Mixing in vitro translated G
with bovine brain
G
allows for the formation of a heterotrimer (Warner et
al., 1996), but this had no effect on the ability of
AlF
to protect in vitro translated G
from proteolysis by trypsin in the
presence or absence of NaCl. Additional evidence that NaCl is not
required for the binding of AlF
to
G
comes from the fact that NaCl is not necessary for the
activation of adenylyl cyclase in membranes (Sternweis and Gilman,
1982; Northup et al., 1983). (
)
In previous
reports investigators have shown by the technique of zonal
sedimentation on sucrose density gradients that
AlF or NaF causes G
subunit
dissociation (Howlett and Gilman, 1980; Hanski et al., 1981;
Sternweis et al., 1981; Northup et al., 1983; Kahn
and Gilman, 1984). However, these experiments were done in the presence
of 100-300 mM NaCl. Based on our results it seemed
likely that G
subunit dissociation would not have occurred
if the sedimentations had been done in the absence of NaCl. In order to
demonstrate this we conducted zonal sedimentation experiments in the
presence and absence of NaCl. G
from S49 cell
membranes sedimented as the free G
subunit in the
presence of NaCl and AlF
and
Mg
. This result corroborated the findings of other
investigators. The experimental design for zonal sedimentation in the
presence of NaCl was based on earlier experiments (Kahn and Gilman,
1984), and therefore the sucrose gradients contained 10 mM MgCl
in addition to 100 mM NaCl and
AlF
, and the centrifugations were done at
4 °C. Substituting 2 mM MgSO
for the
MgCl
caused G
from S49 membranes to sediment at
a rate intermediate between free G
and heterotrimeric
G
. The intermediate rate of sedimentation may have been
caused by using 100 mM NaCl, a concentration used in previous
investigations (Kahn and Gilman, 1984), but one that is not sufficient
to cause complete dissociation of G
subunits when the
Mg
concentration was 2 mM (see Fig. 2). Another possibility is suggested by reports that the
dissociation of heterotrimeric G protein subunits in the presence of
activating ligands is a temperature-dependent process (Codina et
al., 1984). G protein subunits dissociated at 32 °C in the
presence of MgCl
and a nonhydrolyzable GTP analog
subsequently reassociated when the temperature was decreased to 4
°C. In our experiments, a subunit that dissociated when exposed to
NaCl in the presence of AlF
and
Mg
probably would not reassociate during
immunoprecipitation which was done at 24 °C, but might reassociate
during centrifugation at 4 °C, consequently giving rise to a broad
peak sedimenting between free G
and heterotrimeric
G
.
When NaCl was omitted from the sucrose density
gradients, G sedimented as a heterotrimer despite the
presence of AlF
and Mg
.
This result confirmed our prediction that subunit dissociation would
not occur in the absence of NaCl during zonal sedimentation. These data
also support the results of experiments involving immunoprecipitation
which showed that Cl
was required for G
subunit dissociation in the presence of
AlF
and Mg
. Based on
the data presented here, we have concluded that
AlF
, a ligand that is able to activate
heterotrimeric G proteins, can bind to G
without causing
subunit dissociation.