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• W1996457599 abstract "RIC-3 has been identified as a molecule essential for the recruitment of functional nicotinic acetylcholine receptors composed of α7, but it exhibits inhibitory effects on α4β2 or α3β4 receptors. In this study, we investigated the role of RIC-3 in the recruitment of 5-hydroxytryptamine type 3A (5-HT3A) receptors to the cell surface. Although RIC-3 is not essential for the surface transport of 5-HT3A receptors, we found that its presence enhances both receptor transport and function in a concentration-dependent manner. RIC-3 is localized to the endoplasmic reticulum, as evidenced by co-localization with the chaperone molecule, binding protein (BiP). RIC-3 is not detected at significant levels on the cell surface when expressed alone or in the presence of 5-HT3A. RIC-3 and 5-HT3A show a low level interaction that is transient (<4 h). That RIC-3 can interact with an endoplasmic reticulum-retained 5-HT3A construct, combined with the transient interaction observed and lack of significant surface-expressed RIC-3, suggests that RIC-3 may play a role in 5-HT3A receptor folding, assembly, or transport to the cell surface. RIC-3 has been identified as a molecule essential for the recruitment of functional nicotinic acetylcholine receptors composed of α7, but it exhibits inhibitory effects on α4β2 or α3β4 receptors. In this study, we investigated the role of RIC-3 in the recruitment of 5-hydroxytryptamine type 3A (5-HT3A) receptors to the cell surface. Although RIC-3 is not essential for the surface transport of 5-HT3A receptors, we found that its presence enhances both receptor transport and function in a concentration-dependent manner. RIC-3 is localized to the endoplasmic reticulum, as evidenced by co-localization with the chaperone molecule, binding protein (BiP). RIC-3 is not detected at significant levels on the cell surface when expressed alone or in the presence of 5-HT3A. RIC-3 and 5-HT3A show a low level interaction that is transient (<4 h). That RIC-3 can interact with an endoplasmic reticulum-retained 5-HT3A construct, combined with the transient interaction observed and lack of significant surface-expressed RIC-3, suggests that RIC-3 may play a role in 5-HT3A receptor folding, assembly, or transport to the cell surface. The most remarkable morphological feature of the brain is not the highly complex, interconnected neuronal pathways but the enormous number (∼1015) of potentially distinct synaptic connections involved in information transfer between neurons. Moreover, synapses are not static and passive translators of information but can change their efficiency of synaptic transmission (1Collingridge G.L. Isaac J.T Wang Y.T. Nat. Rev. Neurosci. 2004; 5: 952-962Crossref PubMed Scopus (811) Google Scholar). This process is termed “synaptic plasticity” and is thought to lie at the heart of the capacity of the brain for learning and memory. Synaptic plasticity may reside pre- or postsynaptically (or both), modulating neurotransmitter release or neurotransmitter receptor responses, respectively.A fundamental question in neurobiology is how receptor biogenesis is orchestrated. A vast array of receptor-interacting proteins have been identified as participating in ligand-gated ion channel trafficking and localization (1Collingridge G.L. Isaac J.T Wang Y.T. Nat. Rev. Neurosci. 2004; 5: 952-962Crossref PubMed Scopus (811) Google Scholar) and have lead to dramatic advances in our knowledge of synaptic plasticity.The 5-HT3 1The abbreviations used are: 5-HT3, 5-hydroxytryptamine type 3; ER, endoplasmic reticulum; AChR, acetylcholine receptor; nACh, nicotinic acetylcholine; nAChR, nACh receptor; GABAAR, GABAA receptor; HEK293, human embryonic kidney 293; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; BiP, binding protein; BiP, binding protein. 