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Inhibition of Brain Gz GAP and Other RGS Proteins by Palmitoylation of G Protein α Subunits

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Science  07 Nov 1997:
Vol. 278, Issue 5340, pp. 1132-1135
DOI: 10.1126/science.278.5340.1132

Abstract

Palmitoylation of the α subunit of the guanine nucleotide-binding protein Gz inhibited by more than 90 percent its response to the guanosine triphosphatase (GTPase)–accelerating activity of Gz GAP, a Gz-selective member of the regulators of G-protein signaling (RGS) protein family of GTPase-activating proteins (GAPs). Palmitoylation both decreased the affinity of Gz GAP for the GTP-bound form of Gαz by at least 90 percent and decreased the maximum rate of GTP hydrolysis. Inhibition was reversed by removal of the palmitoyl group by dithiothreitol. Palmitoylation of Gαz also inhibited its response to the GAP activity of Gα-interacting protein (GAIP), another RGS protein, and palmitoylation of Gαi1 inhibited its response to RGS4. The extent of inhibition of Gz GAP, GAIP, RGS4, and RGS10 correlated roughly with their intrinsic GAP activities for the Gα target used in the assay. Reversible palmitoylation is thus a major determinant of Gzdeactivation after its stimulation by receptors, and may be a general mechanism for prolonging or potentiating G-protein signaling.

The α subunits of most heterotrimeric G proteins are modified by irreversible lipid amidation of the NH2-terminus and by addition of a palmitoyl thioester at a nearby, conserved cysteine residue (1, 2). Unlike myristoylation, palmitoylation of Gα subunits is reversible, and bound palmitate turns over rapidly in cells. Although virtually nothing is known of the enzymes that catalyze addition and removal of palmitate, palmitate turnover on G-protein α subunits appears to be regulated coordinately with their activation and deactivation. In the case of Gαs (3, 4) and Gαq(5), substantial depalmitoylation occurs upon receptor-promoted activation, and repalmitoylation of Gαscoincides at least roughly with deactivation (3). Treatment with cholera toxin, which prolongs activation of Gs by blocking hydrolysis of bound GTP, also promotes turnover of bound palmitate (6). Conversely, palmitate turnover on Gαi and Gαs is decreased by coexpression of excess Gβγ, which inhibits activation (6, 7).

Palmitoylation is involved in anchoring Gα subunits to the membrane or specifying their membrane localization, or both (1-4, 7-9), by increasing their intrinsic hydrophobicity and, at least for Gαs, by increasing affinity for Gβγ (7). Mutation of the palmitoylated cysteine of Gαz to alanine also potentiated inhibition of adenylyl cyclase in transfected cells (9). Palmitoylation has not yet been linked to alteration of a specific G-protein signaling function, however. It is not required for interaction of Gα subunits with receptors or effectors in vitro (10), and no effect of palmitoylation on the binding or hydrolysis of guanine nucleotides has been reported. Mutation of the palmitoylatable cysteine residue in Gαq or Gαs inhibited signaling (11), but signaling was potentiated by the same mutation in Gαz or Gpa1p, the major Gα subunit in Saccharomyces cerevisiae (9,12). Although palmitoylation may be responsible for such variable effects on different Gα subunits, these results may also arise from effects of mutating the cysteine residue that are unrelated to palmitoylation (10).

We describe the inhibition of the effects of the major GzGTPase activating protein, Gz GAP, by palmitoylation of Gαz. Gz is a relatively rare member of the Gi family that is found in brain, platelets and adrenal medulla, and is therefore suspected to be involved in regulation of secretion (13). Isolated Gαz hydrolyzes bound GTP slowly, such that the half-life of the active, GTP-bound species is about 7 min at 30°C (14, 15). Gz GAP, which we recently purified from bovine brain, accelerates the hydrolysis of Gz-bound GTP over 200-fold (15). GzGAP is a novel member of the RGS family (16), whose members attenuate G-protein signaling at least in part through their GAP activity (17). Gz GAP is most abundant in tissues that also express Gz. It thus appears to be the major determinant of Gz deactivation and, therefore, of the amplitude and duration of Gz-mediated signals.

To examine the effect of palmitoylation of Gαz on its deactivation, we palmitoylated purified Gαz in vitro (18, 19) and measured the rate of hydrolysis of bound [γ-32P]GTP in the presence and absence of Gz GAP (15, 20). Fractional autopalmitoylation of purified Gαz in vitro was nearly complete, 80 ± 10% according to total protein or 120 ± 15% according to the number of GTP-γ-S binding sites (n = 6) (Fig. 1) (19). Treatment with either neutral hydroxylamine or dithiothreitol (DTT) removed the palmitate (Fig. 1), consistent with its addition through a thioester bond. [3H]Palmitate could also be completely removed from Gαz by tryptic proteolysis after protection with GTP-γ-S or Al3+/F (Fig. 1). Because Cys3 is the only cysteine residue before the Arg29 tryptic cleavage site (21), palmitoylation of Cys3 is unique and nearly quantitative under the conditions used here. There was no difference in the rate of autopalmitoylation of Gαz when it was bound to either GDP or GTP-γ-S (22).

