(Received for publication, May 10, 1995; and in revised form, October 6, 1995)
From the
G is a member of the family of trimeric guanine
nucleotide-binding regulatory proteins (G proteins), which plays a
crucial role in signaling across cell membranes. The expression of
G
is predominately confined to neuronal cells and
platelets, suggesting an involvement in a neuroendocrine process.
Although the signaling pathway in which G
participates is
not yet known, it has been linked to inhibition of adenylyl cyclase. We
have found that arachidonate and related unsaturated fatty acids
suppress guanine nucleotide binding to the
subunit of
G
. This inhibition of nucleotide binding by cis-unsaturated
fatty acids is specific for G
; other G protein
subunits are relatively insensitive to these lipids. The IC
for inhibition by the lipids closely corresponds to their
critical micellar concentrations, suggesting that the interaction of
the lipid micelle with G
is the primary event leading
to inhibition. The presence of the acidic group of the fatty acid is
critical for inhibition, as no effect is observed with the
corresponding fatty alcohol. While arachidonic acid produces
near-complete inhibition of both GDP and guanosine
5`-(3-O-thio)triphosphate binding by G
,
release of GDP from the protein was unaffected. Furthermore, the rate
of inactivation of G
by arachidonate is essentially
identical to the rate of GDP release from the protein, indicating that
GDP release is required for inactivation. These observations indicate
that the mechanism of inactivation of G
by unsaturated
fatty acids is through an interaction of an acidic lipid micelle with
the nucleotide-free form of the protein. Although the physiologic
significance of this finding is unclear, similar effects of unsaturated
fatty acids on other proteins involved in cell signaling indicate
potential roles for these lipids in signal modulation. Additionally,
the ability of arachidonate to inactivate this adenylyl
cyclase-inhibitory G protein provides a molecular mechanism for
previous findings that treatment of platelets with arachidonate results
in elevated cAMP levels.
Trimeric guanine nucleotide binding regulatory proteins (G
proteins) ()comprise a class of membrane-associated proteins
that participate in a wide variety of signal transduction pathways by
communicating the external signal from cell surface receptors to
intracellular effector molecules(1, 2) . These G
proteins are
heterotrimers, consisting of two
functional subunits, an
subunit containing bound guanine
nucleotide, and a
complex. In the resting (GDP-bound) state,
a G protein can interact with a liganded receptor in a fashion that
drives the exchange of GDP for GTP on the
subunit. The
-GTP
and
subunits then dissociate, and both subunit complexes can
interact with, and modulate the activity of, downstream effectors. The
signal is terminated by an intrinsic GTPase activity of the
subunit; subsequent reassociation with the
complex returns
the system to its resting state. Effector molecules for G proteins
include adenylyl cyclase, certain subtypes of phospholipase C, and
various ion channels (3, 4) .
G proteins are
classified through the identity of their subunit. The high
sequence homology among these polypeptides has led to the cloning of
several forms for which precise physiologic roles have not yet been
ascribed. One such isotype is
G
(5, 6) . The distribution of
G
is limited primarily to platelets and neurons,
implicating this G protein in some specific role in these
tissues(5, 6, 7, 8) . The protein
has been purified from bovine brain as well as a bacterial expression
system and shown to possess biochemical properties distinct from other
G protein
subunits(9) . For example, nucleotide exchange
by G
is highly dependent on free magnesium
concentrations. At free magnesium concentrations greater than
10
M, GTP binding by G
is
nearly completely suppressed. This effect is not seen with other G
proteins; in fact, the presence of high magnesium concentrations
generally stimulates their rates of nucleotide exchange(2) .
Magnesium-dependent suppression of nucleotide exchange is observed,
however, with members of the monomeric family of GTP-binding proteins, e.g. Ras(10) . G
also has a very slow
intrinsic rate of GTP hydrolysis, more similar to that of Ras and
Ras-related proteins than
subunits(9) . Although G
is formally a member of the G
family, it is
insensitive to ADP-ribosylation catalyzed by pertussis
toxin(9) , a modification that inactivates the other members of
the G
family(11) . A property that G
does share with most members of the G
family is an
ability to mediate inhibition of adenylyl
cyclase(12, 13) . In addition, G
serves as an excellent substrate for activated protein kinase C
both in vitro and in intact platelets(14) , and
evidence has been obtained that this phosphorylation blocks subunit
interactions of this G protein(15) .
