The 5-Hydroxytryptamine(1A) Receptor Is Stably Palmitoylated, and Acylation Is Critical for Communication of Receptor with G i Protein*

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT 1A ) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT 1A receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT 1A receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G i protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G i protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G (cid:1)

functions of the central nervous system as well as the periphery by activating a large family of receptors. With the exception of the 5-HT 3 receptor, which is a transmitter-gated Na ϩ /K ϩ channel, all other 5-HT receptors belong to a large family of receptors that are coupled to different intracellular effectors via heterotrimeric guanine nucleotide-binding proteins (G proteins) (1,2). Structurally, G protein-coupled receptors (GPCRs) possess seven transmembrane domains linked by alternating intracellular (i1-i3) and extracellular (e1-e4) loops. The extracellular receptor surface, including the N terminus, is known to be critically involved in ligand binding. The intracellular receptor surface, including the C-terminal domain and intracellular loops (in particular i2 and i3), is known to be important for G protein recognition and activation (3).
The 5-HT 1A receptor is the most extensively characterized 5-HT receptor. This receptor is coupled to a variety of effectors via pertussis toxin-sensitive heterotrimeric G proteins of the G i/o families (2,4,5). Receptor-dependent activation of G␣ i subunits results in the inhibition of adenylate cyclase and subsequent decrease of cAMP levels in both hippocampal neurons (6,7) and different cell lines expressing the receptor (8 -10). Analysis of G protein specificity for the 5-HT 1A receptor revealed an unexpected complexity. Antisense depletion of different subtypes of the G␣ i subunit revealed that removal of G␣ i1 eliminated 5-HT 1A -induced inhibition of basal cAMP levels, whereas depletion of G␣ i2 and G␣ i3 blocked the 5-HT 1A receptor action on G s -activated adenylyl cyclase (AC) (11). Expression studies in Sf.9 insect cells have also provided the first evidence for possible post-translational modifications of the 5-HT 1A receptor (12). Besides effects mediated by G␣ i/o subunits, activation of the 5-HT 1A receptor leads to a G␤␥-mediated activation of K ϩ current and inhibition of Ca 2ϩ current in hippocampal neurons (13)(14)(15), dorsal raphe nucleus neurons (14) and atrial myocytes (16). In CHO cells, the 5-HT 1A receptor also mediates G␤␥-mediated stimulation of phospholipase C as well as activation of mitogen-activated protein kinase Erk2 (8,17). Considerable interest has been raised from pharmacological studies indicating a role for the 5-HT 1A receptor in regulating anxiety states, and the production of knock-out mice lacking this receptor has confirmed these expectations (18 -20).
The covalent attachment of fatty acids to proteins (acylation) is a widespread post-translational modification (21). Two main modes of acylation have been described: N-myristoylation and palmitoylation (S-acylation). N-myristoylation is a co-translational modification catalyzed by N-myristoyltransferase, which modifies glycine residues located within a consensus sequence at the protein N terminus via an amide linkage (22). Contrary to myristoylation, the addition of long chain fatty acids (mainly palmitic acid) is a post-translational event, which occurs through covalent linkage of palmitate via a labile thioester bond to cysteine residues. In contrast to the myristoylation, the molecular machinery responsible for palmitoylation of proteins is only poorly understood. In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed, and recent reports on protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila melanogaster provided the first glimpse of enzymes that carry out protein palmitoylation (23).
Palmitoylation is unique among lipid modifications as it can be reversible and adjustable. Among the cellular palmitoylated proteins, polypeptides involved in signal transduction e.g. GPCRs, ␣ subunits of G proteins, Ras protein, endothelial nitric-oxide synthase, adenylyl cyclase, phospholypase C, and non-receptor tyrosine kinases, are often targets for such dynamic modification (24 -26). Meanwhile it is widely accepted that repeated cycles of palmitoylation and depalmitoylation can be critically involved in regulation of different signaling processes (27)(28)(29). In the GPCRs, palmitoylation has been shown to be responsible for a wide variety of biological functions (24,27,28,30,31). For example, prevention of palmitoylation of the ␤ 2 -adrenergic receptor leads to an increase of basal receptor phosphorylation and rapid desensitization in response to agonist stimulation (32). Substitution of palmitoylated cysteine residues in the muscarinic acetylcholine m2 receptor reduces its ability to couple to the G i protein (33). We have recently shown that palmitoylation of the 5-HT 4(a) receptor is involved in the modulation of the constitutive receptor activity (34). For several GPCRs palmitoylation has been revealed to be modulated by agonist stimulation (33,35,36), whereas for the human A1 adenosine receptor, the efficacy of palmitoylation was not affected by the agonist (37). Moreover, stimulation of several GPCRs may modulate palmitoylation of receptor-coupled G proteins (38 -41).