1The abbreviations used are: 5-HT3, 5-hydroxytryptamine type 3; ER, endoplasmic reticulum; AChR, acetylcholine receptor; nACh, nicotinic acetylcholine; nAChR, nACh receptor; GABAAR, GABAA receptor; HEK293, human embryonic kidney 293; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; BiP, binding protein; BiP, binding protein. receptors belong to the Cys loop superfamily of ligand-gated ion channels that includes the nicotinic acetylcholine, GABAA, and glycine receptors. The structural relationship (2Brejc K. van Dijk W.N. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1571) Google Scholar, 3Ernst M. Brauchart D. Boresch S. Sieghart W. Neuroscience. 2003; 119: 933-943Crossref PubMed Scopus (138) Google Scholar, 4Nevin S.T. Cromer B.A. Haddrill J.L. Morton C.J. Parker M.W. Lynch J.W. J. Biol. Chem. 2003; 278: 28985-28992Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Reeves D.C. Sayed M.F. Chau P.L. Price K.L. Lummis S.C. Biophys. J. 2003; 84: 2338-2344Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) of the members of this group suggests that their folding and assembly may involve similar posttranslational chaperone-mediated events (6Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 7Connolly C.N. Krishek B.J. McDonald B. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar).The forward transport of these receptors requires the appropriate assembly of specific subunits and release from the endoplasmic reticulum (ER) (6Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (173) Google Scholar,7Connolly C.N. Krishek B.J. McDonald B. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Specific assembly signals have been identified in receptors for GABAA (9Bollan K. Robertson L.A. Tang H. Connolly C.N. Biochem. Soc. Trans. 2003; 31: 875-879Crossref PubMed Scopus (32) Google Scholar), glycine (10Griffon N. Buttner C. Nicke A. Kuhse J. Schmalzing G. Betz H. EMBO J. 1999; 18: 4711-4721Crossref PubMed Scopus (103) Google Scholar), and acetylcholine (11Wang J.M. Zhang L. Yao Y. Viroonchatapan N. Rothe E. Wang Z.Z. Nat. Neurosci. 2002; 5: 963-970Crossref PubMed Scopus (84) Google Scholar). The export of receptors from the ER represents a critical checkpoint for surface expression, with quality control within the lumen of the ER being performed by the resident chaperone proteins (9Bollan K. Robertson L.A. Tang H. Connolly C.N. Biochem. Soc. Trans. 2003; 31: 875-879Crossref PubMed Scopus (32) Google Scholar, 12Wanamaker C.P. Christianson J.C. Green W.N. Ann. N. Y. Acad. Sci. 2003; 998: 66-80Crossref PubMed Scopus (74) Google Scholar). In addition, cytoplasmically exposed ER retention signals within the receptors have been identified as elements that control protein export from the ER (13Boyd G.W. Doward A.I. Kirkness E.F. Millar N.S. Connolly C.N. J. Biol. Chem. 2003; 278: 27681-27687Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The failure to pass these quality control checks results in ER-associated degradation by the proteasome (12Wanamaker C.P. Christianson J.C. Green W.N. Ann. N. Y. Acad. Sci. 2003; 998: 66-80Crossref PubMed Scopus (74) Google Scholar, 14Bedford F.K. Kittler J.T. Muller E. Thomas P. Uren J.M. Merlo D. Wisden W. Triller A. Smart T.G. Moss S.J. Nat. Neurosci. 2001; 4: 908-916Crossref PubMed Scopus (203) Google Scholar, 15Christianson J.C. Green W.N. EMBO J. 2004; 23: 4156-4165Crossref PubMed Scopus (96) Google Scholar).In addition to the generalized chaperone molecules found in the ER, a growing list of selective (to varying degrees) chaperone molecules has been discovered over the last few years. These molecules are responsible for the recruitment of the members of the Cys loop superfamily of ligand-gated ion channels. Critical control steps include receptor folding and assembly, surface transport, synaptic targeting/clustering, and stability. Molecules implicated in receptor folding and assembly include cyclophilin (AChR and 5-HT3 receptor) (16Helekar S.A. Char D. Neff S. Patrick J. Neuron. 