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• W2079882316 abstract "The 5-hydroxytryptamine type 2A (5-HT2A) receptor and the 5-HT2C receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT2A and 5-HT2C receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT2C receptor but not the 5-HT2A receptor binds to the Veli-3·CASK·Mint1 ternary complex and to SAP102. In addition, the 5-HT2C receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT2A receptor. In contrast, a robust interaction between the 5-HT2A receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT2C receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT2A and 5-HT2C receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT2 receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT2A and the 5-HT2C receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties. The 5-hydroxytryptamine type 2A (5-HT2A) receptor and the 5-HT2C receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT2A and 5-HT2C receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT2C receptor but not the 5-HT2A receptor binds to the Veli-3·CASK·Mint1 ternary complex and to SAP102. In addition, the 5-HT2C receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT2A receptor. In contrast, a robust interaction between the 5-HT2A receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT2C receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT2A and 5-HT2C receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT2 receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT2A and the 5-HT2C receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties. Serotonin (5-hydroxytryptamine (5-HT) 1The abbreviations used are: 5-HT, 5-hydroxytryptamine; h-, human; CIPP, channel-interacting PDZ protein; CRIPT, cysteine-rich interactor of PDZ three; GPCR, G-protein-coupled receptor; MAGUK, membrane-associated guanylate kinase; PDZ, PSD-95/Disc-large/Zonula occludens-1; SAP, synapse-associated protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HA, hemagglutinin; DTT, dithiothreitol; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. ) is a major neurotransmitter that is involved in numerous functions of the mammalian central nervous system. These functions are mediated by a large number of receptors. Except for the 5-HT3 receptor, which is a ligand-gated channel, all 5-HT receptors belong to the G-protein-coupled receptor (GPCR) superfamily. Among the GPCRs activated by 5-HT, the 5-HT2 receptor family, namely the 5-HT2A, the 5-HT2B, and the 5-HT2C receptors, continues to raise particular interest. Indeed, they are involved in multiple physiological functions such as the control of endocrine secretion, motor behavior, mood, pain, sleep, thermoregulation, and appetite (1Roth B.L. Willins D.L. Kristiansen K. Kroeze W.K. Pharmacol. Ther. 1998; 79: 231-257Crossref PubMed Scopus (261) Google Scholar). Moreover, a large number of psychoactive drugs, including non-classical antipsychotic drugs, hallucinogens, anxiolytics, and anti-depressants, mediate their action at least in part through activation of 5-HT2 receptors (1Roth B.L. Willins D.L. Kristiansen K. Kroeze W.K. Pharmacol. Ther. 1998; 79: 231-257Crossref PubMed Scopus (261) Google Scholar, 2Meltzer H.Y. Matsubara S. Lee J.C. J. Pharmacol. Exp. Ther. 1989; 251: 238-246PubMed Google Scholar, 3Aghajanian G.K. Marek G.J. Brain Res. Brain Res. Rev. 2000; 31: 302-312Crossref PubMed Scopus (396) Google Scholar, 4Van Oekelen D. Luyten W.H. Leysen J.E. Life Sci. 2003; 72: 2429-2449Crossref PubMed Scopus (178) Google Scholar). Among the 5-HT2 receptor family, the 5-HT2A and the 5-HT2C receptor are widely distributed in the central nervous system, whereas the 5-HT2B receptor is sparse. The 5-HT2A and the 5-HT2C receptors share the highest degree of sequence homology (about 50% overall sequence identity). Thus, it is not surprising that these receptors have very similar pharmacological profiles and that only a few selective ligands are available. Initial studies of 5-HT2A and 5-HT2C receptor signaling showed that both receptors activate phosphatidyl inositol hydrolysis. However, some differences in signal transduction characteristics of these receptors have been reported (5Berg K.A. Clarke W.P. Sailstad C. Saltzman A. Maayani S. Mol. Pharmacol. 