In the present study, we demonstrate that the recombinant 5-HT 1A receptor is modified by covalently attached palmitate. Palmitoylation efficiency was not affected by agonist stimulation, and blockade of protein synthesis by cycloheximide resulted in a significant reduction of the receptor acylation. By site-directed mutagenesis, cysteine residues 417 and 420 located in the cytoplasmic C terminus were identified as acylation sites. Using acylation-deficient mutants, we also were able to verify a functional significance of 5-HT 1A receptor palmitoylation for the coupling to the G␣ i as well as with G␤␥ subunits and for the inhibition of forskolin-stimulated cAMP formation.
Site-directed mutagenesis of the epitope-tagged 5-HT 1A receptor with the substitution of serine for cysteine at position 417 and/or 420 was performed by overlap extension PCR technique using an oligonucleotide containing the mutation(s) corresponding to the above substitutions (43). The recombinant baculoviruses encoding for HA-5-HT 1A mutants were constructed, purified, and amplified as described previously (44). All mutants were verified by dideoxy DNA sequencing of the final plasmid.
Hydroxylamine Treatment and Fatty Acid Analysis-Gels containing the 5-HT 1A receptor labeled with [ 3 H]palmitic acid were fixed (10% acetic acid, 10% methanol) and treated overnight under gentle agitation with 1 M hydroxylamine (pH 7.5) or 1 M Tris (pH 7.5). Gels were then washed in water and rocked for 30 min in dimethyl sulfoxide (Me 2 SO) to wash out cleaved fatty acids before they were processed for fluorography.
For the fatty acid analysis, the [ 3 H]palmitate-labeled 5-HT 1A receptor was purified by immunoprecipitation and SDS-PAGE. The band corresponding to the receptor protein was excised, and fatty acids were cleaved by treatment of the dried gel slices with 6 N HCl for 16 h at 110°C. Fatty acids were then extracted with hexane and separated into individual fatty acid species by thin layer chromatography using acetonitrile/acetic acid (1:1, v/v) as solvent. Radiolabeled fatty acid was visualized by fluorography.
Indirect Immunofluorescence-At 48 h after infection with recombinant 5-HT 1A baculovirus or with baculovirus wild-type, Sf.9 cells grown on coverslips were fixed with paraformaldehyde (3% in PBS) for 15 min. The cells were washed three times with PBS and paraformaldehyde was quenched with 50 mM glycine for 15 min. Cells were then permeabilized with saponin and incubated for 1 h with the anti-HA antibody diluted 1:200 in PBS containing 2% bovine serum albumin. The second antibody (Alexa546 from Alexa diluted 1:1000 in PBS containing 2% bovine serum albumin) was applied for 1 h, and unbound antibodies were washed off at every step with PBS. Finally, coverslips were mounted in 90% (v/v) glycerol. Cells were monitored under a confocal laser-scan microscope LSM510 (Zeiss). Intracellular distribution of the receptors in CHO cells was analyzed as described by Ponimaskin et al. (34).
Assay for [ 35 triphosphate to different G proteins induced by stimulation of 5-HT 1A receptors was performed according to the method described previously (43). Briefly, membranes from Sf.9 cells expressing the 5-HT 1A receptor wild-type or acylation-deficient mutants and G protein ␣ subunits (G i1 , G i2 , G i3 , G s , G 12 , G 13 ) together with ␤ 1 ␥ 2 subunits were resuspended in 55 l of 50 mM Tris/HCl (pH 7.4) containing 2 mM EDTA, 100 mM NaCl, 3 mM MgCl 2 , and 1 M GDP. After adding [ 35 S]GTP␥S (1300 Ci/mmol) to a final concentration of 30 nM, samples were incubated for 5 min at 30°C in the presence or absence of 1 M 5-HT. The reaction was terminated by adding 600 l of 50 mM Tris/HCl (pH 7.5) containing 20 mM MgCl 2 , 150 mM NaCl, 0,5% Nonidet P-40, 200 g/ml aprotinin, 100 M GDP, and 100 M GTP for 30 min on ice. Samples were agitated for 1 h at 4°C after addition of 100 l of 10% suspension of protein A-Sepharose and 10 l of antibodies directed against appropriate G␣ subunits. Antibodies directed against G␣ i , G␣ s , and G␣ 13 were obtained from Santa Cruz Biotechnology. For the precipitation of G␣ 12 subunits, antibody AS1905 (43) was used. Immunoprecipitates were washed three times, boiled in 0.5 ml of 0.5% SDS, and radioactivity was measured by scintillation counting.

