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Originally published In Press as doi:10.1074/jbc.M308177200 on November 6, 2003

J. Biol. Chem., Vol. 279, Issue 5, 3280-3291, January 30, 2004
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The 5-Hydroxytryptamine(1A) Receptor Is Stably Palmitoylated, and Acylation Is Critical for Communication of Receptor with Gi Protein*

Ekaterina Papoucheva{ddagger}§, Aline Dumuis¶, Michèle Sebben¶, Diethelm W. Richter{ddagger}, and Evgeni G. Ponimaskin{ddagger}||

From the {ddagger}Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany and UPR CNRS 9023, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France

Received for publication, July 27, 2003 , and in revised form, November 4, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT1A) 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-HT1A 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-HT1A receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric Gi protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with Gi protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G{alpha}i subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT1A receptor is critical for the enabling of Gi protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G{beta}{gamma}-mediated pathway, in addition to the G{alpha}i-mediated signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Serotonin (5-hydroxytryptamine or 5-HT)1 is a neuromodulator involved in the regulation of many different physiological functions of the central nervous system as well as the periphery by activating a large family of receptors. With the exception of the 5-HT3 receptor, which is a transmitter-gated Na+/K+ 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-HT1A 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 Gi/o families (2, 4, 5). Receptor-dependent activation of G{alpha}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-HT1A receptor revealed an unexpected complexity. Antisense depletion of different subtypes of the G{alpha}i subunit revealed that removal of G{alpha}i1 eliminated 5-HT1A-induced inhibition of basal cAMP levels, whereas depletion of G{alpha}i2 and G{alpha}i3 blocked the 5-HT1A receptor action on Gs-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-HT1A receptor (12). Besides effects mediated by G{alpha}i/o subunits, activation of the 5-HT1A receptor leads to a G{beta}{gamma}-mediated activation of K+ current and inhibition of Ca2+ current in hippocampal neurons (13–15), dorsal raphe nucleus neurons (14) and atrial myocytes (16). In CHO cells, the 5-HT1A receptor also mediates G{beta}{gamma}-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-HT1A 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, {alpha} 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 (27–29). 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 {beta}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 Gi protein (33). We have recently shown that palmitoylation of the 5-HT4(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-HT1A 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-HT1A receptor palmitoylation for the coupling to the G{alpha}i as well as with G{beta}{gamma} subunits and for the inhibition of forskolin-stimulated cAMP formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[9,10-3H(N)]Palmitic acid (30–60 Ci/mmol), [35S]GTP{gamma}S (1300 Ci/mmol), and Tran[35S]-label (>1000Ci/mmol) were purchased from Hartmann Analytic GmbH (Germany). [3H]5-hydroxytryptamine creatinine sulfate, ECL® Western blotting Analysis System and peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences. Antibodies raised against phosphorylated Erk1/2 (phospho-p42/44) and against total Erk (p42/44) were from New England Biolabs. Enzymes used in molecular cloning were obtained from New England Biolab. Protein A-Sepharose CL-4B beads, 5-HT, and F-12 Ham medium were from Sigma. TC-100 insect cell medium, Cellfectin® Reagent, LipofectAMINE 2000® reagent were purchased from Invitrogen. The 8-OH-DPAT was purchased from Tocris. Cell culture dishes were ordered from Nunc. Oligonucleotide primers were synthesized by Invitrogen. AmpliTaq® DNA Polymerase was from PerkinElmer Life Sciences. Anti-hemagglutinin (HA) epitope antibodies were purchased from Santa Cruz Biotechnology.

Recombinant DNA Procedures—All basic DNA procedures were performed as described by Sambrook et al. (42). The m5-HT1A cDNA was kindly provided by Dr. Paul R. Albert (Ottawa, Canada). The m5-HT1A cDNA was amplified with specific primers HA-1A-sence (5'-GGAGTGG TACCCACCAT GGATTACCCA TACGACGTCC C AGACTACGC TATGGATATG TTCAGTCTTGGC-3') and 1A-antisence (5'-CAGGGGGTAC CTATTGAGTG AACAGGAAGGGTC-3') to create 9 amino acids HA tag (YPYDVPDYA) at the N terminus of the receptor. The PCR fragment was ligated to the KpnI site of the multiple cloning sites of pcDNA 3.1(–) or pFastBac plasmid (Invitrogen).