Several reports have
appeared recently, indicating that particular biogenically active
lipids can interact in vitro with signaling proteins and
modulate their activities. For example, arachidonate and related
unsaturated fatty acids physically associate with, and inhibit the
activity of, the Ras GTPase activating protein known as
GAP(16, 17) . Such lipids can also regulate the
association of the Ras-related protein, Rac, with a specific GDP
dissociation inhibitor(18) . Similarly, cis-unsaturated fatty
acids such as oleate and arachidonate have been shown to activate
protein kinase C(19) . While the mechanism by which lipids
modulate the activities of these proteins is not completely defined,
their interaction raises interesting possibilities for the role of
lipids in cellular regulation. In this study, we demonstrate that
cis-unsaturated fatty acids block GTPS binding by
G
. The mechanism of inactivation involves a specific
effect of lipid micelles on the nucleotide-free form of the protein.
These observations are of particular interest since the tissues in
which G
is found are known to accumulate significant
levels of arachidonic acid in response to certain activating
stimuli(20, 21) , and thus the potential exists for
cross-talk between arachidonate-producing pathways and those controlled
by G
.
For experiments assessing the time
course of GDP dissociation from G, 11 pmol of the
protein were incubated at 30 °C for 60 min in the presence of 50
mM HEPES, pH 7.6, 1 mM EDTA, 1 mM DTT, 100
mM NaCl, 0.05% Lubrol, and 0.5 µM [
H]GDP (specific activity,
26,000
cpm/pmol). Arachidonic acid (300 µM) or palmitic acid (300
µM) in 50 mM HEPES, pH 7.6, 1 mM EDTA, 1
mM DTT, and 0.1% Lubrol was added, and samples were incubated
for an additional 2 min. Samples were then spiked with unlabeled GDP
such that the final concentration of GDP in the ``chase'' was
50 µM. The addition of arachidonate and GDP were of small
enough volume as not to significantly perturb the relative
concentration of protein or detergent. At the time points indicated in
the appropriate figure, aliquots of G
were removed to
ice-cold buffer (20 mM Tris-Cl, pH 7.7, 100 mM NaCl,
25 mM MgCl
) and stored on ice until filtration
through BA85 nitrocellulose filters. Filters were dried, and
radioactivity was determined by liquid scintillation spectrophotometry.
For experiments demonstrating the recovery of binding activity with
time, G (7.6 pmol) was incubated under the standard
reaction conditions plus 300 µM arachidonte. After 5 min,
the reaction was diluted 10-fold with 50 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.05% Lubrol, and 2 µM GTP
S. At the times indicated in the appropriate figure,
aliquots were removed from the incubation into ice-cold buffer (20
mM Tris-Cl, pH 7.7, 100 mM NaCl, 25 mM MgCl
), and bound nucleotide was determined.
For
experiments assessing the time course of inactivation of G by arachidonate,
7 pmol of protein were incubated in the
presence or absence of 300 µM arachidonic acid for up to
90 min. At the time points indicated, aliquots of the incubation
mixture containing 0.4 pmol of G
were removed and
immediately subjected to a 60-min GTP
S binding assay. Transferring
the protein from the pre-incubation to the GTP
S binding reaction
effectively diluted the arachidonate to 30 µM in the
samples in which the pre-incubation was performed in the presence of
300 µM lipid. Additional changes from the standard
reaction mixture included the presence of 5 µM GDP during
the pre-incubation to stabilize the G protein and inclusion of 10
µM [
S]GTP
S (specific, activity
10,000 cpm/pmol) in the GTP
S binding mix.
Figure 1:
Effect of
lipids on GTPS binding by G
. GTP
S binding to
purified G
was performed as described under
``Experimental Procedures.'' Briefly, 0.25-1.0 pmol of
G
was incubated at 30 °C for 1 h in the standard
GTP
S reaction mixture supplemented with each lipid at the
concentrations indicated. The lipids examined were arachidonic acid
(
), arachidonyl alcohol (
), linoleic acid (
), oleic
acid (
), arachidic acid (
), and linolenic acid
(
). Binding data are presented as a percentage of the amount of
GTP
S bound in the absence of lipid (the ``control''
value), and the points represent the mean of three separate
determinations. Numeric symbols represent the carbon chain
length and degree of unsaturation of each lipid tested. OH indicates a fatty alcohol.
The ability of arachidonate to suppress
GTPS binding by G
prompted us to examine whether
other unsaturated fatty acids exert the same effect. This was indeed
found to be the case. Oleic acid, linoleic acid, and linolenic acid all
suppressed nucleotide binding by G
in the same
dose-dependent fashion as arachidonic acid (Fig. 1). Oleic acid
and linoleic acid both completely suppressed GTP
S binding by
G
at a concentration of 175 µM, and
linolenic acid was completely inhibitory at 250 µM.