1994; 12: 179-189Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 17Helekar S.A. Patrick J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5432-5437Crossref PubMed Scopus (60) Google Scholar), RIC-3 (AChR) (18Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar, 19Halevi S. Yassin L. Eshel M. Sala F. Sala S. Criado M. Treinin M. J. Biol. Chem. 2003; 278: 34411-34417Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), Plic-1 (GABAA receptor) (14Bedford F.K. Kittler J.T. Muller E. Thomas P. Uren J.M. Merlo D. Wisden W. Triller A. Smart T.G. Moss S.J. Nat. Neurosci. 2001; 4: 908-916Crossref PubMed Scopus (203) Google Scholar), and possibly GRIF-1 (20Beck M. Brickley K. Wilkinson H.L. Sharma S. Smith M. Chazot P.L. Pollard S. Stephenson F.A. J. Biol. Chem. 2002; 277: 30079-30090Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Molecules implicated in receptor transport include rapsyn and 14-3-3eta (AChR) (21Marchand S. Devillers-Thiery A. Pons S. Changeux J.P. Cartaud J. J. Neurosci. 2002; 22: 8891-8901Crossref PubMed Google Scholar, 22Jeanclos E.M. Lin L. Treuil M.W. Rao J. DeCoster M.A. Anand R. J. Biol. Chem. 2001; 276: 28281-28290Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), GABAA receptor-associated protein and Plic-1 (GABAA receptor) (14Bedford F.K. Kittler J.T. Muller E. Thomas P. Uren J.M. Merlo D. Wisden W. Triller A. Smart T.G. Moss S.J. Nat. Neurosci. 2001; 4: 908-916Crossref PubMed Scopus (203) Google Scholar, 23Coyle J.E. Nikolov D.B. Neuroscientist. 2003; 9: 205-216Crossref PubMed Scopus (26) Google Scholar, 24Leil T.A. Chen Z.W. Chang C.S. Olsen R.W. J. Neurosci. 2004; 24: 11429-11438Crossref PubMed Scopus (135) Google Scholar), and gephyrin (glycine receptor) (25Hanus C. Vannier C. Triller A. J. Neurosci. 2004; 24: 1119-1128Crossref PubMed Scopus (71) Google Scholar). Finally, selective molecules involved in synaptic targeting, clustering, and surface stability include rapsyn and neuregulin (AChR) (26Banks G.B. Fuhrer C. Adams M.E. Froehner S.C. J. Neurocytol. 2003; 32: 709-726Crossref PubMed Scopus (75) Google Scholar, 27Ngo S.T. Balke C. Phillips W.D. Noakes P.G. Neuroreport. 2004; 15: 2501-2505Crossref PubMed Scopus (16) Google Scholar), gephyrin and collybistin (glycine receptor) (28Levi S. Logan S.M. Tovar K.R. Craig A.M. J. Neurosci. 2004; 24: 207-217Crossref PubMed Scopus (176) Google Scholar, 29Harvey K. Duguid I.C. Alldred M.J. Beatty S.E. Ward H. Keep N.H. Lingenfelter S.E. Pearce B.R. Lundgren J. Owen M.J. Smart T.G. Luscher B. Rees M.I. Harvey R.J. J. Neurosci. 2004; 24: 5816-5826Crossref PubMed Scopus (210) Google Scholar), and Plic-1, gephyrin, Huntington-associated protein 1, and GABAA receptor-associated protein (GABAA receptor) (30Kneussel M. Brain Res. Brain Res. Rev. 2002; 39: 74-83Crossref PubMed Scopus (85) Google Scholar, 31Fritschy J.M. Schweizer C. Brunig I. Luscher B. Biochem. Soc. Trans. 2003; 31: 889-892Crossref PubMed Scopus (40) Google Scholar, 32Everitt A.B. Luu T. Cromer B. Tierney M.L. Birnir B. Olsen R.W. Gage P.W. J. Biol. Chem. 2004; 279: 21701-21706Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 33Kittler J.T. Thomas P. Tretter V. Bogdanov Y.D. Haucke V. Smart T.G. Moss S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12736-12741Crossref PubMed Scopus (188) Google Scholar).Remarkably, despite the size of this growing list and apart from an implication for cyclophilin (based on modulation of receptor function) (16Helekar S.A. Char D. Neff S. Patrick J. Neuron. 