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 10M, 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.

EXPERIMENTAL PROCEDURES

Production and Purification of Recombinant G Protein Subunits

Lipid Storage and Micelle Preparation

Guanine Nucleotide Binding Assays

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 GTPS. 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 GTPS binding assay. Transferring the protein from the pre-incubation to the GTPS 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]GTPS (specific, activity 10,000 cpm/pmol) in the GTPS binding mix.

Fluorimetric Determination of Critical Micellar Concentrations of Lipids

RESULTS

Cis-unsaturated Fatty Acids Inhibit GTPS Binding by G

Figure 1: Effect of lipids on GTPS binding by G. GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS binding is defined as the amount of GTPS binding observed in the absence of arachidonate under each condition; the presence of Lubrol had a negligible effect on the binding.

Inhibition of Nucleotide Binding by Arachidonate Is Specific for G

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 GTPS 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.

Mechanism of Action

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 GTPS 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 GTPS binding in the presence of increasing GTPS concentrations. However, assessment of the arachidonate-mediated inhibition over a 50-fold range of GTPS 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 GTPS concentrations as high as 25 µM, which is >1000-fold above the K of G protein subunits for GTPS(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 GTPS 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 GTPS 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 GTPS 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 GTPS 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 GTPS (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 GTPS binding reaction mixture containing 10 µM GTPS and a free Mg concentration of 700 nM. For the experiment conducted in the absence of arachidonic acid (), the GTPS reaction mixtures contained an added 30 µM arachidonic acid so that the lipid concentration in all binding assays was held constant. GTPS 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 GTPS 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 GTPS 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.

DISCUSSION

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 GTPS 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 GTPS 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 GTPS. 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 GTPS binding, indicating that GTPS binding was the step being affected. An assessment of the time dependence of arachidonate inhibition of GTPS binding revealed that (a) inhibition of GTPS 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.

FOOTNOTES

*

This work was supported by a Basil O'Connor Scholar Award from the March of Dimes and by American Cancer Society Grants BE-117 (to P. J. C.) and BE-30 (to A. G. Gilman). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 919-613-8613; Fax: 919-613-8642.

()

The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; GTPS, guanosine 5`-(3-O-thio)triphosphate; DTT, dithiothreitol; CMC, critical micellar concentration.

ACKNOWLEDGEMENTS

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