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• W2028583177 abstract "The 5-HT6 receptor (5-HT6R) is one of the most recently cloned serotonin receptors, and it plays important roles in Alzheimer disease, depression, and learning and memory disorders. However, unlike the other serotonin receptors, the cellular mechanisms of 5-HT6R are poorly elucidated relative to its significance in human brain diseases. Here, using a yeast two-hybrid assay, we found that the human 5-HT6R interacts with Jun activation domain-binding protein-1 (Jab1). We also confirmed a physical interaction between 5-HT6R and Jab1 using glutathione S-transferase pulldown, fluorescence resonance energy transfer, co-immunoprecipitation, and immunocyto(histo)chemistry assays. The manipulation of Jab1 expression using Jab1 small interference RNA decreased 5-HT6R-mediated activity and cell membrane expression of 5-HT6R, whereas overexpression of Jab1 produced no significant effect. In addition, we demonstrated that the activation of 5-HT6R induced the translocation of Jab1 into the nucleus and increased c-Jun phosphorylation and the interaction between Jab1 and c-Jun. Furthermore, we found that 5-HT6R and Jab1 were up-regulated in middle cerebral artery occlusion-induced focal cerebral ischemic rats and in cultured cells exposed to hypoxic insults, suggesting possible protective roles for 5-HT6R and Jab1. These findings suggest that Jab1 provides a novel signal transduction pathway for 5-HT6R and may play an important role in 5-HT6R-mediated behavior changes in the brain. The 5-HT6 receptor (5-HT6R) is one of the most recently cloned serotonin receptors, and it plays important roles in Alzheimer disease, depression, and learning and memory disorders. However, unlike the other serotonin receptors, the cellular mechanisms of 5-HT6R are poorly elucidated relative to its significance in human brain diseases. Here, using a yeast two-hybrid assay, we found that the human 5-HT6R interacts with Jun activation domain-binding protein-1 (Jab1). We also confirmed a physical interaction between 5-HT6R and Jab1 using glutathione S-transferase pulldown, fluorescence resonance energy transfer, co-immunoprecipitation, and immunocyto(histo)chemistry assays. The manipulation of Jab1 expression using Jab1 small interference RNA decreased 5-HT6R-mediated activity and cell membrane expression of 5-HT6R, whereas overexpression of Jab1 produced no significant effect. In addition, we demonstrated that the activation of 5-HT6R induced the translocation of Jab1 into the nucleus and increased c-Jun phosphorylation and the interaction between Jab1 and c-Jun. Furthermore, we found that 5-HT6R and Jab1 were up-regulated in middle cerebral artery occlusion-induced focal cerebral ischemic rats and in cultured cells exposed to hypoxic insults, suggesting possible protective roles for 5-HT6R and Jab1. These findings suggest that Jab1 provides a novel signal transduction pathway for 5-HT6R and may play an important role in 5-HT6R-mediated behavior changes in the brain. Neurotransmitters and neurohormones regulate diverse and myriad brain functions ranging from rapid modulation of ligand-gated ion channels to long term modulation such as gene expression, behavior, and mood changes. Serotonin (5-HT) 2The abbreviations used are: 5-HT5-hydroxytryptamine (serotonin)CHO/5-HT6RChinese hamster ovary cells stably expressing human 5-HT6RCTC terminus of 5-HT6RCT-Jab1C-terminal part of Jab1iL2intracellular loop 2 of 5-HT6RiL3intracellular loop 3 of 5-HT6RFRETfluorescence resonance energy transferGPCRG-protein-coupled receptorGSTglutathione S-transferaseHEK/HA-5-HT6RHEK293 cells stably expressing human HA-tagged 5-HT6RHIF-1αhypoxia-inducible