However, the trans-unsaturated fatty acid, elaidic acid, was only
slightly inhibitory at these concentrations (data not shown).
The
steepness of the inhibition curves for the unsaturated fatty acids
indicated that the inhibition was not due to a simple binding event but
rather to some sort of cooperative process. One such process, which is
quite obvious when working with lipids, is the formation of micelles,
which is a highly cooperative aggregation event. Accordingly, we
determined the CMC for the lipids under the same conditions (e.g. ionic strength, Mg concentration) as for the
GTP
S binding experiments. CMC values for the various lipids were
determined using a fluorescence technique(24) , and it was
found that the CMC values corresponded nearly identically to the
observed IC
for the inhibition of GTP
S binding (Table 1). For example, the observed IC
and the CMC
for arachidonic acid were 60 and 73 µM, respectively.
These observations provide strong evidence that the abilities of the
unsaturated fatty acids to suppress GTP
S binding by G
is micelle dependent; i.e. it is an interaction of the
protein with an anionic lipid micelle, which is responsible for the
inhibition.
To facilitate manipulation of the lipid in subsequent
studies, we assessed whether the inhibition by arachidonate of the
ability of G to bind GTP
S occurred when the fatty
acid was present in a mixed micelle. The data in Fig. 2show
that this is the case, as the same type of inhibition is observed in
response to increasing arachidonic acid when the fatty acid is present
in a mixed micelle with the non-ionic detergent, Lubrol. The
dose-response curve is shifted substantially to the right as would be
expected for a process that depends on the mole fraction of lipid in
the micelle(26) . In fact, the IC
for inhibition
of GTP
S binding by G
shifts in proportion to the
mole fraction of the lipid (results not shown).
Figure 2:
Effect of a non-ionic detergent on
arachidonic acid-dependent inhibition of GTPS binding by
G
. G
was subjected to the GTP
S
binding reaction as described under ``Experimental
Procedures'' for 20 min at 30 °C in the presence of the
indicated concentrations of arachidonic acid and 0.05% Lubrol (
).
For comparison, the data obtained in the absence of added Lubrol are
shown (
, see Fig. 1). Data shown are from a single
experiment that has been repeated at least three times. Maximal
GTP
S binding is defined as the amount of GTP
S binding
observed in the absence of arachidonate under each condition; the
presence of Lubrol had a negligible effect on the
binding.
Figure 3:
Comparison of the effect of arachidonic
acid on GTPS binding to G protein
subunits. The indicated
subunits, all purified from bacterial expression systems, were
subjected to a GTP
S binding assay as described under
``Experimental Procedures.'' Assays were carried out in the
absence of lipid (open bars) or in the presence of either 150
µM (hatched bars) or 300 µM arachidonic acid (solid bars). The incubation conditions
were adjusted for each
subunit as described under
``Experimental Procedures.'' Data shown represent the mean of
three separate determinations with the 100% control value being the
binding observed in the absence of added arachidonic acid. AA,
arachidonic acid.
Figure 4:
Effect of arachidonic acid on GDP
dissociation from G. Approximately 1 pmol of
G
was incubated at 30 °C in 50 mM HEPES,
1 mM EDTA, 1 mM DTT, 100 mM NaCl, 0.05%
Lubrol, and 0.5 µM [
H]GDP (26
Ci/mmol). After a 60-min incubation, arachidonic acid (
) or
palmitic acid (
), in 50 mM HEPES, 1 mM EDTA, 1
mM DTT, 0.05% Lubrol, was added to a final concentration of
300 µM. Samples were incubated for an additional 2 min,
and then unlabeled GDP was added to a final concentration of 50
µM. At the time points indicated, 60-µl aliquots were
removed from the reactions to 2 ml of ice-cold 20 mM Tris, pH
7.6, 25 mM MgCl
, 100 mM NaCl. Samples
were filtered through BA85 nitrocellulose filters and dried, and bound
radioactivity was determined. The 100% control value is the amount of
[
H]GDP bound at the initial time point sampled.
Data shown are from a single experiment and are typical of results from
three independent experiments. AA, arachidonic
acid.