1994; 46: 477-484PubMed Google Scholar, 6Berg K.A. Maayani S. Goldfarb J. Scaramellini C. Leff P. Clarke W.P. Mol. Pharmacol. 1998; 54: 94-104Crossref PubMed Scopus (449) Google Scholar). In NIH3T3 cells expressing the 5-HT2C receptor, agonist-independent activity was much more elevated than that measured in cells expressing the same density of 5-HT2A receptors (7Grotewiel M.S. Sanders-Bush E. Naunyn-Schmiedeberg's Arch. Pharmacol. 1999; 359: 21-27Crossref PubMed Scopus (35) Google Scholar). This indicates that the 5-HT2A receptor has lower intrinsic ability to adopt an active conformation than does the 5-HT2C receptor. Different mechanisms of desensitization for the 5-HT2A and 5-HT2C receptor systems have also been described. In Chinese hamster ovary cells, agonist-induced desensitization of the 5-HT2A receptor-mediated phospholipase C activation is inhibited by inhibitors of protein kinase C and Ca2+-calmodulin-dependent protein kinase II (8Berg K.A. Stout B.D. Maayani S. Clarke W.P. J. Pharmacol. Exp. Ther. 2001; 299: 593-602PubMed Google Scholar). In contrast, the 5-HT2C receptor-mediated response is insensitive to these inhibitors. Moreover, the desensitization of the 5-HT2C receptor system but not that of the 5-HT2A receptor is dependent on G-protein receptor kinase activity (8Berg K.A. Stout B.D. Maayani S. Clarke W.P. J. Pharmacol. Exp. Ther. 2001; 299: 593-602PubMed Google Scholar). A large set of recent studies demonstrate that many GPCR functions, such as G-protein-independent signaling, desensitization, internalization, and resensitization, implicate proteins that bind to their intracellular domains (9Brady A.E. Limbird L.E. Cell. Signal. 2002; 14: 297-309Crossref PubMed Scopus (221) Google Scholar, 10Kreienkamp H.J. Curr. Opin. Pharmacol. 2002; 2: 581-586Crossref PubMed Scopus (54) Google Scholar, 11Bockaert J. Marin P. Dumuis A. Fagni L. FEBS Lett. 2003; 546: 65-72Crossref PubMed Scopus (188) Google Scholar). To date, many proteins identified as binding partners of GPCRs are PSD-95/Disc-large/Zonula occludens-1 (PDZ) domain proteins, which recognize a PDZ recognition motif (PDZ ligand) located at their extreme C-terminal extremity (11Bockaert J. Marin P. Dumuis A. Fagni L. FEBS Lett. 2003; 546: 65-72Crossref PubMed Scopus (188) Google Scholar, 12Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar, 13Nourry C. Grant S.G. Borg J.P. Science's STKE. 2003; http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2003/179/re7PubMed Google Scholar). The 5-HT2A and 5-HT2C receptors express a canonical Type 1 PDZ ligand ((S/T)Xϕ, where ϕ is a hydrophobic residue, SCV and SSV, respectively). The similitude of these PDZ ligands, which share residues crucially required for the interaction with a PDZ domain (i.e. valine at the 0 position and serine at the -2 position) but differ in the -1 position and in residues upstream the minimal PDZ ligand, raises the question of whether these receptors can recruit specific or common PDZ domain proteins. The multiple PDZ protein 1 (MUPP1) was the first protein identified as a binding partner of the C-terminal domain of the 5-HT2C receptor by a two-hybrid screen (14Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (150) Google Scholar). Further experiments indicated that this protein is capable of interacting with both the 5-HT2A and 5-HT2C receptors in vitro (15Becamel C. Figge A. Poliak S. Dumuis A. Peles E. Bockaert J. Lubbert H. Ullmer C. J. Biol. Chem. 2001; 276: 12974-12982Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Parker L.L. Backstrom J.R. Sanders-Bush E. Shieh B.H. J. Biol. Chem. 2003; 278: 