factor-1αJab1Jun activation domain-binding protein-1MCAOmiddle cerebral artery occlusionNT-Jab1N-terminal part of Jab1OGDoxygen-glucose deprivationCSNCOP9 signalosomesiRNAsmall interference RNAHAhemagglutininCMVcytomegaloviruseYFPenhanced yellow fluorescent proteineGFPenhanced green fluorescent proteineCYPenhanced cyan fluorescent proteinPBSphosphate-buffered salinerLHRrat lutropin/choriogonadotropin receptorPAR-2protease-activated receptor-2. is known to be one of the key neurotransmitters related to mood changes. The serotonergic system has also been implicated in the neurobiological control of learning and memory. Therefore, it emerges as a key player in affective, cognitive, complex sensory, and motor functions (1.Svenningsson P. Tzavara E.T. Liu F. Fienberg A.A. Nomikos G.G. Greengard P. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 3188-3193Crossref PubMed Scopus (110) Google Scholar). 5-HT mediates its diverse physiological responses through seven distinct receptor families: the 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors. With the exception of the 5-HT3 receptor, all of these receptors are members of the G-protein-coupled receptor (GPCR) superfamily (2.Barnes N.M. Sharp T. Neuropharmacology. 1999; 38: 1083-1152Crossref PubMed Scopus (2736) Google Scholar). Among the 5-HT receptors, the 5-HT6 receptor (5-HT6R) is coupled to a stimulatory Gα (GαS) protein. This receptor increases cAMP formation and then activates cAMP-dependent protein kinase (3.Kohen R. Metcalf M.A. Khan N. Druck T. Huebner K. Lachowicz J.E. Meltzer H.Y. Sibley D.R. Roth B.L. Hamblin M.W. J. Neurochem. 1996; 66: 47-56Crossref PubMed Scopus (367) Google Scholar). 5-HT6R is abundantly distributed in the brain, especially in the limbic region (4.Ward R.P. Hamblin M.W. Lachowicz J.E. Hoffman B.J. Sibley D.R. Dorsa D.M. Neuroscience. 1995; 64: 1105-1111Crossref PubMed Scopus (200) Google Scholar), and it has a high affinity for antipsychotic compounds and tricyclic antidepressants (5.Roth B.L. Craigo S.C. Choudhary M.S. Uluer A. Monsma Jr., F.J. Shen Y. Meltzer H.Y. Sibley D.R. J. Pharmacol. Exp. Ther. 1994; 268: 1403-1410PubMed Google Scholar). These preliminary reports imply that 5-HT6R has a significant role in the control of mood and emotion in the central nervous system. To date, most findings suggest that 5-HT6R plays crucial roles in neurological disorders, including Alzheimer disease, depression, and learning and memory disorders (6.Branchek T.A. Blackburn T.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 319-334Crossref PubMed Scopus (98) Google Scholar, 7.Mitchell E.S. Neumaier J.F. Pharmacol. Ther. 2005; 108: 320-333Crossref PubMed Scopus (203) Google Scholar, 8.Schechter L.E. Ring R.H. Beyer C.E. Hughes Z.A. Khawaja X. Malberg J.E. Rosenzweig-Lipson S. NeuroRx. 2005; 2: 590-611Crossref PubMed Scopus (203) Google Scholar, 9.Svenningsson P. Tzavara E.T. Qi H. Carruthers R. Witkin J.M. Nomikos G.G. Greengard P. J. Neurosci. 2007; 27: 4201-4209Crossref PubMed Scopus (140) Google Scholar). Selective antagonists of 5-HT6R improved memory and cognition in aged rats (10.Foley A.G. Murphy K.J. Hirst W.D. Gallagher H.C. Hagan J.J. Upton N. Walsh F.S. Regan C.M. Neuropsychopharmacology. 2004; 29: 93-100Crossref PubMed Scopus (126) Google Scholar) and in Alzheimer disease patients (11.Upton N. Chuang T.T. Hunter A.J. Virley D.J. Neurotherapeutics. 2008; 5: 458-469Crossref PubMed Scopus (220) Google Scholar). In an effort to elucidate possible underlying mechanisms of 5-HT6R antagonist-mediated cognition enhancement, microdialysis studies have shown that a 5-HT6R blockade elevates cholinergic or glutaminergic neurotransmission in the brain (12.Dawson L.A. Nguyen H.Q. Li P. Neuropsychopharmacology. 