One possibility for the selective effect of arachidonate
on the GTPS binding step is that the lipid micelle could interact
specifically with the unoccupied nucleotide binding site on
G
and effectively compete for GTP
S binding. If
this were the case, inhibition of nucleotide binding by arachidonic
acid should be reduced by increasing the concentration of competing
nucleotide. To explore this possibility, we measured the effect of
arachidonic acid on GTP
S binding in the presence of increasing
GTP
S concentrations. However, assessment of the
arachidonate-mediated inhibition over a 50-fold range of GTP
S
revealed that binding was nearly completely suppressed at all
concentrations of competing nucleotide (Fig. 5). Since
inhibition of nucleotide binding by arachidonic acid was unaffected at
GTP
S concentrations as high as 25 µM, which is
>1000-fold above the K
of G protein
subunits for GTP
S(2) , it is considered highly unlikely
that the lipid micelle is competing for the nucleotide binding site of
the protein.
Figure 5:
Effect
of increasing GTPS concentration on the ability of arachidonic
acid to inhibit nucleotide binding by G
. G
(1.5-3.0 pmol) was subjected to a standard GTP
S
binding assay in the absence (open bars) or presence (hatched bars) of 300 µM arachidonic acid as
described under ``Experimental Procedures.'' Reactions were
initiated by the addition of protein and were allowed to proceed at 30
°C for 60 min. The concentration of GTP
S in the incubation was
varied from 0.5 to 25.0 µM as indicated. Data points
represent means from three separate
determinations.
An alternative explanation for the effect of
arachidonate on GTPS binding, but not on GDP release, by
G
is that the fatty acid could somehow interact with
and inactivate the nucleotide-free form of the G protein that is a
transient intermediate in the exchange process. To examine this
possibility, we assessed the time dependence of the inactivation of
G
by arachidonate. If arachidonate could exert its
effect only on the nucleotide-free form of G
, a
recovery of binding activity should be observed if the protein is
exposed to high arachidonate and then is diluted to an ineffective
concentration. This recovery of binding activity would then reflect the
fraction of the protein that had not yet released its GDP. Furthermore,
if the arachidonic acid is selectively inactivating the nucleotide-free
form of G
, then the rate of inactivation of GTP
S
binding should correspond to the rate of GDP release. Indeed, the
evidence indicates that this is the case (Fig. 6). In the first
experiment (Fig. 6A), binding activity was measured
after G
was first incubated with 300 µM arachidonate for 5 min and then diluted to 30 µM arachidonate. While GTP
S binding activity was detected, the
level of nucleotide binding recovered was significantly less than the
control levels in which only 30 µM fatty acid had been
present throughout.
Figure 6:
Arachidonic acid selectively inactivates
the nucleotide-free form of G. A,
G
was incubated in a batch reaction as described under
``Experimental Procedures'' at 30 °C in the presence of
either 30 µM (
) or 300 (
,
) µM arachidonate. In one of the reaction mixtures containing 300
µM arachidonate (
), the mixture was diluted 10-fold
with 50 mM HEPES, 1 mM EDTA, 1 mM DTT, 2
µM GTP
S (specific activity, 12,000 cpm/pmol) after 5
min of incubation. At the times indicated, aliquots containing
0.5
pmol of G
was removed to ice-cold 20 mM Tris-Cl, 25 mM MgCl
, 100 mM NaCl,
and bound nucleotide was determined. B, G
was
incubated in a batch reaction as described under ``Experimental
Procedures'' at 30 °C in 50 mM HEPES, 10 mM EDTA, 1 mM DTT, 2.65 mM MgCl
, 5
µM GDP, 0.05% Lubrol, and either 0 (
) or 300
µM (
) arachidonate. At the indicated times, aliquots
containing
0.4 pmol of G
were removed and added
to a GTP
S binding reaction mixture containing 10 µM GTP
S and a free Mg
concentration of 700
nM. For the experiment conducted in the absence of arachidonic
acid (
), the GTP
S reaction mixtures contained an added 30
µM arachidonic acid so that the lipid concentration in all
binding assays was held constant. GTP
S binding was performed at 30
°C for 60 min for all data points. Data points represent means of
six separate determinations. AA, arachidonic
acid.
This same type of experiment was performed over
a range of pre-incubation times with 300 µM arachidonate
from 5 to 90 min; in each case, the quantity of G capable of binding nucleotide was assessed after a 10-fold
dilution to the ineffective concentration of the lipid (i.e. 30 µM). The results of this analysis, shown in Fig. 6B, revealed in each case a loss of GTP
S
binding activity that was not recovered by subsequent dilution. This
was not due simply to protein lability, as pre-incubation of
G
in the absence of arachidonate did not result in a
loss of GTP
S binding activity. An equally important finding from
this experiment is that the time dependence in the loss of the binding
activity of G
could be fit to an exponential with a
decay constant of 0.028 min
, which is nearly
identical to the rate constant for GDP release from G
under the same conditions(9) . Taken together, these data
indicate that, in the presence of arachidonate, G
is
able to release GDP normally but is then rapidly inactivated when the
lipid micelle interacts with the nucleotide-free form of the protein.