21576-21583Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Using a proteomic approach associating glutathione S-transferase pull-down experiments and MALDI-TOF mass spectrometry, we have recently identified 15 proteins of a complex interacting with the entire C terminus of the 5-HT2C receptor (17Becamel C. Alonso G. Galeotti N. Demey E. Jouin P. Ullmer C. Dumuis A. Bockaert J. Marin P. EMBO J. 2002; 21: 2332-2342Crossref PubMed Scopus (143) Google Scholar). These proteins include scaffolding proteins that contain PDZ domains. A direct interaction of one of them, PSD-95, with the 5-HT2A receptor has also recently been reported (18Xia Z. Gray J.A. Compton-Toth B.A. Roth B.L. J. Biol. Chem. 2003; 278: 21901-21908Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The present study was carried out to provide an exhaustive identification and comparison of the proteins that interact with the PDZ ligand of the 5-HT2A and 5-HT2C receptors by means of a proteomic approach based on affinity chromatography using synthetic peptides encompassing the C-terminal PDZ ligand of the receptors as baits. This study showed that both receptors interact with specific sets of PDZ proteins in vitro. Additional experiments were performed to 1) identify the molecular determinants located on the C terminus of the receptors involved in these specific interactions and 2) determine whether the specificity of these interactions takes place in vivo. Expression Plasmids and Antibodies—The constructs encoding Veli-3 (Veli-3/PRK7) and the c-Myc-tagged h5-HT2C receptor (5-HT2C/pRK5) have been previously described. The expression vector coding for CIPP fused to an N-terminal FLAG tag (FLAG-CIPPpCI) was a generous gift from Prof. Michel Lazdunski. QuikChange site-directed mutagenesis (Stratagene) was used to engineer a XbaI restriction site upstream the ATG initiation codon of the pBluescript/h5-HT2A construct (provided by Dr. Christoph Ullmer). h5-HT2A was then subcloned into the XbaI site of the PRK5 plasmid. pRK5/h5-HT2A was hemagglutinin (HA) epitope-tagged on the N-terminal domain by polymerase chain reaction amplification with the forward primer 5′-GCTTGATGCGGATCCATGTACCCATACGACGTCCCCGACTATGCTGATATTCTTTGTGAAAATACTTCTTTGA-3′ (the HA sequence is underlined) and the reverse primer 5′-CATGGATCCGCATCAAGCTTCTAGAGGATC-3′. The amplified products were cut BsaB1/AgeI and ligated into the pRK5/h5-HT2A, yielding pRK5/HA-h5-HT2A. The construct was verified by sequencing. The rabbit polyclonal anti-Veli-3 antibody was purchased from Zymed Laboratories Inc. (San Francisco, CA), the mouse monoclonal anti-CASK, anti-Mint1, and anti-5-HT2A receptor antibodies were from Pharmingen, the mouse monoclonal anti-PSD-95 (clone K28/43) antibody was from Upstate Biotechnology (Lake Placid, NY), the mouse monoclonal anti-synapse-associated protein (SAP) 97 was from Stress-Gen Biotechnologies Corp. (Victoria, Canada), the rabbit polyclonal anti-SAP102 antibody was from Oncogene Research Products (Cambridge, MA), the rabbit polyclonal anti-FLAG antibody was from Sigma (Saint Quentin Fallavier, France), and the monoclonal mouse anti-HA antibody (clone 12CA5) was from Roche Applied Science. The rabbit polyclonal anti-5-HT2C receptor antibody was provided by Dr. Abramowski and has been described elsewhere (19Abramowski D. Rigo M. Duc D. Hoyer D. Staufenbiel M. Neuropharmacology. 1995; 34: 1635-1645Crossref PubMed Scopus (209) Google Scholar). The mouse monoclonal anti-c-Myc antibody was a gift from Dr. Bernard Mouillac (INSERM U469 Montpellier, France). Peptide Affinity Chromatography—Synthetic peptides (>95% purity, Eurogentec, Seraing, Belgium) encompassing the 14 C-terminal amino acids of the 5-HT2A and 5-HT2C receptors were coupled via their N-terminal extremity to activated CH-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. MALDI-TOF mass spectrometry analysis indicated the coupling efficacy was higher than 90% for each peptide. Immobilized peptides were