2001; 25: 662-668Crossref PubMed Scopus (227) Google Scholar, 13.Riemer C. Borroni E. Levet-Trafit B. Martin J.R. Poli S. Porter R.H. Bös M. J. Med. Chem. 2003; 46: 1273-1276Crossref PubMed Scopus (184) Google Scholar, 14.Woolley M.L. Marsden C.A. Fone K.C. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 59-79Crossref PubMed Scopus (297) Google Scholar). However, the role of 5-HT6R is not clearly defined in the context of depression due to contradictory results. Although it has been reported that selective 5-HT6R antagonists produced antidepressant-like effects (15.Wesołowska A. Nikiforuk A. Neuropharmacology. 2007; 52: 1274-1283Crossref PubMed Scopus (136) Google Scholar, 16.Wesołowska A. Nikiforuk A. Stachowicz K. Behav. Pharmacol. 2007; 18: 439-446Crossref PubMed Scopus (60) Google Scholar), Svenningsson et al. reported that the 5-HT6R agonist 2-ethyl-5-methoxy-N,N-dimethyltryptamine induced antidepressant-like biochemical and behavioral effects (9.Svenningsson P. Tzavara E.T. Qi H. Carruthers R. Witkin J.M. Nomikos G.G. Greengard P. J. Neurosci. 2007; 27: 4201-4209Crossref PubMed Scopus (140) Google Scholar). Alzheimer disease, depression, and learning and memory disorders are important severe neurological diseases that cause extensive human suffering. Therefore, it is very important to elucidate the mechanisms responsible for 5-HT6R-mediated cognition and mood changes in the brain. However, the cellular mechanisms of 5-HT6R-mediated signal pathways are not well explored except the common GαS-protein-mediated PKA pathway. 5-hydroxytryptamine (serotonin) Chinese hamster ovary cells stably expressing human 5-HT6R C terminus of 5-HT6R C-terminal part of Jab1 intracellular loop 2 of 5-HT6R intracellular loop 3 of 5-HT6R fluorescence resonance energy transfer G-protein-coupled receptor glutathione S-transferase HEK293 cells stably expressing human HA-tagged 5-HT6R hypoxia-inducible factor-1α Jun activation domain-binding protein-1 middle cerebral artery occlusion N-terminal part of Jab1 oxygen-glucose deprivation COP9 signalosome small interference RNA hemagglutinin cytomegalovirus enhanced yellow fluorescent protein enhanced green fluorescent protein enhanced cyan fluorescent protein phosphate-buffered saline rat lutropin/choriogonadotropin receptor protease-activated receptor-2. Jun activation domain-binding protein-1 (Jab1) was initially identified as a protein that interacts with c-Jun and stimulates the binding of c-Jun and JunD to AP-1 sites, potentiating them as transcription factors (17.Claret F.X. Hibi M. Dhut S. Toda T. Karin M. Nature. 1996; 383: 453-457Crossref PubMed Scopus (409) Google Scholar). Jab1 is also known as CSN5, which is the fifth member of the COP9 signalosome (CSN) complex consisting of eight subunits, CSN1–CSN8 (18.Deng X.W. Dubiel W. Wei N. Hofmann K. Mundt K. Trends Genet. 2000; 16: 289Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Jabl exists and functions both as a monomer and as a subunit of the CSN complex. Although Jab1 has been shown to be a key player in plant light signaling, development, cell cycle control, and stability of various proteins in a number of systems (see reviews in Refs. 19.Chamovitz D.A. Segal D. EMBO Rep. 2001; 2: 96-101Crossref PubMed Scopus (152) Google Scholar, 20.Wei N. Serino G. Deng X.W. Trends Biochem. Sci. 2008; 33: 592-600Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), the specific role of Jab1 and its cellular mechanism in the central nervous system still remain obscure. In this study, we discovered a novel interaction between GαS-family GPCR 5-HT6R and Jab1, and we observed Jab1-mediated modulation of the membrane