The 5-Hydroxytryptamine(1A) Receptor Is Stably Palmitoylated, and Acylation Is Critical for Communication of Receptor with Gi Protein
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m308177200
ISSN1083-351X
AutoresEkaterina Papoucheva, Aline Dumuis, Michèle Sebben, Diethelm W. Richter, Evgeni Ponimaskin,
Tópico(s)Neuropeptides and Animal Physiology
ResumoIn 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 Gαi 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 Gβγ-mediated pathway, in addition to the Gαi-mediated signaling. 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 Gαi 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 Gβγ-mediated pathway, in addition to the Gαi-mediated signaling. Serotonin (5-hydroxytryptamine or 5-HT) 1The abbreviations used are: 5-HT5-hydroxytryptamine or serotonin5-HT1Amouse 5-hydroxytryptamine 1A receptorGPCRsG protein-coupled receptorsACadenylyl cyclaseErkextracellular signal-regulated kinase8-OH-DPAT8-hydroxy-N,N-dipropyl-2-aminotetralinPBSphosphate-buffered salineSf.9Spodoptera frugiperda insect cellsGTPγSguanosine 5′-3-O-(thio)triphosphateCHOchinese hamster ovaryWTwild typeHAhemagglutinin. 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) (1Boess F.G. Martin I.L. Neuropharmacology. 1994; 33: 275-317Google Scholar, 2Barnes N.M. Sharp T. Neuropharmacology. 1999; 38: 1083-1152Google Scholar). 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 (3Wess J. Faseb J. 1997; 11: 346-354Google Scholar). 5-hydroxytryptamine or serotonin mouse 5-hydroxytryptamine 1A receptor G protein-coupled receptors adenylyl cyclase extracellular signal-regulated kinase 8-hydroxy-N,N-dipropyl-2-aminotetralin phosphate-buffered saline Spodoptera frugiperda insect cells guanosine 5′-3-O-(thio)triphosphate chinese hamster ovary wild type hemagglutinin. 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 (2Barnes N.M. Sharp T. Neuropharmacology. 1999; 38: 1083-1152Google Scholar, 4Albert P.R. Vitam. Horm. 1994; 48: 59-109Google Scholar, 5Raymond J.R. Mukhin Y.V. Gettys T.W. Garnovskaya M.N. Br. J. Pharmacol. 1999; 127: 1751-1764Google Scholar). Receptor-dependent activation of Gαi subunits results in the inhibition of adenylate cyclase and subsequent decrease of cAMP levels in both hippocampal neurons (6De Vivo M. Maayani S. J. Pharmacol. Exp. Ther. 1986; 238: 248-253Google Scholar, 7Dumuis A. Sebben M. Bockaert J. Mol. Pharmacol. 1988; 33: 178-186Google Scholar) and different cell lines expressing the receptor (8Fargin A. Raymond J.R. Regan J.W. Cotecchia S. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1989; 264: 14848-14852Google Scholar, 9Liu Y.F. Albert P.R. J. Biol. Chem. 1991; 266: 23689-23697Google Scholar, 10Nebigil C.G. Garnovskaya M.N. Casanas S.J. Mulheron J.G. Parker E.M. Gettys T.W. Raymond J.R. Biochemistry. 1995; 34: 11954-11962Google Scholar). Analysis of G protein specificity for the 5-HT1A receptor revealed an unexpected complexity. Antisense depletion of different subtypes of the Gαi subunit revealed that removal of Gαi1 eliminated 5-HT1A-induced inhibition of basal cAMP levels, whereas depletion of Gαi2 and Gαi3 blocked the 5-HT1A receptor action on Gs-activated adenylyl cyclase (AC) (11Liu Y.F. Ghahremani M.H. Rasenick M.M. Jakobs K.H. Albert P.R. J. Biol. Chem. 1999; 274: 16444-16450Google Scholar). Expression studies in Sf.9 insect cells have also provided the first evidence for possible post-translational modifications of the 5-HT1A receptor (12Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Google Scholar). Besides effects mediated by Gαi/o subunits, activation of the 5-HT1A receptor leads to a Gβγ-mediated activation of K+ current and inhibition of Ca2+ current in hippocampal neurons (13Zgombick J.M. Beck S.G. Mahle C.D. Craddock-Royal B. Maayani S. Mol. Pharmacol. 1989; 35: 484-494Google Scholar, 14Clarke W.P. Yocca F.D. Maayani S. J. Pharmacol. Exp. Ther. 1996; 277: 1259-1266Google Scholar, 15Andrade R. Malenka R.C. Nicoll R.A. Science. 