Site-directed mutagenesis of the epitope-tagged 5-HT1A 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-HT1A mutants were constructed, purified, and amplified as described previously (44). All mutants were verified by dideoxy DNA sequencing of the final plasmid.

Metabolic Labeling and Immunoprecipitation—Spodoptera frugiperda (Sf.9) cells were grown in TC-100 medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (complete TC-100). For expression, Sf.9 cells (1.5 x 106) grown in 3.5 mm dishes were infected with recombinant baculovirus encoding for HA-tagged 5-HT1A receptor at a multiplicity of infection (MOI) of 10 pfu per cell. After 48 h, Sf.9 cells were labeled with Tran[35S]-label (50 µCi/ml, >1000) or [3H]-palmitic acid (300 µCi/ml, 30–60 Ci/mmol) for the time periods indicated in figure legends. In some experiments, 5-HT or 8-OH-DPAT were added to the final concentrations as indicated in figure legends. To block protein synthesis, cycloheximide (50 µg/ml) was added 10 min prior to incubation with [3H]palmitate or [35S]methionine. For the pulse-chase experiments cells were subsequently incubated with complete TC-100 medium supplemented with 100 µM unlabeled palmitate and 50 µM sodium pyruvate. After labeling, cells were washed once with ice-cold PBS (140 mM NaCl, 3 mM KCl, 2 mM KH2PO4, 6 mM Na2HPO4, pH 7.4) and lysed in 600 µl of NTEP buffer (0.5% Nonidet P-40, 150 mM NaCl, 50 mM Tris/HCl (pH 7.9), 5 mM EDTA, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride). Insoluble material was pelleted (5 min, 20.000 x g), and anti-HA antibodies were added to the resulting supernatant at a dilution of 1:60. After overnight agitation at 4 °C, 30 µl of protein A-Sepharose CL-4B was added, and samples were incubated under gentle rocking for 2 h. After brief centrifugation, the pellet was washed twice with ice-cold NTEP-buffer, and the immunocomplexes were released from the beads by incubation for 30 min at 37 °C in non-reducing electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 20% glycerol, 6% SDS, and 0.002% bromphenol blue). Radiolabeled polypeptides were analyzed by SDS-PAGE on 12% acrylamide gels under non-reducing condition and visualized by fluorography using Kodak X-Omat AR films. Densitometric analysis of fluorograms was performed by Gel-Pro Analyser Version 3.1 Software.

Hydroxylamine Treatment and Fatty Acid Analysis—Gels containing the 5-HT1A receptor labeled with [3H]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 (Me2SO) to wash out cleaved fatty acids before they were processed for fluorography.