expression and activity of 5-HT6R. In addition, we found that 5-HT6R affects the cytosol/nuclear distribution of Jab1 as well as the interaction between Jab1 and c-Jun, a target protein downstream of Jab1. Furthermore, we demonstrated that 5-HT6R and Jab1 play important roles under conditions of in vitro hypoxia and in vivo cerebral ischemia. Anti-HA, -Myc, -β-tubulin, -histone H3, -c-Jun, and -p-c-Jun antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-5-HT6R was generated by Lab Frontier Co. (Seoul, Korea) or purchased from GeneTex Inc. (San Antonio, TX). Sources for other antibodies were as follows: FLAG (Sigma), GST (Novagen, Madison, WI), His6 (Roche Diagnostics GmbH, Mannheim, Germany), and Jab1 (Novus Biologicals, Littleton, CO). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA) and Abcam (Cambridge, UK). Protease inhibitor mixture was from BioVision (Mountain View, CA). 5-HT, CoCl2, cycloheximide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were from Sigma. Diaminobenzidine was from Roche Applied Science. The ECL kit was from iNtron Biotechnology (Seoul, Korea) and Millipore (Bedford, MA). Fluo-4-AM, Pluronic F-127, and WGA, Alexa Fluor 350 conjugate were from Molecular Probes (Eugene, OR). Human 5-HT6R and HA-tagged human 5-HT6R cDNAs were purchased from UMR cDNA Resource Center (Miner Circle Rolla, MO). The Myc-tagged 5-HT6R plasmid was constructed by recloning the human 5-HT6R cDNA into the EcoRI/XhoI sites of the pCMV-Tag 3B vector. 5-HT6R-eGFP was subcloned (Fw, 5′-CCGCTCGAGATGGTCCCAGAGCC-3′; Rv, 5′-CCGGAATTCGGTTCGTGGGGATGCC-3′) into the XhoI/EcoRI sites of pEGFP-N1 (BD Biosciences, Clontech, Palo Alto, CA). 5-HT6R-eYFP was recloned from 5-HT6R-eGFP into the XhoI/EcoRI sites of pEYFP-N1 (Clontech). cDNA fragments encoding the iL2, iL3, and CT domains of 5-HT6R were subcloned into the EcoRI/BamHI sites of the Gal4 activation domain vector pGBKT7 (Clontech) with the following sets of primers: iL2 (Fw, 5′-CCGGAATTCATGGACCGCTACCTG-3′; Rv, 5′-CGGGATCCGCAGGGCACGCAG-3′), iL3 (Fw, 5′-CCGGAATTCATGTGCAGGATCCTG-3′; Rv, 5′-CGGGATCCGCTTCAGGGCCTTC-3′), and CT (Fw, 5′-CCGGAATTCATGCCACTCTTCATG-3′; Rv, 5′-CGCGGATCCTCAGTTCGTGGG-3′). GST-iL2, -iL3, and -CT were kindly provided by Dr. Yu (Kookmin University, Korea). FLAG-Jab1, GST-Jab1, and eYFP-Jab1 were kindly provided by Drs. J. Song (Sungkyunkwan University, Korea) and K. S. Kwon (Korea Research Institute of Bioscience and Biotechnology). eCFP-Jab1 was recloned from eYFP-Jab1 into the XhoI/BamHI sites of pECFP-C1 (Clontech). His-Jab1 was recloned from GST-Jab1 into the BamHI/EcoRI sites of pET-28a(+) (Novagen, Madison, WI). His-NT-Jab1 (Fw, 5′-CGCGGATCCATGGCAGCTTCCG-3′; Rv, 5′-CCGGAATTCTCAATCAATCCCGGAGAG-3′) and His-CT-Jab1 (Fw, 5′-CGCGGATCCGTTAGTACACAGATGC-3′; Rv, 5′-CCGGAATTCCTAAGCAACGTTAATCTG-3′) were constructed by cloning from His-Jab1 into the BamHI/EcoRI sites of the pET-28a(+) vector (Novagen). siRNA sequences were designed using siRNA target finder software (Turbo si-Designer, Bioneer, Korea), and two single-strand RNA oligonucleotides were chemically synthesized and annealed to form the siRNA duplex. The sequences of siRNA for 5-HT6R and Jab1 genes were as follows: 5-HT6R (sense, 5′- CUGUAACAGCACCAUGAACdTdT-3′; antisense, 5′-GUUCAUGGUGCUGUUACAGdTdT-3′), Jab1 (sense, 5′-CUACAAACCUCCUGAUGAAdTdT-3′; antisense, 5′-UUCAUCAGGAGGUUUGUAGdTdT-3′; or sense, 5′-GCUCAGAGUAUCGAUGAAAdTdT-3′; antisense, 5′-UUUCAUCGAUACUCUGAGCdTdT-3′), and negative control siRNA (sense, 5′-CCUACGCCACCAAUUUCGUdTdT-3′; antisense; 5′-ACGAAAUUGGUGGCGUAGGdTdT-3′). The bait plasmids, pGBKT7/iL2, iL3, or CT of 5-HT6R, were stably expressed in yeast strain AH109 and did not display a self-transcriptional activity. The