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J. Biol. Chem., Vol. 275, Issue 31, 23516-23522, August 4, 2000

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Regulation of G_i Palmitoylation by Activation of the 5-Hydroxytryptamine-1A Receptor^*

Catherine A. Chen and David R. Manning

From the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084

Received for publication, April 21, 2000, and in revised form, May 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nearly all  subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated at cysteine residues near the N terminus. A regulated cycle of palmitoylation could provide a mechanism for modulating G protein signaling by affecting protein interactions and localization of the subunit. In the present studies we utilized both [³H]palmitate incorporation and pulse-chase techniques to address the dynamics of _i palmitoylation in Chinese hamster ovary cells. Both techniques demonstrated a dose- and time-dependent change in [³H]palmitate labeling of _i upon activation of stably expressed 5-hydroxytryptamine-1A receptors by the agonist (+/)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (DPAT), with an EC₅₀ of ~10 nM. For the incorporation assay, DPAT elicited an approximate doubling in labeling at the earliest time point measured. For the pulse-chase assay, DPAT promoted a significant loss of radiolabel almost equally as fast. These data demonstrate that the exchange of palmitate on _i is increased upon stimulation of 5-hydroxytryptamine-1A receptors through the combined processes of depalmitoylation and palmitoylation. These results provide the basis for extending the concept of regulated exchange of palmitate beyond G_s and provide a framework for exploring the specific functional attributes of the palmitoylated and depalmitoylated forms of subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)¹ transduce signals from cell-surface receptors to intracellular effectors via multiple pathways. The phenomenon of signal transduction is relevant to both normal and abnormal physiological processes, prompting significant advances toward understanding how signal amplitude and duration are regulated at the level of receptor, G protein, and effector. Modulation of signaling has been suggested to occur through direct protein interactions (1) and by dynamic modifications that include phosphorylation (2, 3) and palmitoylation (4-9).

Palmitoylation is a reversible post-translational modification that links the fatty acid palmitate to cysteine residues by a thioester bond. Nearly all  subunits of the four G protein families, G_s, G_i, G_q, and G₁₂, are palmitoylated at cysteine residues near the N terminus (10-14). G_i family  subunits, which are palmitoylated at Cys³, are also co-translationally N-myristoylated at Gly² via an irreversible amide bond (9). Protein-bound palmitate has widespread functional significance, influencing protein interactions and membrane localization for G protein  subunits and other proteins including G protein-coupled receptors and kinases (15-17), Ras proteins (18-20), nonreceptor tyrosine kinases (21-23), and endothelial nitric-oxide synthase (24, 25).

For the G_i family  subunits, palmitoylation decreases the affinity of _i and _z for the regulators of G protein signaling (RGS) proteins RGS4, GAIP, and G_z GAP, and decreases the maximal rate of GTP hydrolysis of the  subunit-RGS protein complex (26). Cys³ mutations, which abrogate palmitoylation, have been reported variously to enhance D₂ receptor-mediated inhibition of adenylyl cyclase by _z (27), to have no effect on D₂ receptor-mediated activation of mitogen-activated protein kinase by _z (28), and to inhibit coupling of the _2A-adrenoreceptor to _i (29). Cys³ mutations also decrease the extent of subunit association with membrane (12, 27), which for _i (30) and _z (28) is rescued by overexpression of . It has been proposed that palmitoylation of  subunits maintains membrane association as part of a dual signal (28) bilayer-trapping mechanism (31) similar to that proposed for acylated nonreceptor tyrosine kinases (32) and Ras proteins (20). Furthermore, palmitoylation may maintain  subunits specifically at the plasma membrane and quite possibly in specific subdomains (33, 34) because palmitoylation-deficient _z mutants mistarget to internal membranes (28), as do nonpalmitoylated Gpa1 (yeast  subunit) (35), fyn (22), and Ha-Ras (20).