For the fatty acid analysis, the [3H]palmitate-labeled 5-HT1A 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-HT1A 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 [35S]GTP{gamma}S Binding—Agonist-promoted binding of [35S]guanosine 5'-(3-O-thio)triphosphate to different G proteins induced by stimulation of 5-HT1A receptors was performed according to the method described previously (43). Briefly, membranes from Sf.9 cells expressing the 5-HT1A receptor wild-type or acylation-deficient mutants and G protein {alpha} subunits (Gi1, Gi2, Gi3, Gs, G12, G13) together with {beta}1{gamma}2 subunits were resuspended in 55 µl of 50 mM Tris/HCl (pH 7.4) containing 2 mM EDTA, 100 mM NaCl, 3 mM MgCl2, and 1 µM GDP. After adding [35S]GTP{gamma}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 µM 5-HT. The reaction was terminated by adding 600 µl of 50 mM Tris/HCl (pH 7.5) containing 20 mM MgCl2, 150 mM NaCl, 0,5% Nonidet P-40, 200 µg/ml aprotinin, 100 µM GDP, and 100 µM GTP for 30 min on ice. Samples were agitated for 1 h at 4 °C after addition of 100 µl of 10% suspension of protein A-Sepharose and 10 µl of antibodies directed against appropriate G{alpha} subunits. Antibodies directed against G{alpha}i, G{alpha}s, and G{alpha}13 were obtained from Santa Cruz Biotechnology. For the precipitation of G{alpha}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 [3H]5-HT Binding—The membranes from Sf.9 cells expressing WT or mutated 5-HT1A receptors were dissolved in buffer containing 20 mM Hepes (pH 8.0), 2 mM MgCl2, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. The binding assay with [3H]5-HT was performed as described previously (12, 45). Briefly, 100 µl of binding buffer containing 50 mM Tris (pH 7.7), 0.1% ascorbic acid, 20 µM pargyline, and 1–250 nm of [3H]5-HT was added to 20 µg of the membrane fraction. Nonspecific binding was determined by addition of 100 µM unlabeled 5-HT. After a 30-min incubation at 20 °C, the reaction mixture was loaded on 20-µm 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-HT1A receptor wild-type and acylation-deficient mutants cDNAs were cloned in pcDNA3(–) vector and transfected in NIH3T3 cells by electroporation. Cells were diluted in DMEM (106 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 µCi [3H]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 µM 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-HT1A 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 x 106) grown in 3.5-mm dishes were transfected with recombinant 5-HT1A receptor using LipofectAMINE2000 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 µM 8-OH-DPAT at 37 °C under 5% CO2, 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-[3H]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-[3H]HT binding. Nonspecific binding was determined in the presence of 100-fold excess of specific 5-HT1A receptor agonist 8-OH-DPAT.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Palmitoylation of the 5-HT1A Receptor—A high titer baculovirus stock containing the cDNA of the murine 5-HT1A 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-HT1A receptors were specifically detected by anti-HA antibodies and localized mainly at the cell surface. Labeling with [35S]methionine followed by immunoprecipitation and SDS-PAGE revealed a single protein band with a molecular mass of ~46 kDa (Fig. 1B, left panel). This corresponds to the predicted molecular mass of the 5-HT1A 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-HT1A receptor.



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  		FIG. 1.
Expression and acylation of the 5-HT1A receptor in insect cells. A, Sf.9 cells infected with a baculovirus encoding for recombinant, HA-tagged 5-HT1A receptor or with a baculovirus alone (Bac) were subjected to immunofluorescence staining with anti-HA antibody. B, Sf.9 cells expressing the 5-HT1A receptor were labeled for 2 h either with [35S]methionine (left panel) or with [3H]palmitate (right panel). Cell lysates were subjected to immunoprecipitation with anti-HA antibodies followed by SDS-PAGE and fluorography. The exposure time was 1 day for [35S]methionine and 7 days for [3H]palmitate labeling. The molecular weight marker is indicated between the panels.

 
To examine whether the 5-HT1A receptor is acylated, Sf.9 cells infected with recombinant baculovirus were metabolically labeled with [3H]palmitic acid. Such labeling revealed a single band of 46 kDa (Fig. 1B, right panel) detectable only in cells infected with the recombinant virus. This result demonstrates that the 5-HT1A receptor efficiently incorporates [3H]palmitate.

Having shown that the 5-HT1A 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 [3H]palmitate-derived radioactivity was sensitive to treatment with increasing concentrations of {beta}-mercaptoethanol. Moreover, treatment of [3H]palmitate-labeled 5-HT1A receptors with neutral hydroxylamine resulted in a cleavage of the label from the receptor (Fig. 2B). These results demonstrate that the 5-HT1A 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-HT1A receptor contains only palmitate with no traces of myristic or stearic acid (Fig. 2C).



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  		FIG. 2.
The 5-HT1A receptor is modified with palmitic acid attached via a thioester-type bond. A, 5-HT1A receptor was labeled for 2 h with [3H]palmitate, immunoprecipitated, and treated with increasing concentrations (5, 10, or 15%) of {beta}-mercaptoethanol for 30 min at 37 °C prior to SDS-PAGE and fluorography. B, Sf.9 insect cells expressing the 5-HT1A receptor were labeled with [3H]palmitate and subjected to immunoprecipitation and SDS-PAGE. The gel was treated with 1 M hydroxylamine (right panel) or 1 M Tris-HCl (left panel) prior to fluorography. The fluorogram shown is a representative of two independent experiments. C, 5-HT1A receptor labeled with [3H]palmitate was immunoprecipitated and then subjected to the fatty acid analysis by thin layer chromatography (TLC). The fluorogram of the TLC plate was analyzed with Gel-Pro Analyzer software. The mobility of authentic [3H]palmitate, [3H]stearate, and [3H]myristate standards are indicated by arrows.