Figure 1

Autopalmitoylation of Gαz. GTP-γ-S-activated Gαz (4 μM), purified from Sf9 cells (35), was incubated with 50 μM [3H]Pal-CoA (450 cpm/pmol) for 2 hours at 30°C (19). The extent of palmitoylation in this experiment was 74%, based on total protein. Samples were then incubated for 45 min at 30°C either with no addition, with 0.45 μM trypsin, 0.5 M hydroxylamine, or 15 mM DTT. Samples were analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue (top panel) and then subjected to fluorography (bottom panel). The trypsin lane contains threefold more total sample than the other lanes, because 70% of the initial Gαz was totally proteolyzed even after GTP-γ-S protection.

Palmitoylation of Gαz blocked the action of bovine brain Gz GAP by nearly 90% (87 ± 3.5%, n= 6, in matched experiments) (Fig. 2 and Table 1) (20). Because palmitoylation of Gαz may be incomplete and because GTP bound to residual nonpalmitoylated Gαz will be disproportionately hydrolyzed during a brief GAP assay (Fig. 2A), inhibition of the GAP by palmitoylation of Gαz is underestimated and exceeds 90%. Palmitoylation of Gαzhad no effect on the rate at which it hydrolyzed bound GTP in the absence of GAP (Table 1 and multiple control experiments) or on the rate of binding of GTP-γ-S (22).

Figure 2

Effects of NH2-terminal modification of Gαz on its interaction with Gz GAP. (A) Substrate concentration dependence. Gz GAP activity was assayed as described (15) at the concentrations of Gαz-[γ-32P]GTP shown on the abscissa. The Gαz, purified from Sf9 cells (35), was either untreated (▪), palmitoylated to 0.8 mol/mol based on total protein (▴), or treated with trypsin in the presence of Al3+ and F before binding to [γ-32P]GTP (⧫). The concentration of each substrate was determined according to [γ-32P]GTP binding (15). The concentration of Gz GAP was 50 pM. After subtraction of unstimulated hydrolysis, data for Gαz-GTP were fit to the Michaelis-Menten equation (K m = 2.4 nM, V max = 13.0 fmol/min, k cat = 3.3 min–1). GAP-stimulated hydrolysis for palmitoylated Gαz was fit to a two-component Michaelis-Menten equation using the Marquardt-Levenberg algorithm in the SigmaPlot (Jandel Scientific Software, San Rafael, California) program package. The nonpalmitoylated component, with unchanged K m andV max, reflected 7 to 10% of the total protein-bound [γ-32P]GTP. Because the highest substrate concentration was still below the K m for palmitoyl-Gαz-GTP, the fitted value ofK m varied from 15 to 30 nM andV max varied from 2 to 7 fmol/min depending on initial conditions used in the fit. The drawn line shows simulated values for 90% palmitoylation, K m = 19 nM andV max = 2.6 fmol/min. (If theK m for the palmitoylated Gαz were set equal to 75 nM, the K i from (B), then theV max would be 10 fmol/min) (B) Competitive inhibition of Gz GAP activity by different preparations of Gαz. GAP activity was measured using 2 nM Gαz-[γ-32P]GTP. The substrate Gαz was purified from Sf9 cells. Competing Gαz was bound either to GTP-γ-S (solid symbols) or GDP (open symbols) and added at the concentrations shown. ▪, □: Gαz purified from Sf9 cells, untreated; •: Gαz purified from E. coli (14), untreated; ▴, ▵: palmitoylated Gαz (19); ⧫: Gαz from Sf9 cells treated with trypsin as described for Fig. 1. The extrapolated K i for palmitoyl–Gαz–GTP-γ-S, corrected for substrate concentration, was 76 nM, and the K i for trypsin-treated Gαz–GTP-γ-S was >110 nM. Concentrations of each Gαz–GTP-γ-S species were measured by direct binding assays using trace amounts of [35S]GTP-γ-S. GAP activity without inhibitor was 40 mU.