Regulated palmitoylation could provide a mechanism for modulating G protein signaling by allowing for changes in protein interactions and localization of the  subunit. Despite rigorous efforts, very little is known about the potential palmitoyltransferase (36-38) and palmitoylesterase (39, 40) enzymes that are specific for G protein  subunits; characterization of enzyme regulation awaits successful purification and/or cloning of these proteins. Recent progress toward this end has been made by the identification and purification of a cytoplasmic acyl-protein thioesterase that depalmitoylates G protein  subunits in vitro and in vivo (41). With respect to the addition of palmitate, no thiotransferase enzyme specific for G protein  subunits has been purified to homogeneity, and it is currently unclear as to whether this reaction is an enzymatic or nonenzymatic process (42).

Superimposed on the potential regulation of enzymes that catalyze turnover of palmitate is the possibility that palmitoylation is modulated by the activation state or subcellular location of the  subunit. It has been shown that the palmitoylation/depalmitoylation cycle is activation-dependent for _s (43-45), the ₂-adrenergic receptor (46), and endothelial nitric-oxide synthase (47). An increase in depalmitoylation of _s, as measured by the release of [³H]palmitate in pulse-chase assays, was demonstrated upon activation of G_s through the ₂-adrenergic receptor (43, 44) and by inhibition of GTPase activity by mutagenesis (43). An increase in [³H]palmitate incorporation was also demonstrated following activation of G_s through the ₂-adrenergic receptor (43-45) and cholera toxin (45), presumably due in part to an increase in depalmitoylation, facilitating an exchange of palmitate for [³H]palmitate. Although regulated palmitoylation/depalmitoylation has been clearly demonstrated for the  subunit of G_s, regulated palmitoylation of  subunits belonging to the G_i, G_q, and G₁₂ classes has only begun to be addressed. Recent reports have shown an increase in incorporation of [³H]palmitate for _q/11 (48) and _i (49) upon gonadotropin-releasing hormone receptor activation in pituitary cells; for _q, _i, and _o upon serotonin receptor activation in rat brain membranes in vitro (50); and for _q and _o upon -adrenergic receptor activation in aortic membranes in vitro (51). The concept of palmitate exchange, however, has not been carefully evaluated in an intact cell setting.

In the present studies we have utilized both [³H]palmitate incorporation and pulse-chase techniques to address the dynamics of _i palmitoylation. We demonstrate specifically that the exchange of palmitate on _i is increased upon stimulation of 5-hydroxytryptamine-1A (5-HT_1A) receptors through the combined processes of depalmitoylation and palmitoylation. Our results provide evidence for a regulated cycling of palmitate and provide the basis for extending the concept of regulated exchange beyond the G_s family. The rapid changes in palmitoylation/depalmitoylation suggest a role in transduction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Reagents and supplies were obtained as follows: F-12 nutrient mixture (HAM F-12), Dulbecco's modified eagle medium, Dulbecco's phosphate-buffered saline (CaCl₂-free and MgCl₂-free), and Geneticin^® were from Life Technologies, Inc.; fetal bovine serum was from HyClone Laboratories Inc. (Logan, UT); leupeptin and aprotinin were from Roche Molecular Biochemicals; (+/)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide or 8-OH-DPAT (DPAT), 4-iodo-N-(2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (MPPI), and pertussis toxin (PTX) were from Research Biochemicals International (Natick, MA); fatty acid-free bovine albumin (BSA), cycloheximide, and unlabeled palmitate were from Sigma; [9,10-³H]palmitic acid ([³H]palmitate) and [9,10-³H]myristic acid ([³H]myristate) were from American Radiolabeled Chemicals (St. Louis, MO); [³⁵S]translabel (nominally referred to as [³⁵S]methionine) was from ICN Biomedicals, Inc. (Costa Mesa, CA); enhanced chemiluminescence ECL^TM (ECL) kit, Amplify^TM, Hyperfilm^TM MP, and Sensitize^TM preflash were from Amersham Pharmacia Biotech. DPAT, PTX, and cycloheximide were dissolved in H₂O. MPPI and unlabeled palmitate were dissolved in dimethyl sulfoxide (Me₂SO). [³H]palmitate and [³H]myristate were dried and resuspended in Me₂SO (final of 1% Me₂SO when added to cells). Rabbit antisera 8729 (_i1~_i2>_i3) and 1190 (_s), generated against C-terminal keyhole limpet hemocyanin-conjugated peptides, have been described (52, 53); rabbit nonimmune serum was obtained from Sigma.