 
Activation of the 5-HT1A Receptor Does Not Affect Receptor Palmitoylation—We have previously demonstrated that palmitoylation of the other member of the serotonin receptor family, the 5-HT4(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-HT1A receptor may also be regulated by the agonist, Sf.9 cells expressing the recombinant receptor were treated with increasing concentrations of 5-HT while labeling the cells with [3H]palmitate. The results shown in Fig. 3A demonstrate that stimulation with the agonist does not result in any dose-dependent changes of receptor palmitoylation. Labeling with [35S]methionine done in parallel demonstrated that the expression level of the receptor was not affected upon exposure to the agonist (Fig. 3A).



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  		FIG. 3.
Agonist stimulation does not affect palmitoylation of the 5-HT1A receptor. A, Sf.9 insect cells expressing the 5-HT1A receptor were incubated with [3H]palmitate or [35S]methionine for 60 min in the presence of different concentrations of 5-HT. The receptor was immunoprecipitated, separated by SDS-PAGE and subjected to fluorography. B, Sf.9 cells expressing the 5-HT1A receptor alone or co-expressing Gi protein (G{alpha}i3, {beta}1, {gamma}2 subunits) were incubated with [3H]palmitate in the presence or absence of 1 µM 5-HT for the indicated time periods. The incorporation of radiolabel was detected by immunoprecipitation followed by SDS-PAGE and fluorography. The fluorograms are representative of three independent experiments. Palmitate incorporation into the 5-HT1A receptor after 5-HT stimulation versus vehicle during the time course experiments is shown in C as a average ± S.E. (n = 3).

 
In order to obtain detailed information about the dynamics of palmitoylation, we studied the time-course of agonist-induced incorporation of [3H]palmitic acid into 5-HT1A 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 [3H]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-HT1A receptor with Gi protein (G{alpha}i3, G{beta}1, and G{gamma}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-HT1A receptors were incubated with [35S]methionine or [3H]palmitate in the absence or presence of cycloheximide. Surprisingly, this experiment revealed that blockade of protein synthesis results in nearly complete inhibition of [3H]palmitate incorporation into the receptor (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 [3H]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-HT1A 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.



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  		FIG. 4.
Palmitoylation of the 5-HT1A receptor is stable modification depending on the protein synthesis. A, Sf.9 cells expressing the 5-HT1A receptor were incubated for 60 min with [3H]palmitate or [35S]methionine in the absence or presence of cycloheximide (50 µg/ml). In parallel, 1 µM 5-HT or vehicle (H2O) was added. Cell lysates were then subjected to the immunoprecipitation, SDS-PAGE, and fluorography. The fluorogram is representative of three independent experiments. B, Sf.9 cells were labeled with [3H]palmitate or [35S]methionine for 1 h and then chased with medium containing unlabeled palmitate or methionine for the periods indicated. During the chase time, the cycloheximide (50 µg/ml) was still applied.

 
Identification of Palmitoylation Sites—In order to identify the potential palmitoylation site(s) within the 5-HT1A 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 [35S]methionine or [3H]palmitic acid followed by immunoprecipitation, SDS-PAGE, and fluorography. Labeling with [35S]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 fluorograms after [3H]palmitate labeling in relation to the expression level defined by [35S]methionine labeling. A single substitution of either Cys417 or Cys420 resulted in a significantly decreased, although not completely blocked palmitoylation. The relative value of palmitoylation indicated that incorporation of [3H]palmitate into the C417S and C420S mutants was 19.5 ± 6% (n = 4) and 19.1 ± 9% (n = 4), respectively. A double mutant in which both cysteine residues were replaced by serine did not reveal any detectable incorporation of [3H]palmitate even after prolonged (6 weeks) exposure of the gel (Fig. 5B, lower panel). Thus, we concluded that both cysteine residues Cys417 and Cys420 represent palmitoylation sites on the 5-HT1A receptor.