Assay for [ 3 H]5-HT Binding-
The membranes from Sf.9 cells expressing WT or mutated 5-HT 1A receptors were dissolved in buffer containing 20 mM Hepes (pH 8.0), 2 mM MgCl 2 , 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 2 g/ml aprotinin. The binding assay with [ 3 H]5-HT was performed as described previously (12,45). Briefly, 100 l of binding buffer containing 50 mM Tris (pH 7.7), 0.1% ascorbic acid, 20 M pargyline, and 1-250 nm of [ 3 H]5-HT was added to 20 g of the membrane fraction. Nonspecific binding was determined by addition of 100 M unlabeled 5-HT. After a 30-min incubation at 20°C, the reaction mixture was loaded on 20-m PVDF membranes (Corning, Germany) presoaked in 0.5% polyethylenimine. The membranes were washed with ice-cold binding buffer, and radioactivity was measured by scintillation counter. Data were fitted with the one-site saturation binding model by the Pharmacology module of SigmaPlot 8.02 software (46).
Cell Transfection and cAMP Assay-The 5-HT 1A receptor wild-type and acylation-deficient mutants cDNAs were cloned in pcDNA3(Ϫ) vector and transfected in NIH3T3 cells by electroporation. Cells were diluted in DMEM (10 6 cells/ml) containing 10% dialyzed fetal bovine serum (dFBS) and plated into 12-well clusters. Six hours after transfection, cells were incubated overnight in DMEM without dFBS containing 2 Ci [ 3 H]adenine/ml to label the ATP pool. Cells were washed and then incubated in 1 ml of culture medium containing 0.75 mM IBMX, 50 M forskolin plus the drugs indicated in the figure legends for 15 min at 37°C. The reaction was stopped by replacing the medium with 1 ml of ice-cold 5% trichloroacetic acid. The cAMP accumulation was measured as described previously (7). The amount of the expressed 5-HT 1A receptor was measured as described in Varrault et al. (47).
Erk2 Phosphorylation Assay-CHO cells were grown in F-12 Ham medium supplemented with 10% fetal calf serum and 1% penicillin/ streptomycin. For expression, CHO cells (0.5 ϫ 10 6 ) grown in 3.5-mm dishes were transfected with recombinant 5-HT 1A receptor using Lipo-fectAMINE2000 according to the manufacturer's protocol. Twenty hours after transfection, cells were starved in F-12 Ham medium with 2% bovine serum albumin and 1% penicillin/streptomycin for 16 h. Cells were then stimulated for 5 min with 10 M 8-OH-DPAT at 37°C under 5% CO 2 , washed with PBS and lysed in the loading buffer. Equal amounts of proteins in lysates were separated by SDS-PAGE and then subjected to Western blot. The membranes were probed either with antibodies raised against phosphorylated Erk1/2 (phospho-p42/44; 1:2000 dilution) or against total Erk (p42/44; 1:1000 dilution). To analyze the receptor expression, membranes were probed with antibodies raised against the HA epitope (1:1000). To compare the level of surface expression, binding of 5-[ 3 H]HT was measured in parallel. Amount of the phosphorylated and the total Erk1/2 were quantified by densitometric measurements using GelPro Analyser version 3.1 software. The surface expression of the wild-type and mutant receptors was adjusted to 450 -500 fmol/mg of protein, as accessed by 5-[ 3 H]HT binding. Nonspecific binding was determined in the presence of 100-fold excess of specific 5-HT 1A receptor agonist 8-OH-DPAT.