Cell Culture-- Chinese hamster ovary (CHO) cells expressing the human 5-HT_1A receptor (XbaI-BamHI restriction fragment of G21 (54) in pcDNA1/neo) were provided as a gift from Dr. Perry B. Molinoff. Cells were maintained in monolayer culture in HAM F-12 with L-glutamine supplemented with 10% charcoal-treated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml Geneticin^® at 37 °C in a humidified atmosphere of 5% CO₂. Single 100-mm plates were used for each treatment. Cells were split 2-4 days prior to each experiment and grown to 70-100% confluency.

Radiolabeling-- For [³H]palmitate labeling, cells were incubated with [³H]palmitate (500 µCi/ml in 3 ml, 60 Ci/mmol) at 37 °C in HAM F-12 supplemented with penicillin/streptomycin and 3.6 mg/ml fatty acid-free BSA for 5-120 min (incorporation experiments) or for 3-4 h (pulse-chase experiments). For pulse-chase experiments, cells were subsequently washed once with HAM F-12 and incubated for 5-180 min at 37 °C in HAM F-12 supplemented with penicillin/streptomycin, 3.6 mg/ml fatty acid-free BSA, and 100 µM unlabeled palmitate. For [³H]myristate labeling, cells were incubated with [³H]myristate (300 µCi/ml in 3 ml, 60 Ci/mmol) for 6-7 h at 37° C in HAM F-12 supplemented with penicillin/streptomycin and 3.6 mg/ml fatty acid-free BSA. For [³⁵S]methionine labeling, cells were incubated with [³⁵S]methionine (50 µCi/ml in 3 ml, ~1400 Ci/mmol) for 30 min at 37 °C in L-methionine-free Dulbecco's modified Eagle's medium containing glucose and supplemented with penicillin/streptomycin, 3.6 mg/ml fatty acid-free BSA, and 0.584 mg/ml glutamine.

Cell Fractionation-- Plates were placed on ice, the medium was aspirated, and the cells were washed twice with ice-cold Dulbecco's phosphate-buffered saline followed by harvesting with a cell scraper into 500 µl of ice-cold 20 mM HEPES, pH 7.2, 2 mM MgCl₂, 1 mM EDTA, pH 7.2, 2 µg/ml aprotinin, and 2 µg/ml leupeptin (HME/PI). After 5 min on ice, cells were quick frozen in dry ice/EtOH and stored at 80 °C until use. Following quick thawing, cells were homogenized by 15 passes through a 26-gauge needle/1-cc syringe on ice. Homogenates were centrifuged at 1000 × g for 5 min at 4 °C to pellet nuclei and unbroken cells. The supernatant was centrifuged at 100,000 × g for 60 min at 4 °C to pellet membranes. The pellet was washed twice and resuspended in 500 µl of HME/PI by 10 passes through a 26-gauge needle/1-cc syringe on ice.