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  		FIG. 5.
The 5-HT1A receptor is palmitoylated on two C-terminal cysteine residues, Cys417 and Cys420. A, amino acid sequence of C-terminal domain of the 5-HT1A receptor wild-type and three substitution mutants shown with a single-letter code. The serine residues substituting with the corresponding cysteine residues are shown in bold. The extent of palmitoylation for the substitution mutants was determined by densitometry and calculated as ratio of [3H]palmitate incorporation over the [35S]methionine signal. The value obtained for the wild-type 5-HT1A receptor was set to 100%, data presented in means ± S.E. (n = 4). B, 5-HT1A receptor wild-type and substitution mutants were expressed in Sf.9 cells, labeled either with [3H]palmitate or [35S]methionine and subjected to immunoprecipitation, SDS-PAGE, and fluorography. The exposure time is 3 days for [35S]methionone and 14 days for [3H]palmitate labeling. Representative fluorogram is shown.

 
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-HT1A receptor with different G{alpha} subunits of heterotrimeric G protein by using the GTP{gamma}S coupling assay (43). First, G{alpha} subunits were coexpressed with the wild-type receptor in Sf.9 cells (in all cases the appropriate G{alpha} subunit was co-expressed with {beta}1 and {gamma}2 subunits), and agonist-promoted binding of [35S]GTP{gamma}S to the G{alpha} subunit was accessed by counting radioactivity directly after immunoprecipitation with appropriate antibodies (Fig. 6A). When the wild-type 5-HT1A receptor was co-expressed with G{alpha}i1, G{alpha}i2, or G{alpha}i3, we measured an ~1.7- to 2.5-fold increase in [35S]GTP{gamma}S binding upon stimulation with 1 µM 5-HT. The result confirmed that the 5-HT1A receptor effectively communicates with G proteins of the Gi family. In contrast, there was no coupling after co-expression of the receptor with G{alpha}s, G{alpha}12, or G{alpha}13 subunits.



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  		FIG. 6.
Effect of palmitoylation on the coupling between the 5-HT1A receptor and Gi protein. A, communication of the 5-HT1A receptor with different G proteins. Membranes were prepared from Sf.9 cells expressing G{beta}1{gamma}2 together with G{alpha} subunits as indicated and then incubated with [35S]GTP{gamma}S in the presence of either vehicle (H2O) or 1 µM 5-HT. Immunoprecipitations were performed with appropriate antibodies directed against indicated G{alpha} subunits. Data points represent the means ± S.E. from at least three independent experiments. B, membranes were isolated from Sf.9 cells co-expressing recombinant G{alpha}i3, G{beta}1{gamma}2 subunits together with of either the 5-HT1A receptor wild type or its acylation-deficient mutants. After incubation with [35S]GTP{gamma}S in the presence of vehicle (H2O) or 1 µM 5-HT, membranes were lysed and G{alpha}i3 subunit was immunoprecipitated with specific antibodies. The value obtained for the 5-HT1A receptor wild-type after agonist stimulation were set to 100%. Data points represent the means ± S.E. from at least four independent experiments performed in duplicate. A statistically significant difference between values is noted (*, p < 0.01). Inset, expression analysis for WT and acylation-deficient mutants. Samples from parallel infections were used for Western blot analysis with G{alpha}i3- or HA-specific antibody. C, saturation binding of 5-[3H]HT with WT and palmitoylation-deficient 5-HT1A 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 ± S.E. from at least four independent experiments performed in triplicate.

 
We then tested the ability of palmitoylation-deficient receptor mutants to couple to one G{alpha}i subunit, G{alpha}i3. In the case of single mutants C417S and C420S, agonist-dependent GTP{gamma}S binding was significantly decreased, compared with the WT receptor. However, some activation of G{alpha}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{alpha}i3 subunit after agonist stimulation was completely abolished (Fig. 6B). It is notable that the WT 5-HT1A receptor, all mutants and G{alpha}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-[3H]HT for wild-type 5-HT1A receptors (KD = 140 ± 66 nM), was similar to that obtained for the C417S (KD = 101 ± 44 nM), C420S (KD = 110 ± 23 nM), and C417/420S (KD = 109 ± 23 nM) mutants. Taken together, these data indicate a functional importance of 5-HT1A receptor palmitoylation for the coupling to Gi protein.