RESULTS
Expression and Palmitoylation of the 5-HT 1A Receptor-A high titer baculovirus stock containing the cDNA of the murine 5-HT 1A receptor tagged with an HA epitope at the N terminus was prepared as described under "Experimental Procedures" and used for infection of Sf.9 insect cells. In order to monitor the expression and subcellular distribution of the receptor, infected Sf.9 cells were subjected to immunofluorescence analysis (Fig. 1A). The HA-tagged 5-HT 1A receptors were specifically detected by anti-HA antibodies and localized mainly at the cell surface. Labeling with [ 35 S]methionine followed by immunoprecipitation and SDS-PAGE revealed a single protein band with a molecular mass of ϳ46 kDa (Fig. 1B, left panel). This corresponds to the predicted molecular mass of the 5-HT 1A receptor. The absence of specific bands in the immunoprecipitates from non-infected or baculovirus wild-type-infected Sf.9 cells confirmed that the 46-kDa band indeed represents the 5-HT 1A receptor.
To examine whether the 5-HT 1A receptor is acylated, Sf.9 cells infected with recombinant baculovirus were metabolically labeled with [ 3 H]palmitic acid. Such labeling revealed a single band of 46 kDa (Fig. 1B, right panel)  Having shown that the 5-HT 1A receptor is acylated, we went on to analyze the chemical nature of the fatty acid bond in order to distinguish between amide-type and ester-type fatty acid linkages. As shown in Fig. 2A, the [ 3 H]palmitate-derived radioactivity was sensitive to treatment with increasing concen-trations of ␤-mercaptoethanol. Moreover, treatment of [ 3 H]palmitate-labeled 5-HT 1A receptors with neutral hydroxylamine resulted in a cleavage of the label from the receptor (Fig.  2B). These results demonstrate that the 5-HT 1A receptor contains thioester-linked acyl groups and no fatty acids linked through amide or hydroxyester bonds. To determine the identity of receptor-bound fatty acids, the receptor was subjected to the fatty acid analysis. For that, fatty acids were hydrolyzed from the gel-purified protein and separated by thin layer chromatography (TLC). Analysis of the TLC data revealed that the 5-HT 1A receptor contains only palmitate with no traces of myristic or stearic acid (Fig. 2C).
Activation of the 5-HT 1A Receptor Does Not Affect Receptor Palmitoylation-We have previously demonstrated that palmitoylation of the other member of the serotonin receptor family, the 5-HT 4(a) receptor, is a dynamic process and that receptor stimulation by agonists increases the rate of palmitate turnover (36). To test whether palmitoylation of the 5-HT 1A recep-  (Fig. 3A).
In order to obtain detailed information about the dynamics of palmitoylation, we studied the time-course of agonist-induced incorporation of [ 3 H]palmitic acid into 5-HT 1A receptors. As shown in Fig. 3B (control), the intensity of radiolabel incorporation into the receptor increased steadily during the labeling period, reflecting a basal level of palmitoylation. The kinetic of [ 3 H]palmitate incorporation was then studied in the presence of 5-HT. The results shown in Fig. 3B reveal that exposure to the agonist does not influence the efficiency of radiolabel incorporation over the whole labeling period. The effect of the agonist on receptor palmitoylation was further analyzed after coexpression of the 5-HT 1A receptor with G i protein (G␣ i3 , G␤ 1 , and G␥ 2 subunits). In such coupled system, agonist stimulation has no effect on the efficiency receptor palmitoylation (Fig. 3B).
To evaluate the role of protein synthesis in receptor palmitoylation, Sf.9 cells expressing 5-HT 1A receptors were incubated with [   ceptor (Fig. 4A). The inhibitory effect of cycloheximide was not changed in the presence of the agonist. To analyze whether receptor-bound fatty acids undergo a rapid turnover, long time pulse-chase experiments were performed. As illustrated in Fig.  4B, the lifetime of [ 3 H]palmitate labeling corresponds to the lifetime of the receptor itself, demonstrating that no cleavage of fatty acid from the receptor occurs during the chase period. Taken together, these data suggest that palmitoylation of the 5-HT 1A receptor is not sensitive to the agonist stimulation and that acylation is a stable modification rather being limited to a pool of the newly synthesized receptors.