stored at 4 °C in Tris-HCl (50 mm, pH 7.4) and dithiothreitol (DTT, 10 mm) to prevent cysteine oxidation at the -1 position in the 5-HT2A sequence. Brains of Swiss mice (obtained from Janvier, Le Genest-St. Isle, France) were thoroughly washed in phosphate-buffered saline (PBS), homogenized with a Polytron homogenizer, and centrifuged at 200 × g for 3 min. Pellets were resuspended in ice-cold lysis buffer containing Tris-HCl (50 mm, pH 7.4), EDTA (1 mm), and a mixture of protease inhibitors (Roche Applied Science), homogenized 20 times on ice with a glass Teflon homogenizer, and centrifuged at 10,000 × g for 30 min. The membrane pellets were resuspended in CHAPS extraction buffer (50 mm Tris-HCl, pH 7.4, 0.05 mm EDTA, 10 mm CHAPS, and protease inhibitors) for 3 h in rotation at 4 °C. Then samples were centrifuged for 1 h at 10,000 × g. Solubilized proteins (2 mg/condition) were incubated overnight at 4 °C with 2 μg of immobilized peptide. Samples were washed twice with 1 ml of extraction buffer and then 4 times with PBS. Proteins were eluted with either 350 μl of isoelectrofocusing medium containing urea (7 m), thiourea (2 m), CHAPS (4%), ampholines (preblended, pI 3.5–9.5, 8 mg/ml, Amersham Biosciences), DTT (100 mm), tergitol NP7 (0.2%, Sigma), and traces of bromphenol blue for two-dimensional electrophoresis analysis or 50 μl of SDS sample buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 mm DTT, and bromphenol blue) for immunoblotting. Two-dimensional Electrophoresis and Two-dimensional Gel Protein Pattern Analysis—Proteins were first separated according to their isoelectric point along linear immobilized pH gradient strips (pH 3–10, 18 cm long, Amersham Biosciences). Sample loading for the first dimension was performed by passive in-gel re-swelling. After the first dimension the immobilized pH gradient strips were equilibrated for 10 min in a buffer containing urea (6 m), Tris-HCl (50 mm, pH 6.8), glycerol (30%), SDS (2%), DTT (10 mg/ml), and bromphenol blue and then for 15 min in the same buffer containing 15 mg/ml iodoacetamide instead of DTT. For the second dimension, the strips were loaded onto vertical 12.5% SDS-polyacrylamide gels. The gels were silver-stained according to the procedure of Shevchenko et al. (20Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). Gels to be compared were always processed and stained in parallel. Gels were scanned using a computer-assisted densitometer. Spot detection, gel alignment, and spot quantification were performed using the Image Master 2-D Elite software (Amersham Biosciences). Quantification of proteins was expressed as volumes of spots. To correct for variability resulting from silver staining, results were expressed as relative volumes of total spots in each gel. MALDI-TOF Mass Spectrometry and Protein Identification—Proteins of interest were excised and digested in gel using trypsin (Gold, Promega, Charbonnières, France), and tryptic peptides were extracted from the gels as previously described (20Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar, 21Becamel C. Galeotti N. Poncet J. Jouin P. Dumuis A. Bockaert J. Marin P. Biol. Proced. Online. 2002; 4: 94-104http://www.biologicalprocedures.com/bpo/arts/1/39/m39.htmCrossref PubMed Scopus (34) Google Scholar). Digest products were completely dehydrated in a vacuum centrifuge and resuspended in 10 μl of formic acid (2%), desalted using Zip Tips C18 (Millipore, Bedford, MA), eluted with 10 μl of acetonitrile-trifluoroacetic acid (60–0.1%), and concentrated to a volume of 2 μl. Aliquots (0.5 μl) were loaded onto the target of an Ultraflex MALDI-TOF mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) and mixed with the same volume of α-cyano-4-hydroxy-trans-cinnamic acid (Sigma, 10 mg/ml in acetonitrile-trifluoroacetic acid, 50–0.1%). Analysis was performed in reflectron mode with an accelerating voltage of 20 kV and a delayed extraction of 400 ns. Spectra were analyzed using the XTOF software (Bruker-Franzen Analytik), and auto-proteolysis products of trypsin (Mr, 842.51, 1045.56, and 2211.10) were used as internal calibrates. Identification of proteins was performed using both Mascot and PeptIdent software (available on line at www.matrixscience.com and www.expasy.org/tools/peptident.html, respectively), as previously described (21Becamel C. Galeotti N. Poncet J. Jouin P. Dumuis A. Bockaert J. Marin P. Biol. Proced. Online. 