Table 1

Inhibition of Gz GAP activity after palmitoylation of Gαz. Hydrolysis of [γ-32P]GTP bound to Gαz (from Sf9 cells, 1.8 nM) was measured over 2 min in the presence or absence of 400 pM Gz GAP (100% is 246 mU) (15, 20). Before binding to [γ-32P]GTP, the Gαz was treated in the order shown with Pal-CoA (19), with 15 mM DTT at 30°C for 40 min, or with trypsin after Al3+/F protection (21). Al3+ and F were removed by gel filtration after binding of [γ-32P]GTP (15).

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Palmitoylation of Gαz inhibited its response to Gz GAP by decreasing both its affinity for the GAP and the maximal rate of hydrolysis of the GAP–Gαz-GTP complex. The dependence of GAP activity on the concentration of palmitoylated Gαz was biphasic (Fig. 2A). The first phase indicates the presence of about 10% residual nonpalmitoylated Gαz-GTP, which displays unaltered substrate kinetics. The second phase reveals that palmitoylated Gαz-GTP has both a 7- to 15-fold increase in Michaelis constant (K m) combined with a 50 to 80% decrease in maximum V(V max). Essentially identical results were obtained in two other experiments with separately palmitoylated batches of Gαz. The precision of the fit for the palmitoylated component is limited because its concentration could not be increased above the high apparent K m. To confirm the effect of palmitoylation on the affinity of Gαz for the GAP, we measured the ability of palmitoyl–Gαz–GTP-γ-S to compete with nonpalmitoylated Gαz-GTP in a standard GAP assay (Fig. 2B). The data were again biphasic. They indicate that palmitoylation of Gαz was 92% complete and that palmitoyl–Gαz–GTP-γ-S bound to the GAP with an inhibition constant (K i) of about 75 nM, about 30-fold greater than that of the nonpalmitoylated protein.

Treatment of palmitoylated Gαz with 15 mM DTT restored Gz GAP activity to more than 85% of that displayed with Gαz that had not been palmitoylated (Table 1). Restoration of activity is consistent with the removal of more than 90% of the palmitate from Gαz by the identical treatment (Fig. 1). Treatment with DTT also restored the affinity of Gαz for the GAP (22). Both these effects of DTT on palmitoylated Gαz required prolonged incubation at 30°C and were not observed if DTT was simply added to the GAP assay. DTT treatment had no effect on the basal rate of hydrolysis of Gαz-bound GTP with or without palmitate, but usually slightly enhanced GAP-stimulated hydrolysis by nonpalmitoylated Gαz (<10%; Table 1) (23).

Gz GAP is a member of the RGS protein family (16), many of which have GAP activity toward members of the Gi and Gq families (17, 24, 25). Palmitoylation of other Gα subunits also inhibited their responses to the GAP activities of RGS proteins (Table2). The extent of inhibition depended on which Gα was used in the assay. RGS proteins are selective among individual members of the Gαi and Gαqfamilies, including Gαz (24, 25), and fractional blockade of GAP activity by Gα palmitoylation was generally greatest when a GAP was assayed with a good Gα-GTP substrate. For example, GAIP (26) displays somewhat lower GAP activity toward Gαz than does brain GzGAP, and palmitoylation of Gαz inhibited the GAP activity of GAIP by about 45% (corrected for substoichiometric palmitoylation). RGS4 (27) and RGS10 (28) are much less active on Gαz, and their GAP activities were inhibited by only about 20%. When RGS4 was assayed with Gαi1 as substrate, however, its activity was inhibited by 65 to 70%. For each RGS protein and Gα substrate, inhibition was reversed by removal of palmitate by DTT. These data suggest that palmitoylation is a general mechanism for protecting GTP- activated G-proteins against GAP-accelerated deactivation.

Table 2

Inhibited response to GAIP and RGS4 after palmitoylation of Gα subunits. GAP activity was assayed as described (15, 20) with either 2.2 nM Gαz–[γ-32P]GTP or 5 nM Gαi1-[γ-32P]GTP as substrate, either palmitoylated or not. The data show percent inhibition by palmitoylation relative to parallel assays with a nonpalmitoylated Gα control at the same concentration. For assays using Gαzsubstrate, the specific activity of each RGS protein is given in standard units (15), with molar amounts of each GAP calculated according to total protein. The maximum specific activity of purified RGS10 was somewhat higher than that of the preparation used for these experiments. For RGS4 and Gαi1, the assay underestimates GAP activity for kinetic reasons (15, 25), and inhibition of RGS4 by palmitoylation of the Gαi1-GTP substrate is therefore also underestimated. Assays contained 0.15 nM Gz GAP or GAIP, 6 nM RGS10 or either 1.5 nM or 1.0 nM RGS4 (for Gαz or Gαi1, respectively). GAIP (26), RGS4 (27), and RGS10 (28), all His6-tagged, were purified fromE. coli (25). Preliminary data indicate that GAIP expressed in E. coli, Sf9 cells, or HEK 293 cells displays similar enzymatic properties. Data for Gαi1 are corrected for substoichiometric palmitoylation: 70 and 75% in the experiments from which data were averaged. Data for Gαzare not corrected, but palmitoylation of Gαz is usually ≥90%. Data are averages of at least two experiments, with duplicate determinations.