1994; 12: 179-189Abstract Full Text PDF PubMed Scopus (99) Google Scholar), no molecule has been implicated in the recruitment of 5-HT3 receptors. As RIC-3 has been proposed to inhibit 5-HT3 receptor expression (19Halevi S. Yassin L. Eshel M. Sala F. Sala S. Criado M. Treinin M. J. Biol. Chem. 2003; 278: 34411-34417Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), we addressed the role of RIC-3 on 5-HT3A transport to the cell surface. RIC-3 was identified in a genetic screen for molecules that are required to maintain AChR function (18Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar). Characterization of RIC-3 confirmed that it is required for the production of functional AChRs (18Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar, 19Halevi S. Yassin L. Eshel M. Sala F. Sala S. Criado M. Treinin M. J. Biol. Chem. 2003; 278: 34411-34417Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The predominant localization of RIC-3 to the ER and not at synapses, combined with the observation that AChRs failed to exit the neuronal cell body in the absence of RIC-3 (19Halevi S. Yassin L. Eshel M. Sala F. Sala S. Criado M. Treinin M. J. Biol. Chem. 2003; 278: 34411-34417Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), is highly suggestive of a role in AChR transport from the ER to the surface. However, a contradictory study (34Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2004; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) reports that RIC-3 is not involved in the transport of AChR α7 receptors to the cell surface. Instead, the function of RIC-3 appears to be at the level of protein folding and is essential for the generation of the α7 ligand (α-bungarotoxin) binding site. Furthermore, RIC-3 was found at the cell surface associated with α7 (34Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2004; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), raising the question regarding the location of RIC-3 chaperone activity.We investigated the role of RIC-3 on the trafficking of 5-HT3A receptors and discovered a significant role in the enhancement of receptor recruitment to the cell surface. The interaction of RIC-3 with 5-HT3A occurs within the ER and is transient, and RIC-3 is not detected at the cell surface. We propose that RIC-3 plays a role in the transport of 5-HT3A receptors, possibly by enhancing protein folding and/or stabilization.MATERIALS AND METHODSCell Culture and Transfection—Simian COS-7 cells (ATCC CRL 1651) and human embryonic kidney (HEK293) cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2 mm glutamine, 1 mm sodium pyruvate, 100 μg/ml streptomycin, and 100 units/ml penicillin in an atmosphere of 5% CO2. Exponentially growing cells were transfected by electroporation (400 V, infinity resistance, 125 farads, Bio-Rad Gene Electropulser II). 10 μg of DNA was used for each transfection (2 × 106 cells) with equimolar ratios of expression constructs. Cells were analyzed 12-48 h after transfection.DNA Constructs—Human 5-HT3A subunit cDNAs were expressed from the mammalian expression vector pGW1 (8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar). The 5-HT3A-Myc and 5-HT3A-HA were tagged with the Myc epitope (EQKLISEEDL) or the hemagglutinin (HA) epitope (YPYDVPDYA) between amino acids 5 and 6 (Thr29-tag-Thr30 in 5-HT3A) by site-directed mutagenesis as reported previously (8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar, 13Boyd G.W. Doward A.I. Kirkness E.F. Millar N.S. Connolly C.N. J. Biol. Chem. 2003; 278: 27681-27687Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Human RIC-3 was a kind gift from M. Treinin (Hebrew University) and was subcloned into pGW1 by PCR (HindIII/XbaI sites).Antibodies—Anti-HA and anti-Myc monoclonal antibodies were used directly as supernatant (20 μg/ml) or purified on immobilized protein A. Antiserum to RIC-3 was raised in sheep using either an extracellular/luminal (depending on whether RIC-3 is expressed on the cell surface) epitope (RIC-3b, SDGQTPGARFQRSHL) or an intracellular epitope (RIC-3a, KAYTGSMLRKRNP) as the antigen. RIC-3b was used for immunofluorescence/ELISA, and RIC-3a was used for immunoprecipitations. Neither RIC-3 antibody produced a significant signal by immunofluorescence on rat neurons (hippocampal). The secondary antibodies, goat anti-mouse Alexa Fluor 488/568 and donkey anti-sheep Alexa Fluor 488, were purchased from Molecular probes and used at 1 μg/ml. The secondary