Immunoprecipitation-- The resuspended membrane fraction was combined with an equal volume (500 µl) of 100 mM NaPO₄, pH 7.2, 2% deoxycholate, 2% Triton X-100, 1% SDS, 300 mM NaCl, 4 mM EDTA, pH 7.2, 4 µg/ml aprotinin, and 4 µg/ml leupeptin (2× RIPA) and solubilized for 60 min on ice. Samples were "precleared" by incubation with protein A-Sepharose and rabbit nonimmune serum (1:500) for 60 min at 4 °C followed by centrifugation at 10,000 × g for 5 min at 4 °C. _i or _s was immunoprecipitated from the supernatant by incubation with protein A-Sepharose and _i- or _s-directed antisera (1:100) overnight at 4 °C. Immunoprecipitates were collected by centrifugation at 10,000 × g for 5 min at 4 °C. Pellets were washed three times with 50 mM NaPO₄, pH 7.2, 0.5% Triton X-100, 150 mM NaCl, and 2 mM EDTA, pH 7.2, and residual buffer was removed with a 26-gauge needle/1-cc syringe. Pellets were incubated for 1 min at 75 °C in 100 mM Tris, pH 6.8, 2% SDS, 20 mM dithiothreitol, 10% glycerol, and bromphenol blue and then loaded onto SDS-polyacrylamide gels.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Fluorography, and Densitometry-- Samples and prestained molecular weight markers were electrophoresed on 9% SDS-polyacrylamide gels. Gels were stained with 50% methanol, 10% acetic acid, and 0.25% Coomassie^® Blue R-250 and destained with 30% methanol and 10% acetic acid followed by 10% acetic acid alone. For ³H experiments, gels were subsequently incubated in Amplify^TM for 30 min at room temperature. For all experiments, gels were then dried at 80 °C for 60 min, exposed to preflashed Hyperfilm^TM (³H experiments at 80 °C, ³⁵S experiments at room temperature), and quantified by densitometry using multiple exposures and comparisons to standard curves to verify that all bands were within film linear range.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To address the hypothesis that activation of a G_i-coupled receptor induces changes in palmitoylation of _i, we carried out experiments using CHO cells stably expressing the 5-HT_1A receptor. The pharmacological properties of the 5-HT_1A receptor have been extensively characterized, and it is quite clear from direct measurements of G protein activation (55) and PTX sensitivity of effector regulation that the 5-HT_1A receptor couples primarily to members of the G_i family. The CHO cells used here contain endogenous _i but not _o or _z as determined by Western blotting (56). Receptor levels were ~3 pmol/mg as determined by Scatchard analysis of [¹²⁵I]p-MPPI saturation binding (57).

To evaluate the possibility of an agonist-promoted incorporation of palmitate into _i, CHO cells were incubated for 60 min with [³H]palmitate in the absence or presence of increasing concentrations of DPAT, a 5-HT_1A receptor-selective agonist (58). Incorporation of radiolabel was assessed by immunoprecipitation of _i from subsequently prepared membranes followed by SDS-PAGE and fluorography. As shown in Fig. 1, DPAT elicited a dose-dependent increase in radiolabel incorporation into _i; the EC₅₀ for DPAT was ~10⁸ M. DPAT was used at 1 µM in all subsequent studies.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   DPAT increases incorporation of [3H]palmitate into i in a dose-dependent manner. CHO cells stably expressing 5-HT1A receptors were incubated with [3H]palmitate for 60 min in the presence of vehicle (H2O) (open circle) or increasing concentrations of DPAT (closed circle). Incorporation of radiolabel was assessed by immunoprecipitation of i from subsequently prepared membranes followed by SDS-PAGE/fluorography (inset) and densitometry. A representative of two independent experiments is shown.