Identification of Palmitoylation Sites-In order to identify the potential palmitoylation site(s) within the 5-HT 1A receptor, we constructed a series of mutant receptors in which C-terminal cysteine residues 417 and/or 420 were substituted by serines (Fig. 5A). All mutants were expressed in Sf.9 insect cells by the baculovirus system and labeled with either [ 35 S]methionine or [ 3 H]palmitic acid followed by immunoprecipitation, SDS-PAGE, and fluorography. Labeling with [ 35 S]methionine revealed that the mutated receptors were efficiently expressed along with the WT receptor (Fig. 5B, upper  panel). We also quantified the level of palmitate incorporation for individual mutants by densitometric analysis of fluoro-grams after [ 3 H]palmitate labeling in relation to the expression level defined by [ 35 S]methionine labeling. A single substitution of either Cys 417 or Cys 420 resulted in a significantly decreased, although not completely blocked palmitoylation. The relative value of palmitoylation indicated that incorporation of [ 3 H]palmitate into the C417S and C420S mutants was 19.5 Ϯ 6% (n ϭ 4) and 19.1 Ϯ 9% (n ϭ 4), respectively. A double mutant in which both cysteine residues were replaced by serine did not reveal any detectable incorporation of [ 3 H]palmitate even after prolonged (6 weeks) exposure of the gel (Fig. 5B,  lower panel). Thus, we concluded that both cysteine residues Cys 417 and Cys 420 represent palmitoylation sites on the 5-HT 1A receptor.
Role of Palmitoylation in Coupling of the Receptor with G proteins-To test for the functional significance of receptor palmitoylation, we analyzed interaction of the 5-HT 1A receptor with different G␣ subunits of heterotrimeric G protein by using the GTP␥S coupling assay (43). First, G␣ subunits were coexpressed with the wild-type receptor in Sf.9 cells (in all cases the appropriate G␣ subunit was co-expressed with ␤ 1 and ␥ 2 subunits), and agonist-promoted binding of [ 35 S]GTP␥S to the G␣ subunit was accessed by counting radioactivity directly after immunoprecipitation with appropriate antibodies (Fig.   FIG. 4. Palmitoylation of

Functional Role of Palmitoylation of the 5-HT 1A Receptor
6A). When the wild-type 5-HT 1A receptor was co-expressed with G␣ i1 , G␣ i2 , or G␣ i3 , we measured an ϳ1.7to 2.5-fold increase in [ 35 S]GTP␥S binding upon stimulation with 1 M 5-HT. The result confirmed that the 5-HT 1A receptor effectively communicates with G proteins of the G i family. In contrast, there was no coupling after co-expression of the receptor with G␣ s , G␣ 12 , or G␣ 13 subunits.
We then tested the ability of palmitoylation-deficient receptor mutants to couple to one G␣ i subunit, G␣i 3 . In the case of single mutants C417S and C420S, agonist-dependent GTP␥S binding was significantly decreased, compared with the WT receptor. However, some activation of G␣ i over the basal level was still detectable (Fig. 6B). In contrast, when the non-palmitoylated receptor mutant C417/420S was expressed, the relative activation of G␣ i3 subunit after agonist stimulation was completely abolished (Fig. 6B). It is notable that the WT 5-HT 1A receptor, all mutants and G␣ i3 subunits were expressed in a similar level, as assessed by Western blot experiments (Fig. 6B, inset). We also analyzed the pharmacological profile for the WT receptor and found that it was similar to that previously reported for this receptor expressed in insect cells (48). More importantly, analysis of the mutants revealed that replacement of palmitoylated cysteines does not change their pharmacological properties (Fig. 6C). The binding affinity of 5-[ 3 H]HT for wild-type 5-HT 1A receptors (K D ϭ 140 Ϯ 66 nM), was similar to that obtained for the C417S (K D ϭ 101 Ϯ 44 nM), C420S (K D ϭ 110 Ϯ 23 nM), and C417/420S (K D ϭ 109 Ϯ 23 nM) mutants. Taken together, these data indicate a functional importance of 5-HT 1A receptor palmitoylation for the coupling to G i protein.
Mutation of Palmitoylated Cysteines Affects the Capacity of the 5-HT 1A Receptor to Inhibit the Adenylyl Cyclase Activity in NIH3T3 Cells-The experiments with Sf.9 insect cells demonstrate the importance of acylation for interaction of the receptor with G i protein (Fig. 6). Therefore, we tested the functional role of 5-HT 1A receptor palmitoylation in a mammalian cell system. We analyzed the ability of WT and mutant receptors to inhibit the forskolin-stimulated cAMP accumulation upon application of the specific 5-HT 1A receptor agonist 8-OH-DPAT (49). As a model system we used NIH3T3 cells that do not  (47). These cells were transfected with the pcDNA3.1(Ϫ) plasmid containing cDNA encoding for wild-type and the acylationdeficient mutants of the 5-HT 1A receptor. The total expression level for the WT and all mutants was adjusted to 1500 -1650 fmol/mg protein, which allowed for a quantitative analysis of results.