prey plasmid, human brain cDNA library/pACT2, was transformed into yeast strain Y187. All yeast two-hybrid screening was performed as described previously (21.Yun H.M. Kim S. Kim H.J. Kostenis E. Kim J.I. Seong J.Y. Baik J.H. Rhim H. J. Biol. Chem. 2007; 282: 5496-5505Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). GST, GST-tagged proteins, and His-tagged proteins were transformed into BL21 (DE3) and induced by adding 0.4 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C during the midlog phase. The cells were sonicated in lysis buffer (1× PBS, pH 7.4, 1 mm dithiothreitol, 0.01% Triton X-100, and protease inhibitor mixture). All His-tagged proteins formed inclusion bodies except GST and GST-tagged protein. The inclusion bodies were isolated and dissolved as previously described (22.Kang H. Lee W.K. Choi Y.H. Vukoti K.M. Bang W.G. Yu Y.G. Biochem. Biophys. Res. Commun. 2005; 329: 684-692Crossref PubMed Scopus (35) Google Scholar). After GST, the GST-tagged proteins and the His-tagged proteins were purified and GST pulldown assays were performed using the Profound Pulldown GST Protein:Protein Interaction kit (Pierce). In detail, after GST, GST-iL2, GST-iL3, and GST-CT were expressed in Escherichia coli, the proteins were immobilized on glutathione gel, and purified His-tagged Jab1 was incubated with the glutathione gel bound to GST or GST-tagged proteins. GST pulldown assays using CHO/K1 cells and rat brain lysates were performed as described previously (21.Yun H.M. Kim S. Kim H.J. Kostenis E. Kim J.I. Seong J.Y. Baik J.H. Rhim H. J. Biol. Chem. 2007; 282: 5496-5505Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). HEK393 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. HEK293 cell lines stably expressing HA-5-HT6R (HEK/HA-5-HT6R) were selected with 800 μg/ml G-418 and maintained with 400 μg/ml G-418. CHO cells stably expressing 5-HT6R (CHO/5-HT6R) cells were kindly provided by Dr. M. Teitler (Albany Medical College, Albany, NY), and the cells were maintained with 400 μg/ml G-418. Cultured hippocampal neurons were dissected from 16- to 18-day fetal Sprague-Dawley rats and dissociated using trypsin and trituration through a Pasteur pipette. The neurons were plated on coverslips coated with poly-l-lysine in Neurobasal/B27 medium (Invitrogen) containing 0.5 mml-glutamine, 25 μm 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin. Experiments were carried out on neurons after 14 days in vitro. For transient transfection, cells were transfected using Lipofectamine 2000 (Invitrogen). HEK293 cells were transfected with eCFP-Jab1 (donor) and 5-HT6R-eYFP (acceptor), fixed 24 h after transfection in 4% paraformaldehyde and PBS for 20 min at room temperature, and then washed three times in PBS. The coverslips were mounted on slides using CRYSTAL/MOUNTTM (Biomeda Corp., Foster City, CA) and examined under Olympus FluoView FV1000 confocal microscope (Tokyo). A fluorescence resonance energy transfer (FRET) signal is defined as the light emitted by 5-HT6R-eYFP at 535–565 nm in response to the light emitted at 480–495 nm by eCFP-Jab1. The background FRET signal was detected when eYFP and eCFP vectors were expressed. FRET signal was also detected by the acceptor photobleaching method as previously reported (23.Hwang C.Y. Ryu Y.S. Chung M.S. Kim K.D. Park S.S. Chae S.K. Chae H.Z. Kwon K.S. Oncogene. 2004; 23: 8868-8875Crossref PubMed Scopus (64) Google Scholar, 24.Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (263) Google Scholar). This method measures the release of donor quenching after acceptor photobleaching, thus providing a measurement of close co-localization of the two fusion proteins. HEK293 cells were gently lysed with lysis buffer (1× PBS, pH 7.4, 10 mm NaF, 5 mm dithiothreitol, 0.5% Triton X-100, 0.5% Nonidet P-40, and protease inhibitor mixture) for 30 min on ice and then centrifuged at 20,000 × g and 4 °C for 15 min. The supernatant was then collected. Rat brain was homogenized in lysis buffer added to 0.32 mm sucrose. Cell and brain lysates were precleared with 50 μl of ImmunoPure immobilized Protein G Plus (Pierce) for 2 h, and the precleared lysates were incubated with 2 μg of each specific antibody overnight at 4 °C. The lysates were then were incubated with 50 μl of ImmunoPure immobilized Protein G Plus for 4 h at 4 °C and were washed six times. Immune complexes were eluted by boiling for 10 min at 95 °C in SDS sample buffer, followed by immunoblotting. The immunocytochemistry procedure was performed as described previously (21.Yun H.M. Kim S. Kim H.J. Kostenis E. Kim J.I. Seong J.Y. Baik J.H. Rhim H. J. Biol. Chem. 2007; 282: 5496-5505Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In detail, HEK293 or CHO/K1 cells were cultured and fixed with 4% paraformaldehyde in PBS 24 h after transfection. Cultured hippocampal neurons were grown until 14 days in vitro and then fixed with 4% paraformaldehyde in PBS. For immunohistochemistry, adult male Sprague-Dawley rats were perfused with saline and then fixed with 4% paraformaldehyde in PBS. Brains were sectioned at 10 μm in a cryostat at −20 °C. Sections were collected on a coated glass slide and dried at room temperature before being returned to −70 °C for storage. Slides were post-fixed with acetone for 30 min on ice. 5-HT6R activity was measured using an FDSS6000 96-well fluorescence plate reader (Hamamatsu Photonics, Japan) as previously described (21.Yun H.M. Kim S. Kim H.J. Kostenis E. Kim J.I. Seong J.Y. Baik J.H. Rhim H. J. Biol. Chem. 2007; 282: 5496-5505Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 25.Kim H.J. Yun H.M. Kim T. Nam G. Roh E.J. Kostenis E. Choo H.Y. Pae A.N. Rhim H. Comb. Chem. High Throughput Screen. 2008; 11: 316-324Crossref PubMed Scopus (16) Google Scholar). Briefly, HEK293 cells were transiently transfected with human 5-HT6R and Gα15 protein using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were seeded into 96-well black wall clear-bottom plates, and 5-HT6R activity was measured the next day. After the cells were loaded with 5 μm Fluo-4/AM and 0.001% Pluronic F-127 for 60 min at 37 °C in an HEPES-buffered solution (115 mm NaCl, 5.4 mm KCl, 0.8 mm MgCl2, 20 mm HEPES, and 13.8 mm glucose, pH 7.4), cells were assayed with the FDSS6000 system. After determination of a short baseline, 10 μm or one of various indicated doses of 5-HT was added to HEK293 cells, and the Ca2+ response was measured at 480 nm. All data were collected and analyzed using the FDSS6000 system and related software (Hamamatsu Photonics). After CHO/5-HT6R cells were treated with 5-HT in serum-free Dulbecco's modified Eagle's medium, the cells were harvested by centrifugation at 600 × g for 5 min at 4 °C. Nuclear and cytoplasmic fractions were separated using the Nuclear/Cytosol Fractionation Kit (BioVision), following the manufacturer's protocol. Sprague-Dawley rats weighing 250–300 g were used for a rat model of focal cerebral ischemia. The animals were anesthetized by inhalation of 1.5% isoflurane and were submitted to 2 h of ischemia by occlusion of the middle cerebral artery with intraluminal Mononylon 4.0 sutures introduced through the internal cervical carotid artery form. After 2 h of MCAO, the intraluminal filament was withdrawn from the internal cervical carotid artery to allow reperfusion for 1 day. HEK293 cells and HEK/HA-5-HT6R cells were exposed to various doses of CoCl2 or oxygen-glucose deprivation (OGD) as described previously (26.Chan P.H. J. Cereb. Blood Flow Metab. 