The role of lipids in cellular signaling has received increasing attention in recent years(27) . It is now clear that lipids such as arachidonic acid and diacylglycerol actively participate as second messengers in signaling pathways(28, 29) . Examples are also beginning to emerge of arachidonate and other cis-unsaturated fatty acids directly modulating activities of signaling proteins. For example, these fatty acids can associate with and alter the activity of Ras-GAP (17, 30) . Cis-unsaturated fatty acids have also been shown to regulate association between the monomeric G protein, Rac, and its GDP dissociation inhibitor(18) . Arachidonate and other unsaturated fatty acids have also been shown to activate certain isozymes of the protein kinase, protein kinase C(19) .
In this report, we have
identified an additional effect of cis-unsaturated fatty acids on a
signaling protein, that being the inactivation of a G protein
subunit, specifically G
. Arachidonate-dependent
inactivation of GTP
S binding by G
was quite
specific for this
subunit, as treatment of a number of other
subunits had only minimal effects on their abilities to bind
nucleotide. The inactivation was dependent upon the presence of an
acidic group on the lipid and correlated with the formation of a lipid
micelle. Several cis-unsaturated fatty acids were potent inhibitors of
GTP
S binding by G
with a dose dependence that
matched the lipid's respective CMC. This suggests that it is an
interaction between the charged surface of a micelle with G
that is required for its inhibition. These results are similar to
the results of Serth et al.(17) , who observed the
inhibition of Ras-GAP in the presence of fatty acids and acidic
phospholipids but not in the presence of neutral lipids, and only under
conditions in which the active lipids formed micellar structures.
The inhibition of GTPS binding by G
seen upon
the addition of arachidonic acid could have been exerted at either of
two distinct steps in the process, these being dissociation of bound
GDP or association of the GTP
S. The former step was initially
considered the most likely, as GDP dissociation from G proteins is
10
-fold slower than association of guanine nucleotides (31) . To identify the step in G
nucleotide
exchange affected by arachidonate, we directly determined the effect of
arachidonic acid on the rate of GDP release. Quite surprisingly, GDP
release was virtually unaffected by concentrations of arachidonate,
which essentially completely suppress GTP
S binding, indicating
that GTP
S binding was the step being affected. An assessment of
the time dependence of arachidonate inhibition of GTP
S binding
revealed that (a) inhibition of GTP
S binding was not
reversible even after 60 min of incubation and (b) the rate of
this inactivation corresponded precisely with that of GDP release by
G
. Taken together, these experiments indicate that the
effect of arachidonate on the ability of G
to bind guanine
nucleotides is dependent on an association of the lipid micelle with
the nucleotide-free form of the protein, resulting in an alteration of
the protein that renders it inactive.
While lipid-mediated
modulation of G protein activity by irreversible inactivation seems an
unlikely mode of regulation in the cell, the selectivity of the process
for G over other G proteins, as well as the unique
distribution of G
, provides strong reasons to suspect that
the process is physiologically relevant. As noted above, the cell types
in which G
is found, such as platelets and chromaffin
cells, are known to possess high levels of phospholipase A
activity(20, 21) . These cells are also known to
produce substantial levels of arachidonate in response to external
stimuli(20, 32, 33) . Also of note in this
regard are previous studies showing that treatment of platelets with
high levels of exogenous arachidonate results in increased
intracellular cAMP accompanied by reduced
aggregation(34, 35) . In one of these studies,
treatment of platelets with an adenylyl cyclase inhibitor restored
aggregation in the presence of arachidonate, indicating that the fatty
acid was exerting its effect at or upstream of adenylyl
cyclase(35) . The finding that arachidonate can inactivate a G
protein that is both present in platelets and implicated in the
inhibition of adenylyl cyclase thus provides a potential molecular
mechanism for these effects.
Finally, it is certainly possible that
in the context of an intact cell an increase in the concentration of
arachidonic acid might be only transiently inhibitory, i.e. the cellular environment could provide protection of the
apoprotein form of G from permanent inactivation by
the lipid. Possibilities here include a protective factor in these
cells that associates with G
or one that reverses the
association between G
and inhibitory lipids.
Identification of the pathway in which G
participates
will likely shed some light on these results and on the possibilities
for the novel means of G protein regulation they may represent.