2002; 4: 94-104http://www.biologicalprocedures.com/bpo/arts/1/39/m39.htmCrossref PubMed Scopus (34) Google Scholar). Immunoblotting—Proteins, resolved on 12.5% gels, were transferred electrophoretically onto nitrocellulose membranes (Hybond-C, Amersham Biosciences). Membranes were blocked in blocking buffer (Tris-HCl, 50 mm, pH 7.5, 200 mm NaCl, Tween 20, 0.1 and 5% skimmed dried milk) for 1 h at room temperature and incubated overnight with primary antibodies (anti-Veli-3, 1:500; anti-CASK, 1:500; anti-Mint1, 1:250; anti-PSD-95, 1:5000; anti-SAP102, 1:350, anti-SAP97, 1:500; anti-5-HT2A receptor, 1:250) in blocking buffer. Blots were washed three times with blocking buffer and incubated with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (1:2,000 in blocking buffer) for 1 h at room temperature. Immunoreactivity was detected with an enhanced chemiluminescence method (Renaissance Plus, PerkinElmer Life Sciences). Co-immunoprecipitation—CHAPS-soluble proteins from brain extracts (500 μg per experiment) were incubated overnight at 4 °C either with the anti-5-HT2A receptor antibody, the anti-PSD-95 antibody, or the anti-Veli-3 antibody (1 μg each). Samples were incubated for 1 h at 4 °C with 50 μl of protein A-Sepharose beads (Amersham Biosciences). After 5 washes with extraction buffer, immunoprecipitated proteins were eluted in SDS sample buffer, resolved on 12.5% polyacrylamide gels, and detected by immunoblotting. Immunocytochemistry and Confocal Microscopy—Subconfluent COS-7 cells, plated onto poly-l-ornithine (15 μg/ml, Mr = 40,000, Sigma)-coated glass coverslips (12-mm diameter) in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% dialyzed fetal calf serum, were transfected with LipofectAMINE™ 2000 (Invitrogen) according to the manufacturer's instructions using 0.5 μg of each cDNA per coverslip. Twenty-four hours after transfection, cells were washed in PBS and fixed in paraformaldehyde (4% (w/v) in PBS) for 15 min at room temperature. They were washed 3 times with glycine (0.1 m) and permeabilized with 0.1% (w/v) Triton X-100 for 5 min. Cells were then washed 3 times with blocking buffer (gelatin 0.2% in PBS) and incubated overnight at 4 °C with the primary antibody (anti-Myc, 1:500; anti-HA, 1:400; anti-FLAG, 1:250; and anti-Veli-3, 1:500) in blocking buffer. Cells were washed 3 times with blocking buffer and incubated for 1 h at room temperature with either an Alexa green-labeled anti-mouse or a Cy3-labeled anti-rabbit antibody (1:1000 dilution in blocking buffer). After three washes, the cells were mounted on glass slides using gel mount (Biomeda Corp., Foster City, CA). Confocal laser-scanning microscopy was performed using a DMIRB Leica confocal inverted microscope. A series of optical sections were collected with a step of 0.30 μm. Images were collected sequentially to avoid cross-contamination between the fluorochromes and scanned at 1024 × 1024-pixel resolution. Immunohistochemistry and Electron Microscopy—Swiss mice were deeply anesthetized with sodium pentobarbital (50 mg/kg). Animals were perfused through the ascending aorta with PBS, pH 7.4, followed by 300 ml of fixative composed of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4. Brains were then dissected out and fixed by immersion in the same fixative without glutaraldehyde for 12 h at 4 °C. The forebrain was then sliced frontally with a vibratome (VT 1000S, Leica) into 40–50-μm thick sections. After washing in PBS, sections were successively incubated 1) for 48 h