If regulated palmitoylation plays a role in signal transduction, we predicted that it would be evident at time points that correlate with effector regulation. CHO cells were incubated for 5, 30, 60, and 120 min with [³H]palmitate in the absence and presence of DPAT to evaluate the time course of [³H]palmitate incorporation into _i. As shown in Fig. 2A, incorporation of radiolabel increased over time in the absence of agonist (vehicle), representing "basal" turnover of palmitate. DPAT resulted in an increase in radiolabel incorporation at all time points. The data are expressed as percent change in Fig. 2B. Here, DPAT is seen to elicit the maximal difference beginning at the earliest measurable time point (5 min); this difference was sustained up to 60 min. The narrowing between stimulated and unstimulated values by 120 min probably represents an approach to equilibrium. Control experiments in which cells were pretreated with DPAT for 120 min prior to labeling excluded the possibilities that DPAT instability or 5-HT_1A receptor desensitization accounted for the converging values.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   DPAT-promoted incorporation of [3H]palmitate into i is inhibited by MPPI and PTX. CHO cells stably expressing 5-HT1A receptors were incubated with [3H]palmitate for 5-120 min in the presence of vehicle (H2O plus Me2SO), 1 µM DPAT, 1 µM DPAT plus 10 µM MPPI (30-90-min MPPI pretreatment), or 1 µM DPAT plus 500 ng/ml PTX (7-h PTX pretreatment). Incorporation of radiolabel was assessed by immunoprecipitation of i from subsequently prepared membranes followed by SDS-PAGE/fluorography and densitometry. A representative experiment is shown in A and percentage change (versus vehicle for DPAT and DPAT/MPPI; versus PTX alone for PTX/DPAT) in B as an average ± S.E., where n = 4-7 independent experiments for DPAT, n = 3 for DPAT/MPPI, and n = 3 for DPAT/PTX. * indicates p < 0.05, and ** indicates p < 0.01 as compared with DPAT by two-way ANOVA (analysis of variance) followed by Fisher's PLSD post hoc test.

To establish the specificity of the agonist-promoted incorporation of [³H]palmitate into _i, CHO cells were incubated for varying lengths of time with [³H]palmitate in the presence of DPAT and MPPI, a 5-HT_1A receptor-selective antagonist (59), or DPAT and PTX, a bacterial toxin that ADP ribosylates _i in the heterotrimeric form and prevents receptor-G protein coupling. MPPI blocked the increase in incorporation of radiolabel elicited by DPAT at all time points (Fig. 2A). MPPI alone resulted in a small decrease in incorporation (data not shown), which may have resulted from its weak inverse-agonist activity or from inhibition of residual serotonin activity in the charcoal-treated serum. PTX similarly inhibited the increase in incorporation of radiolabel elicited by DPAT, suggesting that receptor-G protein coupling is important for regulated palmitate turnover. Of note, when cells were treated with PTX alone, radiolabel incorporation was reduced below basal for _i, though not _s (data not shown), suggesting that receptor-G protein coupling also contributes to basal palmitate turnover itself. Labeling was nevertheless observed to some extent despite the complete ADP ribosylation of _i (as indicated by a shift in electrophoretic mobility), therefore factors beyond receptor-G protein coupling likely contribute to basal turnover. Both MPPI and PTX significantly reduced the percent change elicited by DPAT (Fig. 2B). Taken together, these data demonstrate that the agonist-dependent increase in palmitate turnover on _i is because of 5-HT_1A receptor activation and is dependent on receptor-G protein coupling.

As shown in Fig. 3, several controls attested to the specificity of [³H]palmitate incorporation. No signal was detected when pre-immune antibody was used instead of the _i-directed antibody (Fig. 3A, lanes 1 and 2). There was no significant change in incorporation of radiolabel into membrane proteins other than _i (data not shown) or into immunoprecipitated _s (lanes 10 and 11), indicating that DPAT does not alter equilibration of radiolabel within available intracellular pools. DPAT did not increase _i synthesis as measured by [³⁵S]methionine labeling (lanes 7 and 9), nor did it alter the amount of _i in the immunoprecipitate as determined by immunoblotting (data not shown). To further exclude the possibility of an increase in _i synthesis, we tested whether the increase in incorporation of [³H]palmitate was evident in the absence of protein synthesis. Cycloheximide was sufficient to block protein synthesis as measured by [³⁵S]methionine (lanes 7 and 8) but did not affect the increase in _i radiolabeling promoted by DPAT (lanes 5 and 6). These data indicate that de novo palmitoylation of newly synthesized _i was not a contributing factor, and further that 5-HT_1A receptor up-regulation (57) did not account for our observations.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   DPAT-promoted incorporation of [3H]palmitate into i does not require protein synthesis and occurs via a thioester bond. CHO cells stably expressing 5-HT1A receptors were incubated with [3H]palmitate for 30 min, [35S]methionine for 30 min, or [3H]myristate for 7 h in the presence of vehicle (H2O), 1 µM DPAT, or 1 µM DPAT plus 50 µg/ml cycloheximide (30-min cycloheximide pretreatment). Incorporation of radiolabel was assessed by immunoprecipitation of i (except A, lanes 1 and 2, where preimmune serum was added and lanes 10 and 11, where s antiserum was added) from subsequently prepared membranes followed by SDS-PAGE and fluorography or autoradiography. In B, gels were incubated overnight in either 1 M Tris-HCl, pH 7.0, or 1 M hydroxylamine-KOH, pH 7.0, prior to fluorography as indicated. A representative of three independent experiments is shown.