Expression of the WT 5-HT 1A receptor resulted in significant inhibition of forskolin-promoted cAMP formation upon receptor stimulation with 8-OH-DPAT in a dose-dependent manner (Fig. 7A). Replacement of any of the two palmitoyla-tion sites was accompanied by a significant decrease in the capacity of mutated receptors to inhibit forskolin-stimulated cAMP formation. While the maximal inhibition of cAMP formation obtained for the WT 5-HT 1A receptor was 32 Ϯ 3.6% (n ϭ 4), for the palmitoylation-deficient mutants C417S and C420S this value was reduced to 17 Ϯ 2.4% and 22 Ϯ 4%, respectively (Fig. 7B). In the case of the non-acylated 5-HT 1A receptor mutant, the inhibitory potential of the receptor was completely abolished and exposure to agonists had no effect on the intracellular cAMP level (Fig. 7). Analysis of the dose-dependent inhibition of cAMP formation upon applica- Immunoprecipitations were performed with appropriate antibodies directed against indicated G␣ subunits. Data points represent the means Ϯ S.E. from at least three independent experiments. B, membranes were isolated from Sf.9 cells co-expressing recombinant G␣ i3 , G␤ 1 ␥ 2 subunits together with of either the 5-HT 1A receptor wild type or its acylationdeficient mutants. After incubation with [ 35 S]GTP␥S in the presence of vehicle (H 2 O) or 1 M 5-HT, membranes were lysed and G␣ i3 subunit was immunoprecipitated with specific antibodies. The value obtained for the 5-HT 1A receptor wild-type after agonist stimulation were set to 100%. Data points represent the means Ϯ S.E. from at least four independent experiments performed in duplicate. A statistically significant difference between values is noted (*, p Ͻ 0.01). Inset, expression analysis for WT and acylationdeficient mutants. Samples from parallel infections were used for Western blot analysis with G␣ i3 -or HA-specific antibody. C, saturation binding of 5-[ 3 H]HT with WT and palmitoylation-deficient 5-HT 1A receptors was performed on membranes prepared from infected Sf.9 cells. Nonspecific binding did not exceed 5% of specific one and is subtracted from the total counts. Finally, data were fitted to the one-site saturation model. Data points represent the means Ϯ S.E. from at least four independent experiments performed in triplicate.
tion of 8-OH-DPAT revealed that the EC 50 value for the single mutants was ϳ2.5 times higher than that obtained for the WT 5-HT 1A receptor. We calculated an EC 50 of 127 Ϯ 4 nM for the C417S, 140 Ϯ 7 nM for the C420S mutants and 52 Ϯ 6 nM for the WT. These data confirmed the results obtained for G␣ i3 coupling in Sf.9 insect cells and point to a high functional significance of 5-HT 1A receptor palmitoylation in the G␣ i -mediated signaling.

Intracellular Distribution of Wild-type and Mutant Receptors
Expressed in CHO Cells-To examine the intracellular localization of the wild-type and mutated 5-HT 1A receptors, the genes encoding for the appropriate proteins were cloned in a pcDNA3.1 plasmid and expressed in CHO cells. To monitor expression and intracellular distribution of receptors, transfected cells were subjected to immunofluorescence with anti-HA antibody. As seen in Fig. 8, there were no apparent differences in the immunostaining between wild-type and mutated receptors. This suggests that palmitoylation did not crit- Erk1/2 Activation by 5-HT 1A Wild-type and Acylation-deficient Mutants-In addition to G␣ i -mediated inhibition of the AC, the 5-HT 1A receptor may modulate the activity of Erk via a G␤␥-mediated pathway (17). Therefore, we analyzed the ability of 5-HT 1A receptor WT and its acylation-deficient mutants to activate Erk by Western blot analysis with antibodies directed against phosphorylated form of Erk1/2. In parallel, the expression level of Erk and 5-HT 1A receptor was verified by a Western blot with antibodies against total Erk or against HA epitope. The surface expression level for the WT and all mutant receptors was adjusted to 450 -500 fmol/mg protein, which allowed for a quantitative analysis of results. Fig. 8 demonstrates that agonist treatment of CHO cells transiently transfected with WT 5-HT 1A receptor resulted in an ϳ8-fold increased phosphorylation and thus activation of Erk. For the single acylation mutants C417S and C420S we obtained partial decrease of agonist-induced activation of Erk in comparison with the wild-type receptor. In the case of non-acylated mutant C417/420S, treatment with agonist induced only a very weak