at 4 °C with the primary antibodies against the 5-HT2A receptor, the 5-HT2C receptor, or PSD-95 (1:500 dilution each), 2) for 12 h at 4 °C with a peroxidase-labeled Fab fragment of goat anti-mouse or anti-rabbit IgG (Biosys, Compiègne, France, 1:1000 dilution), and 3) with 0.1% 3,3′ diaminobenzidine diluted in 50 mm Tris-HCl, pH 7.3, in the presence of 0.2% H2O2. The primary and secondary antibodies were diluted in PBS containing 1% BSA, 1% normal goat serum, and 0.1% saponin. Immunostained sections were either mounted in Permount (Biomeda, Foster City, CA) and observed under a light microscope or further treated for electron microscopy. The sections were carefully rinsed in 0.1 m cacodylate buffer, pH 7.3, and postfixed in 1% OsO4 in the same buffer. The sections were then dehydrated in graded concentrations of ethanol and embedded in araldite. Punches of 1.5-mm diameter were cut through the hippocampus or the frontal cortex and mounted on araldite blocks. After slicing into ultrathin sections, they were observed in an electron microscope (Hitachi H 7110) without counterstaining. Recruitment of Distinct Sets of PDZ Proteins by the PDZ Ligands of the 5-HT2A and 5-HT2C Receptors—Proteins interacting with the PDZ ligand of the 5-HT2A and 5-HT2C receptors were isolated by affinity chromatography using synthetic peptides encompassing the 14 C-terminal residues of the receptors as bait. Proteins from whole brain extracts were incubated with this bait because both receptors are widely distributed in the mammalian CNS. Two-dimensional gel analysis of proteins retained by the affinity chromatography showed a similar protein pattern recruited by both peptides, with some differences (Fig. 1). To specifically identify the proteins that were recruited through a PDZ-based mechanism, differential analyses of gels obtained with wild type peptides and peptides in which the C-terminal valine was mutated into alanine were conducted. This mutation is known to drastically reduce the binding to PDZ proteins (13Nourry C. Grant S.G. Borg J.P. Science's STKE. 2003; http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2003/179/re7PubMed Google Scholar). Seven spots (or groups of spots) that were apparent in the gels obtained with C-terminal peptide of the 5-HT2C receptor were undetectable in the gels obtained with the mutant peptide (indicated by arrows, Fig. 1B). These proteins were identified by MALDI-TOF mass spectrometry. They include activin receptor-interacting protein 1 (spot 1), a membrane-associated guanylate kinase (MAGUK) with inverted domain structure (this protein is also called MAGI2), several proteins associated to the post-synaptic density, SAP97 (spot 2), SAP102 (spot 3), and PSD-95 (spots 4 and 5), MPP-3 (spot 6), a member of the P55 MAGUK subfamily, and Veli-3, one of the vertebrate homologues of LIN7 (spot 7) (22Fujita A. Kurachi Y. Biochem. Biophys. Res. Commun. 2000; 269: 1-6Crossref PubMed Scopus (80) Google Scholar, 23Lin L. Peters L.L. Ciciotte S.L. Chishti A.H. Biochim. Biophys. Acta. 1998; 1443: 211-216Crossref PubMed Scopus (11) Google Scholar, 24Borg J.P. Straight S.W. Kaech S.M. de Taddeo-Borg M. Kroon D.E. Karnak D. Turner R.S. Kim S.K. Margolis B. J. Biol. Chem. 1998; 273: 31633-31636Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 25Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 26Jo K. Derin R. Li M. Bredt D.S. J. Neurosci. 