antibody, sheep anti-mouse horseradish peroxidase (HRP), was purchased from Amersham Biosciences and used at 1/1000.Immunofluorescence—COS7 cells were fixed in 3% paraformaldehyde (in PBS), washed twice in 50 mm NH4Cl (in PBS), and blocked (10% FBS, 0.5% bovine serum albumin in PBS) for 30 min. Subsequent washes and antibody dilutions were performed in PBS containing 10% FBS and 0.5% bovine serum albumin. Following surface immunofluorescence, cells were permeabilized by the addition of 0.5% Triton X-100 (10 min), and the immunofluorescence protocol was repeated from the NH4Cl step. Where applicable, the fluorophores, Alexa 488 and Alexa 568, were used to detect surface or total receptor populations, respectively. Cells were examined using a wide-field imaging system (Improvision).Quantification of Cell Surface Expression—HEK293 cells were plated into 96-well dishes. Eight transfections (2 μg of 5-HT3A-HA and 0-8 μg of RIC-3, total DNA maintained at 10 μg using GABAA receptor α-1 cDNA) were used in each dish (with each condition in sextuplet). Cells were fixed in 3% paraformaldehyde (in PBS). Cell surface detection was performed in the absence of detergent, and total expression levels were determined following Triton X-100 treatment (0.5% for 15 min). Cells were washed twice in 50 mm NH4Cl (in PBS) and blocked (10% FBS, 0.5% bovine serum albumin in PBS) for 1 h. Subsequent washes were performed in block. Receptor expression was determined using an HRP-conjugated secondary antibody and assayed using 3,3′,5,5′-tetramethylbenzidine (Sigma) as the substrate, with detection at 450 nm after 30 min following the addition of 0.5 m H2SO4. The reaction rate was determined to remain linear for up to 1 h.Membrane Potential Assay—HEK293 cells were plated into 96-well dishes (Molecular Devices). Eight transfections (as above) were used for each dish. After 24 h, cells were incubated in Red Membrane Potential® assay solution diluted 1:10 (Molecular Devices) for 1 h at room temperature. A range of concentrations of 5-HT (in Hanks' solution) was added automatically by the Flexstation II® apparatus and responses recorded over 90 s using SoftMax Pro 4.6 to analyze the results and plot dose-response curves according to the manufacturer's instructions. Each point is the average of at least 6 wells monitored over a total area of ∼6 mm2.Immunoprecipitation—HEK293 cells were l-methionine-starved for 30 min before being labeled with [35S]methionine (0.2 mCi/6-cm dish, Translabel ICN/Flow) for 4 h. Where appropriate, cells were chased in DMEM/FBS in the presence of 100 μg/ml cycloheximide for time indicated. Cells were lysed in 10 mm sodium phosphate buffer containing 5 mm EDTA, 5 mm EGTA, 50 nm sodium fluoride, 50 mm sodium chloride, 1 mm sodium orthovanadate, 5 mm sodium pyrophosphate, 2% Triton X-100, 0.5% deoxycholate, 0.1 mm phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml antipain, 10 mg/ml pepstatin, and 0.1 mg/ml aprotinin (lysis buffer). Immunoprecipitations were performed as described previously (8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar) and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.Cell Surface Biotinylation—Cells were washed three times in cold PBS, and cell surface proteins were biotinylated using 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 30 min on ice followed by quenching in cold PBS containing 100 mm glycine. Cells were lysed in PBS containing 1% Triton X-100, and biotinylated proteins were isolated overnight using NeutrAvidin-agarose (Pierce) at 4 °C. Beads were washed five times and bound protein eluted in SDS-PAGE sample buffer.RESULTSTo examine the subcellular distribution of 5-HT3A receptors and RIC-3, heterologous expression in COS-7 cells was utilized. COS-7 cells were chosen because of their flattened phenotype, offering higher morphological resolutions of intracellular structures. To facilitate biochemical and morphological analyses, the 5-HT3A subunit was