increase (ϳ1.8-fold) in phosphorylation of Erk. These data suggest the importance of receptor palmitoylation for signaling through the G␤␥-mediated pathway, in addition to the G␣ imediated signaling. DISCUSSION Covalent attachment of palmitic acid to proteins is often a reversible modification, and dynamic acylation has been demonstrated for a number of signaling proteins. Moreover, palmitoylation of several GPCRs, including ␤ 2 -and ␣ 2A -adrenergic, dopamine D1, and muscarinic acetylcholine m2 receptors, have been shown to be regulated by the agonist (24, 33, 50 -52). For the 5-HT 4(a) receptor, we have also recently demonstrated that agonist stimulation increases the turnover rate of the receptorbound palmitate (36). In the present work we demonstrate palmitoylation of other member of the 5-HT receptor family, the 5-HT 1A receptor ( Figs. 1 and 2). On the contrary to the data obtained for the 5-HT 4(a) receptor, agonist stimulation of the recombinant 5-HT 1A receptor did not cause any changes in its palmitoylation efficiency (Fig. 3). Since it has been reported that the recombinant 5-HT 1A receptor effectively couples to endogenous G o -like proteins in insect cells (53), we suggest that agonist-independent palmitoylation obtained here reflects real physiological situation. Moreover, results obtained after coinfection of Sf.9 cells with recombinant G i protein, further confirming agonist-independence of 5-HT 1A palmitoylation also in a coupled system. Treatment of cells with an inhibitor of protein synthesis, cycloheximide, lead to abolished incorporation of [ 3 H]palmitate into the receptor, indicating no significant turnover of receptor-bound palmitate (Fig. 4A). Furthermore, results of long time pulse-chase experiments indicated that the majority of fatty acids were stably attached to the receptor (Fig.  4B), suggesting that palmitoylation of the 5-HT 1A receptor is a rather irreversible modification. Such a stable and agonistindependent palmitoylation is still unusual for GPCRs, which generally undergo repeated cycles of palmitoylation/depalmitoylation. Interestingly, the 5-HT 1A receptor possesses a very short C terminus composed of only 18 amino acids, and this could be a possible reason for the absence of a specific motif required for the recognition by the depalmitoylation enzyme(s) thioesterase. Alternatively, the orientation of the palmitate group within the membrane together with the composition of a neighboring amino acids (see below) may render them inaccessible to the enzyme.
From the analysis of palmitoylated GPCRs it is known that acylation occurs on cysteine residues located in the C-terminal cytoplasmic domain of the receptors (54). For the 5-HT 1A receptor we also identified C-terminal cysteine residues Cys 417 and Cys 420 as palmitoylation sites (Fig. 5). Characterization of acylation-deficient 5-HT 1A mutants revealed that palmitoylation at either Cys 417 or Cys 420 was still sufficient to maintain interaction of receptor with the G i protein, although to a significantly lower extent than the WT receptor (Figs. 6, 7, and 9). Mutation of both palmitoylation sites completely abolishes signaling, indicating that palmitoylation of the 5-HT 1A receptor is critically involved in activation of the G␣ i protein. This is consistent with recent reports demonstrating the importance of palmitoylation of ␤ 2 -adrenergic and endothelin-B receptors for an agonist-stimulated coupling to G␣ s and to both G␣ q and G␣ i proteins, respectively (32,55,56). Recent data on CCR5 and prostacyclin receptors also demonstrated that receptor palmitoylation is significantly involved in efficient activation of intracellular signaling pathways (57,58). On the other hand, this is in contrast with the results we obtained for the 5-HT 4(a) receptor. Here we found that palmitoylation was not critically involved in the coupling between receptor and G s protein after agonist-stimulation. Similar results have been also reported for the ␣ 2 -adrenergic receptor, which functionally couples to both G s as well as G i (59). In the case of the m 2 -muscarinic receptor, it has also been shown that C-terminal Cys 457 is not required for receptor-mediated inhibition of AC activity (60) although its mutation decreased ability of the receptor to activate G i (33). These opposing findings show that there is no common acylation function applicable to all GPCRs, and thorough analysis of each individual receptor is therefore necessary.