1999; 19: 4189-4199Crossref PubMed Google Scholar). These proteins possess one or several PDZ domains (Table I), validating our approach to identify PDZ domain partners of membrane-bound receptors. Only three of the binding partners of the 5-HT2C receptor PDZ ligand (PSD-95, MPP-3, and Veli-3) were identified in our previous proteomic study based on a glutathione S-transferase pull-down assay using the entire C terminus of the 5-HT2C receptor as bait. This indicates that the approach using short synthetic peptides is more sensitive than that using the entire C-terminal domain fused to glutathione S-transferase to detect PDZ-based interactions.Table IProteomic analysis of proteins interacting with the C terminus of the 5-HT2Aand the 5-HT2C receptor SWISS-PROT and TrEMBL accession numbers are listed. Proteins that were recruited by both receptor subtypes were identified by MALDI-TOF mass spectrometry from both the gels obtained with the 5-HT2A and 5-HT2C receptor C-terminal peptides. For these proteins, the results of MALDI-TOF analyses that yielded the larger sequence coverage are indicated.Position in gels in Fig. 1Protein nameAccession numberMALDI-TOF MSPDZ domains5-HT2A5-HT2CPeptidesCoverage%1ARIP-1 (activin receptor-interacting protein 1)Q9WVQ14163.86++++2SAP97 (synapse-associated protein 97)Q624021428.03++++3SAP102 (synapse-associated protein 102)P701751823.43-++4PSD-95 (post-synaptic density protein-95)Q621083444.63+++5PSD-95 (post-synaptic density protein-95)Q621083444.63+++6MPP-3 (MAGUK p55 subfamily member-3)O889102043.01+++7Veli-3 (vertebrate homolog of LIN 7)O88952849.71+/-++8CIPP (channel-interacting PDZ domain protein)O704711652.64+++/-9AOP-2 (antixoxidant protein 2)O087091148.40++- Open table in a new tab Surprisingly, trypsin digestion of the two major forms of PSD-95 (spots 4 and 5, Fig. 1B) detected on silver-stained two-dimensional gels, which showed large differences in isoelectric points (∼0.6) and molecular masses (∼ 15 kDa), generated identical peptide mass fingerprints (34 peptides representing 45% overall sequence coverage). Two alternatively spliced isoforms of PSD-95 have recently been identified in rodents and human, a short isoform containing a pair of N-terminal cysteines that can be palmitoylated and designated PSD-95α and a form containing a longer N terminus, designated PSD-95β (27Chetkovich D.M. Bunn R.C. Kuo S.H. Kawasaki Y. Kohwi M. Bredt D.S. J. Neurosci. 2002; 22: 6415-6425Crossref PubMed Google Scholar). The N-terminal peptide of PSD-95α (MD-CLCIVTTKK, Mr = 1254.62) was identified in the tryptic digest of all isoforms of PSD-95 detected in the two-dimensional gels, indicating that they do not result from the N-terminal alternative splicing but differ in other regions that were not covered by the peptides identified in our mass spectrometry analyses. We failed to detect any palmitoylation of the cysteines located on the N-terminal peptide because the palmitoylated form of PSD-95 is insoluble in non-ionic detergents such as CHAPS, which was used in our experiments (28El-Husseini Ael D. Schnell E. Dakoji S. Sweeney N. Zhou Q. Prange O. Gauthier-Campbell C. Aguilera-Moreno A. Nicoll R.A. Bredt D.S. Cell. 2002; 108: 849-863Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Five proteins that interacted with the PDZ ligand of the 5-HT2A receptor were also identified (Fig. 1A). They include activin receptor-interacting protein 1, SAP97, PSD-95, MPP-3, and CIPP (spot 8), a protein containing four PDZ domains that was recently identified as a binding partner of Kir4.0 potassium channel family members, N-methyl-d-aspartate receptor NR2 subunits, neurexins, neuroligins, and acid-sensing ionic channel 3 (29Kurschner C. Mermelstein P.G. Holden W.T. Surmeier D.J. Mol. Cell. Neurosci. 1998; 11: 161-172Crossref PubMed Scopus (101) Google Scholar, 30Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Differential analysis of the proteins recruited by the C-terminal peptides of the 5-HT2A and the 5-HT2C receptors showed a specific recruitment of an additional protein spot by the C terminus of the 5-HT2A receptor through a PDZ-independent mechanism (spot 9, Fig. 1A). Indeed, mutating the C-terminal valine into alanine did not inhibit the binding of this protein (data not shown). This protein was identified as antioxidant protein-2. Antioxidant protein-2 belongs to the thio" @default.
• W2079882316 created "2016-06-24" @default.
• W2079882316 creator A5000827888 @default.
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