The cycloheximide data also suggest that the increase in _i labeling promoted by DPAT is not because of an increase in co-translational myristoylation of newly synthesized proteins with [³H]myristate metabolized from [³H]palmitate. To confirm that our measurements were reflective of palmitoylation, gels containing immunoprecipitated labeled _i were treated with hydroxylamine. The incorporation of label was almost completely sensitive to hydroxylamine (Fig. 3B, lanes 4 and 5), which indicates fatty acid linkage via a thioester bond and is consistent with palmitate incorporation. As a negative control, [³H]myristate, which is linked via an amide bond to _i, was resistant to hydroxylamine as expected (lanes 3 and 6).

We went on to evaluate [³H]palmitate release from _i by pulse-chase techniques to ascertain whether the increase in [³H]palmitate incorporation in fact represented turnover of palmitate. CHO cells were incubated with [³H]palmitate for 4 h (pulse) followed by a 2-h incubation with 100 µM unlabeled palmitate in the absence or presence of increasing concentrations of DPAT (chase). The remaining [³H]palmitate was assessed by immunoprecipitation of _i from membranes followed by SDS-PAGE and fluorography. As presented in Fig. 4, DPAT caused a dose-dependent enhancement of release of radiolabel from _i. The EC₅₀ for DPAT was ~10⁸ M, similar to that obtained from the incorporation assay.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   DPAT increases release of [3H]palmitate from prelabeled i in a dose-dependent manner. CHO cells stably expressing 5-HT1A receptors were incubated with [3H]palmitate for 4 h (pulse) followed by a 2-h chase with 100 µM unlabeled palmitate in the presence of vehicle (H2O) (open circle) or increasing concentrations of DPAT (closed circle). Remaining radiolabel was assessed by immunoprecipitation of i from subsequently prepared membranes followed by SDS-PAGE/fluorography (inset) and densitometry. A representative of two independent experiments is shown.