tagged using the epitopes of HA or Myc. These epitope tags were added to the amino terminus of 5-HT3A between amino acids 5 and 6 of the mature polypeptide (downstream from the predicted signal sequence cleavage site) to create 5-HT3A-HA or 5-HT3A-Myc. No functional effects of these tags were evident (8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar).It has been reported previously (8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar) that 5-HT3A-HA can access the cell surface and function as a homomeric 5-HT3 receptor. In support of this assertion, surface immunofluorescence of the HA epitope can be detected in the absence of detergent, where it is evenly distributed in a punctate pattern (Fig. 1B). Intracellular staining is also evident (Fig. 1A). In higher expressing cells (not shown), receptors often exhibited localization to filopodial structures, as observed elsewhere (32Everitt A.B. Luu T. Cromer B. Tierney M.L. Birnir B. Olsen R.W. Gage P.W. J. Biol. Chem. 2004; 279: 21701-21706Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Upon permeabilization, the majority of intracellular receptors are observed in an ER-like pattern (8Boyd G.W. Low P. Dunlop J.I. Ward M. Vardy A.W. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (66) Google Scholar). In addition, small punctate spots are observed, indicative of their presence in transport vesicles (36Ilegems E. Pick H.M. Deluz C. Kellenberger S. Vogel H. J. Biol. Chem. 2004; 279: 53346-53352Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Upon the co-expression of 5-HT3A-HA and RIC-3, a more robust cell surface expression of 5-HT3A-HA is observed (Fig. 1D). Moreover, a striking localization to filopodia is observed, similar to that reported previously (32Everitt A.B. Luu T. Cromer B. Tierney M.L. Birnir B. Olsen R.W. Gage P.W. J. Biol. Chem. 2004; 279: 21701-21706Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The more dramatic localization to filopodia upon co-expression is most likely a result of increased expression rather than a distinct targeting mechanism. In addition to increased surface staining, a significant increase in immunofluorescence is observed in permeabilized cells (Fig. 1C).Given the apparent increase in cell surface levels of 5-HT3A, we endeavored to explore the subcellular localization of recombinantly expressed RIC-3 using the antibody raised against the extracellular/luminal (depending on localization to the surface/intracellular compartments, respectively) region. It should be noted that although earlier analysis programs predicted two transmembrane domains (18Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar), more recent data bases such as those for SignalP 2.0 (34Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2004; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and SignalP 3.0 suggest that the first transmembrane region is a signal sequence. Regardless, the epitope used in this study is predicted to be extracellular/luminal in either model. In the presence of detergent, a high level of RIC-3 is detected, showing a highly clustered distribution as well as an ER-like pattern (Fig. 1E). No peripheral staining, which would indicate surface expression, was observed. In support of this observation, only very low levels of surface staining, similar to background levels in untransfected cells, were evident (Fig. 1F). To confirm RIC-3 localization to the ER, co-staining for BiP, an ER marker (7Connolly C.N. Krishek B.J. McDonald B. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), was performed. Both RIC-3 and BiP staining show similar but not identical patterns (Fig. 1, G and H). It has been reported that RIC-3 is significantly localized to the plasma membrane in a cell line expressing the nicotinic acetylcholine receptors (nAChR) α7 subunit (34Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2004; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We investigated, therefore, whether RIC-3 was similarly localized when 5-HT3A was present. Upon the co-expression of RIC-3 with 5-HT3A-HA, significant surface staining (not shown) or the presence of surface biotinylated protein (see Fig. 4D) for RIC-3 was still not evident, suggesting that RIC-3 may be functioning within the ER in the folding, assembly, or transport of 5-HT3A receptors rather than as a plasma membrane clustering/anchoring molecule. However, we cannot rule out a transient presence of RIC-3 at the cell surface.Fig. 4Determination of interactions between 5-HT3A and RIC-3 by co-immunoprecipitation.A, HEK293 cells expressing 5-HT3A-HA and/or RIC-3 (as indicated) were [35S]methionine-labeled (4 h), and cells were lysed and immunoprecipitated (IP) using antibodies against the HA epitope of 5-HT3A (3A) or RIC-3 as indicated using protein G-agarose. B, HEK293 cells expressing 5-HT3A′CRAR′-HA (3A*) and/or RIC-3 were analyzed as above. C, HEK293 cells expressing 5-HT3A-HA and RIC-3 were [35S]methionine-labeled (3 h) and chased for the times indicated in DMEM/10% FBS containing 100 μg/ml cycloheximide. 5-HT3A-HA receptors were immunoprecipitated using antibodies against HA. Proteins were separated on a 7.5% SDS-polyacrylamide gel and analyzed by autoradiography. D, HEK293 cells expressing RIC-3 (lanes 1 and 2) or RIC-3 and 5-HT3A-HA (lane 3). Total RIC-3 expression was determined by Western blotting 3% of the sample (one dish). Surface RIC-3 was analyzed following surface biotinylation, purification of biotinylated proteins using NeutrAvidin-agarose, and probing of 30% of sample (one dish) with anti-RIC-3 antibodies by Western blotting. The arrow indicates the location of the RIC-3 band (A-D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To quantify the apparent increased cell surface expression observed by immunofluorescence, we used the whole cell ELISA approach to quantify cell surface and total receptor expression. This approach is similar to immunofluorescence, except that an HRP-conjugated antibody is used to provide quantitative results. It is not possible to equate these values with an actual percentage distribution of a receptor, as the estimation of total receptors is consistently underestimated in the presence of detergent. In HEK293 cells expressing 5-HT3A-HA, positive signals are apparent for cell surface receptors (mock-transfected cell values have been subtracted) (Fig. 2). Increasing amounts of RIC-3 cDNA (2-8 μg) were co-transfected with 2 μg of 5-HT3A-HA. In support of a role in the recruitment of 5-HT3A receptors to the cell surface by RIC-3, cell surface levels of 5-HT3A-HA were enhanced as RIC-3 was increased. The most significant enhancement occurred at equimolar or 1:2 ratios, with diminishing additional effects at higher ratios. An examination of the ratio between surface and total receptor levels supported this observation and suggests that the transport pathway may become limiting for some other factor. Interestingly, a small increase in total receptor expression was observed, becoming significant when higher amounts of RIC-3 were co-transfected. This result suggests that RIC-3 may be exerting a stabilizing effect on 5-HT3A.Fig. 2Quantification of 5-HT3A-Myc expression levels in the presence of increasing amounts of RIC-3. HEK293 cells transfected with 2 μg of cDNA encoding 5-HT3A-Myc and varying (0-8 μg) amounts of RIC-3 are shown. GABAA receptor α-1 cDNA was included to ensure that equal amounts of cDNA (10 μg) were used in all transfections. Surface receptor levels were detected using antibodies to Myc followed by secondary antibodies conjugated to HRP in the absence of detergent. Total receptor levels were determined as above, in the presence of dete" @default.
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• W1996457599 title "Cell Surface Expression of 5-Hydroxytryptamine Type 3 Receptors Is Promoted by RIC-3" @default.
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