How can palmitoylation of the 5-HT 1A receptor mediate communication between receptor and G i protein? Since the surface expression level, intracellular distribution as well as pharmacological properties of palmitoylation-deficient mutants were quite similar to those obtained for the receptor WT, we exclude the differences in intracellular receptor trafficking and agonist binding as possible reasons for impaired G i protein activation. Alternatively, there are two ways in which palmitoylation could affect the receptor functions: (i) palmitoylation may be required for the receptor to assume the proper conformation needed either for the receptor/G protein recognition or binding process and/or for receptor-mediated G protein activation or (ii) FIG. 8. Role of palmitoylation for the intracellular 5-HT 1A receptor distribution. CHO cells were transfected either with wild-type or mutant 5-HT 1A receptor cDNAs. Twenty-four hours post-transfection, cells were fixed, permeabilized, and then subjected to immunofluorescence analysis with an anti-HA antibody. After incubation with the fluorescent second antibodies, cells were subjected to the confocal microscopy with appropriated filters set at ϫ630 magnification. the palmitoylation may be essential for receptor trafficking and/or localization to the membrane subdomains, like rafts.
It has been proposed that palmitoylation of GPCRs may provide a lipophilic membrane anchor to create an additional fourth intracellular loop in the C-terminal region of the receptor (27,30). More recently, direct evidence for this idea has been obtained for rhodopsin (61,62). Since the 5-HT 1A receptor possesses double acylation site within the C-terminal domain (Fig. 5), complete receptor palmitoylation may result in the formation of an additional small intracellular loop as proposed in Fig. 10. The fact that the 5-HT 1A receptor remains in a continuous palmitoylation state suggests a tight and irreversible association with the plasma membrane. Such membrane association may be further stabilized by basic amino acids surrounding palmitoylated cysteine residues (Fig. 10). According to the two-signal model for membrane binding (21,63), combination of the palmitate plus basic motif provides stable and essentially irreversible binding of the intracellular C-terminal domain with the plasma membrane. Functionally, the resulting conformation of the C-terminal domain may represent a structural determinant important for the communication with G i proteins. Mutation of single acylation site will result in more transient interaction of the receptor C terminus with the membrane. Although such conformations will be still sufficient for interaction with G i protein in some extent, the general coupling efficiency will be reduced. Replacement of both palmitoylated cysteines may destroy the fourth loop and therefore abolish the G i -mediated receptor activity.
An intriguing alternative mechanism could be the involve- The seventh transmembrane domain as well as the amino acid sequence of C-terminal cytoplasmic tail of the 5-HT 1A receptor are shown schematically. The basic residues are marked by ϩ. A cluster of basic residues can provide electrostatic interaction with the inner leaflet of the membrane bilayer. Two palmitate moieties provide additional hydrophobic interaction with membrane, therefore resulting in more persistent association. In combination, this two signals can contribute to formation of a stable intracellular loop.
FIG. 9. Expression and activation of Erk trough WT and acylation-deficient mutants of the 5-HT 1A receptor. A, CHO cells transiently transfected with 5-HT 1A receptor WT or mutants were treated with 10 M 8-OH-DPAT or vehicle for 5 min. Proteins were separated by SDS-PAGE and then subjected to Western blot. The membranes were probed either with antibodies raised against total (upper panel) or phosphorylated (middle panel) Erk. To analyze 5-HT 1A receptor expression, membranes were probed with antibodies raised against HA epitope (lower panel). Fluorograms are representative of four independent experiments. B, quantification of Erk phosphorylation by the WT and substitution mutants was performed by densitometry and calculated as the ratio of total Erk expression over the Erk phosphorylation signal. Each value represents the mean Ϯ S.E. (n ϭ 4). A statistically significant difference between values is noted (*, p Ͻ 0.05; **, p Ͻ 0.01) ment of 5-HT 1A receptor palmitoylation in trafficking to the specific membrane subdomains. Indeed, palmitoylation has been reported to be important for the enrichment of acylated proteins in detergent-resistant membranes (DRM), like caveolae and lipid rafts (64 -71). Assuming that palmitoylation of the 5-HT 1A receptor may represent a signal for DRM trafficking, it could be suggested that the removal of one or both palmitate chains from the 5-HT 1A receptor will reduce its association with DRMs. Since such specific membrane subdomains have been proposed to maintain different components of a particular signaling system together, therefore representing "hot spots" for signaling (72), non-DRM localization of the 5-HT 1A receptor may result in uncoupling from G i /adenylyl cyclase signaling pathway. Additional studies will be necessary to establish whether stable palmitoylation of the 5-HT 1A receptor plays a role in the "right" receptor structure or whether it is involved in DRM trafficking.