Mutation of Palmitoylated Cysteines Affects the Capacity of the 5-HT1A 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 Gi protein (Fig. 6). Therefore, we tested the functional role of 5-HT1A 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-HT1A receptor agonist 8-OH-DPAT (49). As a model system we used NIH3T3 cells that do not contain any detectable [3H]8-OH-DPAT binding sites (47). These cells were transfected with the pcDNA3.1(–) plasmid containing cDNA encoding for wild-type and the acylation-deficient mutants of the 5-HT1A 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-HT1A 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 palmitoylation 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-HT1A receptor was 32 ± 3.6% (n = 4), for the palmitoylation-deficient mutants C417S and C420S this value was reduced to 17 ± 2.4% and 22 ± 4%, respectively (Fig. 7B). In the case of the non-acylated 5-HT1A 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 application of 8-OH-DPAT revealed that the EC50 value for the single mutants was ~2.5 times higher than that obtained for the WT 5-HT1A receptor. We calculated an EC50 of 127 ± 4 nM for the C417S, 140 ± 7 nM for the C420S mutants and 52 ± 6 nM for the WT. These data confirmed the results obtained for G{alpha}i3 coupling in Sf.9 insect cells and point to a high functional significance of 5-HT1A receptor palmitoylation in the G{alpha}i-mediated signaling.



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  		FIG. 7.
Effect of 5-HT1A receptor palmitoylation on the inhibition of forskolin-stimulated adenylyl cyclase activity. A, intracellular cAMP level was measured at increasing concentrations of 8-OH-DPAT in transiently transfected NIH3T3 cells. Data points represent the means ± S.E. from four independent experiments performed in triplicate. B, the effect of saturating concentration (10–5 M) of 8-OH-DPAT was evaluated in NIH3T3 cells transiently expressing the 5-HT1A receptor wild-type, C417S, C420S, and C417/420S mutants. Levels of cAMP accumulation were measured after a 15-min incubation and expressed as a percentage of cAMP accumulation in mock-transfected cells. The percentage conversion of [3H]ATP to [3H]cAMP in mock-transfected NIH3T3 cells was 0.115 ± 0.013. Each value is the mean ± S.E. of at least four independent experiments, each performed in triplicate. In all cases the level surface expression for receptors was adjusted to 1500–1650 fmol/mg.

 
Intracellular Distribution of Wild-type and Mutant Receptors Expressed in CHO Cells—To examine the intracellular localization of the wild-type and mutated 5-HT1A 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 critically contribute to the intracellular distribution of the recombinant 5-HT1A receptors.



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  		FIG. 8.
Role of palmitoylation for the intracellular 5-HT1A receptor distribution. CHO cells were transfected either with wild-type or mutant 5-HT1A 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 x630 magnification.