2001; 21: 2-14Crossref PubMed Scopus (1450) Google Scholar) with a minor modification. Briefly, the cells were washed two times with a glucose-free HEPES-buffered solution and then immersed in deoxygenated glucose-free HEPES-buffered solution bubbled with nitrogen gas for 30 min to remove residual oxygen. The cells were transferred to an anaerobic chamber (Billups-Rothenberg Inc., Del Mar, CA), exposed to the anaerobic gas mixture (5% CO2, 10% H2, and 85% N2), and maintained at 37 °C for 6 h. After incubation, the cells were removed from the anaerobic chamber, and the media was replaced with normal Dulbecco's modified Eagle's medium-based culture media. Cells were allowed to recover for 12 h before being analyzed. Cell viability was measured by the detection of dehydrogenase activity retained in living cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (50 μl, 5 mg/ml) was added to the cells, and the formazan was dissolved by adding DMSO after the media was removed. The optical densities were measured at 570 nm. The optical intensity was measured using the AlphaEase program (Version 5.1, Alpha Innotech, San Leandro, CA) and was analyzed using the Prism Version 4 program (GraphPad Software Inc., San Diego, CA). All numeric values are represented as the mean ± S.E. The statistical significance of the data was determined using a Student's unpaired t test. We previously demonstrated that the C-terminal region of human 5- HT6R interacts with the Fyn tyrosine kinase. We also characterized the signaling pathways downstream of 5-HT6R activation via a direct interaction with Fyn (21.Yun H.M. Kim S. Kim H.J. Kostenis E. Kim J.I. Seong J.Y. Baik J.H. Rhim H. J. Biol. Chem. 2007; 282: 5496-5505Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In the present study, we found a new binding protein, Jab1, that bound to 5-HT6R in a yeast two-hybrid screening assay using a human brain cDNA library. We found that Jab1 binds to both the intracellular loop 3 (iL3) and the C-terminal (CT) region of human 5-HT6R based on the yeast two-hybrid screening assay (Fig. 1A). To verify a specific interaction between Jab1 and 5-HT6R, we attempted to determine whether Jab1 selectively binds to 5-HT6R using a GST pulldown assay. As shown in Fig. 1B, His-tagged Jab1 specifically interacted with GST-iL3 or GST-CT, which contain the iL3 and CT regions of 5-HT6R, respectively, but Jab1 did not interact with GST alone or with GST-iL2 (intracellular loop 2 of 5-HT6R). We then sought to identify the specific residues within Jab1 that are responsible for binding to 5-HT6R. After Jab1 was arbitrarily divided into two constructs based on amino acid size (the N-terminal portion of Jab1, NT-Jab1: amino acids 1–151 and the C-terminal portion of Jab1, CT-Jab1: amino acids 152–334, Fig. 1A), and GST pulldown assays were again performed using His-tagged NT- Jab1 or His-tagged CT-Jab1. The Jab1 fragments were detected with anti-His antibodies. As shown in Fig. 1C, we found that CT-Jab1 and NT-Jab1 selectively interacted with GST-iL3 and GST-CT of 5-HT6R, respectively. These GST pulldown results were also confirmed in a mammalian system using CHO/K-1 cells transiently transfected with FLAG-tagged Jab1 and in rat brain lysates. Endogenous and full-length exogenous Jab1 from CHO/K-1 cells (Fig. 1D) and endogenous Jab1 from rat brain (Fig. 1E) bound to both GST-iL3 and GST-CT of 5-HT6R, whereas no signal was detected using GST alone. To show the relevance of the specific binding of Jab1 to 5-HT6R, it is necessary to demonstrate the association of the two full-length proteins in mammalian cells using an in vivo