1986; 234: 1261-1265Google Scholar), dorsal raphe nucleus neurons (14Clarke W.P. Yocca F.D. Maayani S. J. Pharmacol. Exp. Ther. 1996; 277: 1259-1266Google Scholar) and atrial myocytes (16Karschin A. Ho B.Y. Labarca C. Elroy-Stein O. Moss B. Davidson N. Lester H.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5694-5698Google Scholar). In CHO cells, the 5-HT1A receptor also mediates Gβγ-mediated stimulation of phospholipase C as well as activation of mitogen-activated protein kinase Erk2 (8Fargin A. Raymond J.R. Regan J.W. Cotecchia S. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1989; 264: 14848-14852Google Scholar, 17Garnovskaya M.N. van Biesen T. Hawe B. Casanas Ramos S. Lefkowitz R.J. Raymond J.R. Biochemistry. 1996; 35: 13716-13722Google Scholar). 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 (18Ramboz S. Oosting R. Amara D.A. Kung H.F. Blier P. Mendelsohn M. Mann J.J. Brunner D. Hen R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14476-14481Google Scholar, 19Heisler L.K. Chu H.M. Brennan T.J. Danao J.A. Bajwa P. Parsons L.H. Tecott L.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15049-15054Google Scholar, 20Parks C.L. Robinson P.S. Sibille E. Shenk T. Toth M. Proc. Natl. Acad. Sci. U. S. A. 1998; Vol. 95: 10734-10739Google Scholar). The covalent attachment of fatty acids to proteins (acylation) is a widespread post-translational modification (21Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Google Scholar). 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 (22Boutin J.A. Cell Signal. 1997; 9: 15-35Google Scholar). 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 (23Linder M.E. Deschenes R.J. Biochemistry. 2003; Vol. 42: 4311-4320Google Scholar). 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, α 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 (24Bouvier M. Moffett S. Loisel T.P. Mouillac B. Hebert T. Chidiac P. Biochem. Soc. Trans. 1995; 23: 116-120Google Scholar, 25Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Google Scholar, 26Bijlmakers M.J. Marsh M. Trends Cell Biol. 2003; 13: 32-42Google Scholar). Meanwhile it is widely accepted that repeated cycles of palmitoylation and depalmitoylation can be critically involved in regulation of different signaling processes (27Ross E.M. Curr. Biol. 1995; 5: 107-109Google Scholar, 28Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Google Scholar, 29Cramer H. Schmenger K. Heinrich K. Horstmeyer A. Boning H. Breit A. Piiper A. Lundstrom K. Muller-Esterl W. Schroeder C. Eur. J. Biochem. 2001; 268: 5449-5459Google Scholar). In the GPCRs, palmitoylation has been shown to be responsible for a wide variety of biological functions (24Bouvier M. Moffett S. Loisel T.P. Mouillac B. Hebert T. Chidiac P. Biochem. Soc. Trans. 1995; 23: 116-120Google Scholar, 27Ross E.M. Curr. Biol. 1995; 5: 107-109Google Scholar, 28Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Google Scholar, 30Bouvier M. Chidiac P. Hebert T.E. Loisel T.P. Moffett S. Mouillac B. Methods Enzymol. 1995; 250: 300-314Google Scholar, 31Bouvier M. Loisel T.P. Hebert T. Biochem. Soc. Trans. 1995; 23: 577-581Google Scholar). For example, prevention of palmitoylation of the β2-adrenergic receptor leads to an increase of basal receptor phosphorylation and rapid desensitization in response to agonist stimulation (32Moffett S. Mouillac B. Bonin H. Bouvier M. EMBO J. 1993; 12: 349-356Google Scholar). Substitution of palmitoylated cysteine residues in the muscarinic acetylcholine m2 receptor reduces its ability to couple to the Gi protein (33Hayashi M.K. Haga T. Arch. Biochem. Biophys. 1997; 340: 376-382Google Scholar). We have recently shown that palmitoylation of the 5-HT4(a) receptor is involved in the modulation of the constitutive receptor activity (34Ponimaskin E.G. Heine M. Joubert L. Sebben M. Bickmeyer U. Richter D.W. Dumuis A. J. Biol. Chem. 2002; 277: 2534-2546Google Scholar). For several GPCRs palmitoylation has been revealed to be modulated by agonist stimulation (33Hayashi M.K. Haga T. Arch. Biochem. Biophys. 