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Inhibition of the response to GAPs by palmitoylation is apparently highly specific, not simply the result of increased NH2-terminal hydrophobicity of the Gα substrate (29). In contrast to palmitoylation, myristoylation of the NH2-terminal amine of Gαz enhanced Gz GAP activity. Although myristoylation had no effect on the basal rate of hydrolysis of Gz-bound GTP (22), the response to Gz GAP of 2.2 nM nonmyristoylated Gαz-GTP, purified from Escherichia coli, was only 31% that of myristoylated Gαz. This difference primarily reflects the lower affinity of nonmyristoylated Gαz for Gz GAP, reflected in an increase in K d of three- to fivefold relative to myristoylated Gαz (Fig. 2B). A similar but smaller difference was obtained with RGS4; the GAP accelerated rate for unmodified Gαz was 76% that of myristoyl-Gαz. Myristoylation of Gαi1 and Gαo also increased their affinities for GzGAP (15). Because autopalmitoylation of α subunits requires prior NH2-terminal myristoylation (18), we were unable to determine the effect of palmitoylation of nonmyristoylated Gαz on its activity as a GAP substrate. Regardless, although palmitoylation of Cys3 markedly inhibits the binding of Gαz to Gz GAP, myristoylation enhances affinity for the GAP.

Because both palmitoylation and myristoylation occur near the NH2-terminus, we examined the interaction of GzGAP with Gαz from which the NH2-terminal α helix was removed by tryptic cleavage at Arg29 (Fig. 1). NH2-terminal truncation of Gαz essentially abolished GAP activity (Table 1 and Fig. 2). Remaining activity was largely accounted for by incomplete proteolysis, because incubation of trypsin-treated Gαz with palmitoyl–coenzyme A (Pal-CoA) further inhibited the low residual GAP activity by about 50% (Table 1). NH2-terminal proteolysis of Gαi1also inhibited its response to the GAP activity of RGS4 (22). As was the case for palmitoyl-Gαz, the insensitivity of proteolyzed Gαz to Gz GAP reflected a grossly diminished affinity (K d>100 nM; Fig. 2A). Proteolysis reproducibly increased the intrinsic rate at which Gαz hydrolyzed bound GTP by two- to threefold (Table 1).

Because three different NH2-terminal modifications of Gαz and Gαi1—palmitoylation, myristoylation, and proteolysis—all modulate their responses to the GAP activities of several RGS proteins, this region of Gα subunits is apparently crucial for RGS protein recognition. However, no contact between RGS4 and the NH2-terminus of its Gαi1substrate was observed in a crystal structure of the RGS4-Gαi1 complex (30). The NH2-terminus did contact an adjacent RGS4 molecule, but this was judged to be an artifact of crystal packing and it is unlikely that relevant contact could take place even if the NH2-terminus were freed of packing constraints when in solution. The Gα NH2-terminus may regulate sensitivity to GAPs but not lie at the protein interface. Alternatively, because only the central portion of RGS4 was defined in the crystallographic structure of the complex (30), it is also possible that the Gα NH2-terminus binds to the RGS protein outside of its central, conserved domain (17). Such an interaction is consistent with the idea that unconserved regions of RGS proteins are important for the specificity of their interactions with G proteins and is also consistent with our finding that palmitoylation inhibits most strongly when a GAP is assayed with a preferred Gα target.

Because inhibition of the action of RGS proteins by palmitoylation of their Gα substrates correlates roughly with GAP activity and can be virtually complete for a specific RGS-Gα pair, palmitoylation has the capacity to totally inhibit the GAP activity of RGS proteins for their correct cellular targets. Thus, the palmitoylation-depalmitoylation cycle may control both the signal amplitude and the temporal response in G-protein pathways. Palmitoylation can amplify G protein–mediated signals or, alternatively, regulated depalmitoylation could serve as either an off-switch or signal attenuator. Such controls may be G protein–specific, and their complete elucidation awaits better understanding of the control of palmitate addition and removal. Regardless, any of these mechanisms would be compatible with the enhanced binding of palmitoylated Gα to Gβγ (7), which would serve to lower background signaling in the absence of stimulation. Regulation of GAP activity may be a major function of Gα palmitoylation.

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