co-immunoprecipitation assay. After HA-tagged full-length 5-HT6R was transiently transfected into HEK293 cells, cell lysates were prepared, immunoprecipitated with anti-HA antibodies, and subsequently immunoblotted with anti-Jab1 antibodies. As shown in Fig. 1F, the transfected HA-5-HT6R was able to bind to endogenous Jab1 (the middle lane of the second row) in HEK293 cells, whereas no signal was detected from nontransfected cells. When co-immunoprecipitation was performed in reverse with anti-Jab1 antibodies, followed by immunoblotting with anti-HA antibodies, the same result was obtained (the last lane of the first row in Fig. 1F). We also detected the association of 5-HT6R and Jab1 in native tissues using rat brain lysates. As shown in Fig. 1G, endogenous 5-HT6R selectively binds to endogenous Jab1 (third lane) in the rat brain, whereas no signal was detected in immunoprecipitates using control IgG antibody (second lane). These results strongly demonstrate that Jab1 selectively interacts with human 5-HT6R in mammalian cell lines and in native tissues. FRET has been used as a spectroscopic ruler to measure molecular proximity (27.Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1978) Google Scholar). Monitoring energy transfer between versatile green fluorescent proteins has frequently been used to detect molecular interactions between proteins. We therefore utilized FRET techniques using enhanced cyan fluorescence protein (eCFP)-tagged Jab1 (eCFP-Jab1) and enhanced yellow fluorescence protein (eYFP)-tagged 5-HT6R (5-HT6R-eYFP). When eCFP and eYFP control constructs were co-expressed in HEK293 cells and imaged, no FRET signal was observed (Fig. 2A). However, when eCFP-Jab1 and 5-HT6R-eYFP were co-expressed and imaged under the same experimental conditions, excitation of the eCFP component of Jab1 allowed detection of eYFP with FRET, which is represented by pseudocolor images (the last column in Fig. 2A). This FRET-based molecular proximity was also confirmed using an acceptor photobleaching method (23.Hwang C.Y. Ryu Y.S. Chung M.S. Kim K.D. Park S.S. Chae S.K. Chae H.Z. Kwon K.S. Oncogene. 2004; 23: 8868-8875Crossref PubMed Scopus (64) Google Scholar, 24.Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (263) Google Scholar). If eCFP and eYFP are in close proximity, the donor (eCFP) fluorescence should increase in the region where the acceptor (eYFP) has been bleached. After bleaching eYFP-5-HT6R, we observed increased fluorescence of eCFP-Jab1 in the region of acceptor bleaching when compared with the pre-bleaching images (Fig. 2B and supplemental Fig. S1A). These results indicate that 5-HT6R-eYFP and eCFP-Jab1 were in close proximity in the cells. We next examined the co-localization of 5-HT6R and Jab1 using double immunofluorescence staining in various mammalian cell lines. In HEK293 and CHO/K1 cells, we examined the co-localization of endogenous Jab1 and exogenously transfected 5-HT6R. As shown in Fig. 2C and supplemental Fig. S1B, HA-5-HT6R was predominantly localized at the plasma membrane, and Jab1 was shown to be diffusely distributed in the cytosol and nucleus, as well as at the plasma membrane. The merged images showed a prominent co-localization between 5-HT6R and Jab1 at or near the plasma membrane in HEK293 and CHO-K1 cells. In cultured rat hippocampal neurons, we also examined the co-localization of the two proteins their endogenous native forms. Although the sta" @default.
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• W2028583177 title "Physical Interaction of Jab1 with Human Serotonin 6 G-protein-coupled Receptor and Their Possible Roles in Cell Survival" @default.
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