To determine the time course and specificity of the agonist-promoted release of [³H]palmitate, CHO cells were incubated with [³H]palmitate for 3-4 h followed by incubation with 100 µM unlabeled palmitate in the presence of either DPAT or DPAT plus MPPI for varying lengths of time. As shown in Fig. 5A, in the absence of agonist (vehicle) radiolabel was released from _i over time, representative of basal depalmitoylation. DPAT promoted a greater loss of [³H]palmitate at all time points beyond 5 min. This effect was blocked by MPPI. The data are expressed as percent change versus vehicle in Fig. 5B. Here, MPPI significantly inhibited the percent change elicited by DPAT. The somewhat slower attainment of the maximal response elicited by DPAT, as compared with that measured by the [³H]palmitate incorporation experiments, was possibly due to the continued incorporation of residual [³H]palmitate at the onset of the chase. The pulse-chase experiments demonstrate an agonist-promoted depalmitoylation of _i. These data, taken together with the incorporation experiments that demonstrated an agonist-promoted incorporation of radiolabel, strongly support an increase in palmitate turnover upon _i activation.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   DPAT-promoted release of [3H]palmitate from prelabeled i is inhibited by MPPI. CHO cells stably expressing 5-HT1A receptors were incubated with [3H]palmitate for 3-4 h (pulse) followed by a 5-180-min chase with 100 µM unlabeled palmitate and vehicle (H2O plus Me2SO), 1 µM DPAT, or 1 µM DPAT plus 10 µM MPPI (30-90-min MPPI pretreatment). Remaining radiolabel was assessed by immunoprecipitation of i from subsequently prepared membranes followed by SDS-PAGE/fluorography and densitometry. A representative experiment is shown in A and percentage change versus vehicle in B as an average ± S.E. where n = 5-8 independent experiments for DPAT and n = 3-5 for DPAT/MPPI. * indicates p < 0.05, and ** indicates p < 0.01 as compared with DPAT by two-way ANOVA followed by Fisher's PLSD post hoc test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Palmitoylation of G protein  subunits is emerging as a key player in modulating subunit location and subunit-protein interactions. The roles of palmitoylation have been investigated mainly by a loss of function approach involving subunits that lack the cysteine acceptor site for palmitate or subunits that are depalmitoylated by chemical or enzymatic means. However, the regulation of _i palmitoylation in an activation-dependent manner has not been critically assessed. We demonstrate here that palmitoylation of _i is subject to regulation through the 5-HT_1A receptor and that this regulation specifically entails an enhanced exchange of palmitate through the combined processes of depalmitoylation and palmitoylation. These data provide the basis for extending the concept of regulated exchange of palmitate beyond G_s and provide a framework for exploring the specific functional attributes of the palmitoylated and depalmitoylated forms of subunit.

Incorporation and pulse-chase experiments demonstrated a dose- and time-dependent change in [³H]palmitate labeling of _i upon activation of the 5-HT_1A receptor by DPAT. For both types of experiments, the EC₅₀ for DPAT was ~10 nM and maximal changes were achieved by 1 µM. These values are consistent with those previously reported for effector regulation through the 5-HT_1A receptor (56, 60, 61). For the incorporation assay, DPAT elicited an increase in labeling with the maximal percent change evident by 5 min. For the pulse-chase assay, DPAT promoted a loss of radiolabel with the maximal percent change evident by 120 min. The changes in labeling of _i were specific to receptor stimulation, as they were inhibited by the antagonist MPPI. They were additionally specific to receptor-G_i coupling, as they were inhibited by PTX. Nonspecific labeling, changes in _i synthesis or immunoprecipitation, and alterations in radioligand uptake were excluded. The increase in labeling did not represent co-translational myristoylation or compensatory up-regulation of the 5-HT_1A receptor. Sensitivity to hydroxylamine confirmed the nature of palmitate attachment as a thioester bond.

The combined use of incorporation and pulse-chase techniques was important. The incorporation assay does not distinguish an enhanced exchange of palmitate from a net increase in palmitate. Nor does the pulse-chase assay distinguish an enhanced exchange from a net decrease. Detection of agonist-promoted changes by both assays, however, supports an enhanced exchange. These observations nevertheless do not preclude a simultaneous change in stoichiometry. For _s, however, the stoichiometry was reported to be unaffected by agonist (62).