 
Erk1/2 Activation by 5-HT1A Wild-type and Acylation-deficient Mutants—In addition to G{alpha}i-mediated inhibition of the AC, the 5-HT1A receptor may modulate the activity of Erk via aG{beta}{gamma}-mediated pathway (17). Therefore, we analyzed the ability of 5-HT1A 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-HT1A 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-HT1A receptor resulted in an ~8-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 (~1.8-fold) in phosphorylation of Erk. These data suggest the importance of receptor palmitoylation for signaling through the G{beta}{gamma}-mediated pathway, in addition to the G{alpha}i-mediated signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 {beta}2- and {alpha}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-HT4(a) receptor, we have also recently demonstrated that agonist stimulation increases the turnover rate of the receptor-bound palmitate (36). In the present work we demonstrate palmitoylation of other member of the 5-HT receptor family, the 5-HT1A receptor (Figs. 1 and 2). On the contrary to the data obtained for the 5-HT4(a) receptor, agonist stimulation of the recombinant 5-HT1A receptor did not cause any changes in its palmitoylation efficiency (Fig. 3). Since it has been reported that the recombinant 5-HT1A receptor effectively couples to endogenous Go-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 Gi protein, further confirming agonist-independence of 5-HT1A palmitoylation also in a coupled system. Treatment of cells with an inhibitor of protein synthesis, cycloheximide, lead to abolished incorporation of [3H]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-HT1A receptor is a rather irreversible modification. Such a stable and agonist-independent palmitoylation is still unusual for GPCRs, which generally undergo repeated cycles of palmitoylation/depalmitoylation. Interestingly, the 5-HT1A 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-HT1A receptor we also identified C-terminal cysteine residues Cys417 and Cys420 as palmitoylation sites (Fig. 5). Characterization of acylation-deficient 5-HT1A mutants revealed that palmitoylation at either Cys417 or Cys420 was still sufficient to maintain interaction of receptor with the Gi 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-HT1A receptor is critically involved in activation of the G{alpha}i protein. This is consistent with recent reports demonstrating the importance of palmitoylation of {beta}2-adrenergic and endothelin-B receptors for an agonist-stimulated coupling to G{alpha}s and to both G{alpha}q and G{alpha}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-HT4(a) receptor. Here we found that palmitoylation was not critically involved in the coupling between receptor and Gs protein after agonist-stimulation. Similar results have been also reported for the {alpha}2-adrenergic receptor, which functionally couples to both Gs as well as Gi (59). In the case of the m2-muscarinic receptor, it has also been shown that C-terminal Cys457 is not required for receptor-mediated inhibition of AC activity (60) although its mutation decreased ability of the receptor to activate Gi (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.



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  		FIG. 9.
Expression and activation of Erk trough WT and acylation-deficient mutants of the 5-HT1A receptor. A, CHO cells transiently transfected with 5-HT1A receptor WT or mutants were treated with 10 µM 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-HT1A 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 ± S.E. (n = 4). A statistically significant difference between values is noted (*, p < 0.05; **, p < 0.01)

 
How can palmitoylation of the 5-HT1A receptor mediate communication between receptor and Gi 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 Gi 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) 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-HT1A 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-HT1A 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 Gi 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 Gi 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 Gi-mediated receptor activity.



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  		FIG. 10.
Proposed structure for the C-terminal domain of the 5-HT1A receptor. The seventh transmembrane domain as well as the amino acid sequence of C-terminal cytoplasmic tail of the 5-HT1A receptor are shown schematically. The basic residues are marked by +. 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.

 
An intriguing alternative mechanism could be the involvement of 5-HT1A 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-HT1A receptor may represent a signal for DRM trafficking, it could be suggested that the removal of one or both palmitate chains from the 5-HT1A 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-HT1A receptor may result in uncoupling from Gi/adenylyl cyclase signaling pathway. Additional studies will be necessary to establish whether stable palmitoylation of the 5-HT1A receptor plays a role in the "right" receptor structure or whether it is involved in DRM trafficking.


   FOOTNOTES
 
* These studies were supported by the fund of the Medical School at the University of Göttingen and by the Deutsche Forschungsgemeinschaft through the Center of Molecular Physiology of the Brain (to E. G. P.) and Grant PO 732/1-2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a Georg-Christoph Lichtenberg doctoral fellowship from Lower Saxony, Germany. Back

|| To whom correspondence should be addressed. Tel.: 49-551-395939; Fax: 49-551-396031; E-mail: evgeni{at}ukps.gwdg.de.

1 The abbreviations used are: 5-HT, 5-hydroxytryptamine or serotonin; 5-HT1A, mouse 5-hydroxytryptamine 1A receptor; GPCRs, G protein-coupled receptors; AC, adenylyl cyclase; Erk, extracellular signal-regulated kinase; 8-OH-DPAT, 8-hydroxy-N,N-dipropyl-2-aminotetralin; PBS, phosphate-buffered saline; Sf.9, Spodoptera frugiperda insect cells; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; CHO, chinese hamster ovary; WT, wild type; HA, hemagglutinin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Paul Albert from the Department of Neuroscience at the University of Ottawa for providing the m5-HT1A receptor cDNA.



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