1997; 340: 376-382Google Scholar, 35Hawtin S.R. Tobin A.B. Patel S. Wheatley M. J. Biol. Chem. 2001; 276: 38139-38146Google Scholar, 36Ponimaskin E.G. Schmidt M.F. Heine M. Bickmeyer U. Richter D.W. Biochem. J. 2001; 353: 627-634Google Scholar), whereas for the human A1 adenosine receptor, the efficacy of palmitoylation was not affected by the agonist (37Gao Z. Ni Y. Szabo G. Linden J. Biochem. J. 1999; 342: 387-395Google Scholar). Moreover, stimulation of several GPCRs may modulate palmitoylation of receptor-coupled G proteins (38Gurdal H. Seasholtz T.M. Wang H.Y. Brown R.D. Johnson M.D. Friedman E. Mol. Pharmacol. 1997; 52: 1064-1070Google Scholar, 39Mumby S.M. Muntz K.H. Biochem. Soc. Trans. 1995; 23: 156-160Google Scholar, 40Chen C.A. Manning D.R. J. Biol. Chem. 2000; 275: 23516-23522Google Scholar, 41Stevens P.A. Pediani J. Carrillo J.J. Milligan G. J. Biol. Chem. 2001; 276: 35883-35890Google Scholar). 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 Gαi as well as with Gβγ subunits and for the inhibition of forskolin-stimulated cAMP formation. Materials—[9,10-3H(N)]Palmitic acid (30–60 Ci/mmol), [35S]GTPγS (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. (42Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: a Laboratory Manual. 2 Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). 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 (43Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F. FEBS Lett. 1998; 429: 370-374Google Scholar). The recombinant baculoviruses encoding for HA-5-HT1A mutants were constructed, purified, and amplified as described previously (44Veit M. Nurnberg B. Spicher K. Harteneck C. Ponimaskin E. Schultz G. Schmidt M.F. FEBS Lett. 1994; 339: 160-164Google Scholar). 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 × 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 × 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. (34Ponimaskin E.G. Heine M. Joubert L. Sebben M. Bickmeyer U. Richter D.W. Dumuis A. J. Biol. Chem. 2002; 277: 2534-2546Google Scholar). Assay for [35S]GTPγS 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 (43Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F. FEBS Lett. 1998; 429: 370-374Google Scholar). Briefly, membranes from Sf.9 cells expressing the 5-HT1A receptor wild-type or acylation-deficient mutants and G protein α subunits (Gi1, Gi2, Gi3, Gs, G12, G13) together with β1γ2 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]GTPγS (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 Gα subunits. Antibodies directed against Gαi, Gαs, and Gα13 were obtained from Santa Cruz Biotechnology. For the precipitation of Gα12 subunits, antibody AS1905 (43Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F. FEBS Lett. 1998; 429: 370-374Google Scholar) 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 (12Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Google Scholar, 45Hall M.D. el Mestikawy S. Emerit M.B. Pichat L. Hamon M. Gozlan H. J. Neurochem. 1985; 44: 1685-1696Google Scholar). 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 (46SigmaPlot 8.02. (2002) SPSS Inc., ChicagoGoogle Scholar). 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 (7Dumuis A. Sebben M. Bockaert J. Mol. Pharmacol. 1988; 33: 178-186Google Scholar). The amount of the expressed 5-HT1A receptor was measured as described in Varrault et al. (47Varrault A. Journot L. Audigier Y. Bockaert J. Mol. Pharmacol. 1992; 41: 999-1007Google Scholar). 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 × 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. 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. 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 β-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). 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 (36Ponimaskin E.G. Schmidt M.F. Heine M. Bickmeyer U. Richter D.W. Biochem. J. 2001; 353: 627-634Google Scholar). 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). In order to obtain detailed information about the dynamics of palmitoylation, we studied the time-course of agonist-induced incorporation of [3H]pa
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