Our results parallel previous observations of an increased turnover of palmitate on _s upon activation through the ₂-adrenergic receptor (43, 44) and by inhibition of GTPase activity by mutagenesis (43). Also consistent with our results are recent reports of increases in [³H]palmitate incorporation for _q/11 (48), _s, and _i (49) upon gonadotropin-releasing hormone receptor activation in pituitary cells. The changes in these cells, however, may have been attributable to an increase in subunit synthesis, as demonstrated subsequently for _q/11 in gonadotropes (63). Increases in [³H]palmitate incorporation have also been observed for _q, _s, _i, and _o upon incubation of rat brain membranes with serotonin in vitro (50), and for _q and _o upon incubation of aortic membranes with phenylephrine in vitro (51); however, neither study addressed the dynamics of palmitoylation in intact cells. In studies pertaining to _i, specificity, de novo palmitoylation, and depalmitoylation were not directly evaluated. Agonist stimulation of the D₂ dopamine receptor in CHO-K1 cells did not promote depalmitoylation of wild-type _z, another member of the G_i family (28). Differences in receptor expression and coupling efficiencies may contribute to the differences in results, or regulated palmitate turnover may not occur for all G_i family  subunits.

The status of  subunit palmitoylation may contribute positively or negatively to signal transduction. Recent data for G_i family  subunits suggest an interplay between palmitoylation and the GTPase activity promoted by RGS proteins. Palmitoylation of _i1 inhibits the affinity of RGS4 for _i1, as well as the GAP activity for the subunit; similar results were obtained with GAIP and _i and with G_z GAP and _z (26). Data for _s subunits suggest that palmitoylation contributes to subunit distribution between plasma membrane and cytosol. A temporal correlation, for example, between activation-dependent depalmitoylation (43) and increases in the soluble/cytosolic subunit (64, 65) has been demonstrated. Consistent with these data, nonpalmitoylated _s has a reduced hydrophobicity and a reduced affinity for in vitro (66), and palmitoylation-defective (C3S) (65, 67) and constitutively active (R201C) (64, 65) _s mutants are localized to cytosol; in contrast however, other mutants maintain membrane association (10, 68), and activation-dependent changes in solubility are not always detected (62). For _i, membrane association is maintained for activated subunits (68), similar to that seen for _o (69) and _z (27), and depalmitoylation of _i by acyl-protein thioesterase does not alter cytosolic levels (68). These data suggest that _i may remain at the plasma membrane upon depalmitoylation but do not preclude alterations in localization of the subunit to microdomains, which may function to attenuate or promote signaling. Several investigators have reported that G proteins are concentrated in caveolin-enriched domains (33, 34), as well as other subdomains with low buoyant density and distinct immunofluorescence patterns (68, 70).

Possible mechanisms for activation-dependent depalmitoylation may involve changes in  subunit conformation or accessibility upon binding or releasing or guanine nucleotide. The current model favors an increase in susceptibility to a constitutively active palmitoylesterase upon dissociation of  from (41, 43). In vitro biochemical data support this model in that protects _s-GDP but not _s-GTP from depalmitoylation by a recombinant esterase (66). Moreover, heterotrimeric G that is purified (41) or present in cell extracts (43) exhibits an increase in depalmitoylation upon AlF₄ activation. Monomeric _s-GTP and monomeric _s-GDP are depalmitoylated at the same rate in vitro, suggesting that the nucleotide-dependent changes in  subunit conformation are not contributing factors (66). Despite compelling evidence for this model, these data do not preclude the possibility of additional regulation of palmitoylation downstream of  and dissociation (71).

	FOOTNOTES

^* These studies were supported by National Institutes of Health Grants GM53156 and MH14654.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-1775; Fax: 215-573-2236; E-mail: manning@pharm.med.upenn.edu.

Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.M003439200

	ABBREVIATIONS

The abbreviations used are: G proteins, heterotrimeric GTP-binding regulatory proteins; RGS, regulators of G protein signaling; 5-HT_1A, 5-hydroxytryptamine-1A; HAM F-12, F-12 nutrient mixture; Me₂SO, dimethyl sulfoxide; DPAT, (+/)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide or 8-OH-DPAT; MPPI, 4-iodo- N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride; PTX, pertussis toxin; BSA, bovine albumin; CHO, Chinese hamster ovary; ECL, enhanced chemiluminescence; [³H]palmitate, [9,10-³H]palmitic acid; [³H]myristate, [9,10-³H]myristic acid; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

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