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• W2051003806 abstract "RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT3 receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse “slicks” and bright “halo” structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition. RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT3 receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse “slicks” and bright “halo” structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition. The ligand-gated ion channels are critical participants in cellular communication, playing a major role in synaptic transmission. The ligand-gated ion channels include receptors for acetylcholine (nACh) 3The abbreviations used are: nAChnicotinic acetylcholineERendoplasmic reticulum5-HT35-hydroxytryptamine type 3FRAPfluorescence recovery after photobleachingYFPyellow fluorescent proteinCFPcyan fluorescent protein.3The abbreviations used are: nAChnicotinic acetylcholineERendoplasmic reticulum5-HT35-hydroxytryptamine type 3FRAPfluorescence recovery after photobleachingYFPyellow fluorescent proteinCFPcyan fluorescent protein., γ-aminobutyric acid type A, serotonin (5-HT), and glycine (the cys-loop superfamily) and N-methyl-d-aspartic acid, kainate, and AMPA (glutamate receptors). A vast array of receptor-interacting proteins have been identified as participating in receptor trafficking and localization (1Collingridge G.L. Isaac J.T. Wang Y.T. Nat. Rev. 2004; 5: 952-962Crossref Scopus (820) Google Scholar) and have led to dramatic advances in our knowledge of synaptic architecture and plasticity. nicotinic acetylcholine endoplasmic reticulum 5-hydroxytryptamine type 3 fluorescence recovery after photobleaching yellow fluorescent protein cyan fluorescent protein. nicotinic acetylcholine endoplasmic reticulum 5-hydroxytryptamine type 3 fluorescence recovery after photobleaching yellow fluorescent protein cyan fluorescent protein. Chaperone molecules play an important role in the intracellular transport of receptors. However, a fundamental question in neurobiology remains: How is receptor biogenesis orchestrated? Despite the fact that receptor composition determines receptor function and their pharmacological repertoire, little information is available regarding how this may be achieved. Specific assembly signals exist that can determine a preference for particular subunit partners (2Connolly C.N. Wafford K.A. Biochem. Soc. Trans. 2004; 32: 529-534Crossref PubMed Scopus (160) Google Scholar). However, few selective chaperone molecules have been implicated in the process of receptor biogenesis. General chaperone proteins such as BiP, calnexin, calreticulin, and PDI do operate on the ligand-gated ion channels but offer no specificity. In contrast, molecules such as stargazin (3Vandenberghe W. Nicoll R.A. Bredt D.S. J. Neurosci. 2005; 25: 1095-1102Crossref PubMed Scopus (98) Google Scholar), PSD-95 (4Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), 14-3-3 (5Exley R. Moroni M. Sasdelli F. Houlihan L.M. Lukas R.J. Sher E. Zwart R. Bermudez I. J. Neurochem. 2006; 98: 876-885Crossref PubMed Scopus (35) Google Scholar), and RIC-3 (6Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar) (7Halevi 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 (143) Google Scholar, 8Ben-Ami H.C. Yassin L. Farah H. Michaeli A. Eshel M. Treinin M. J. Biol. Chem. 2005; 280: 28053-28060Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 9Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2005; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 10Cheng A. McDonald N.A. Connolly C.N. J. Biol. Chem. 2005; 280: 22502-22507Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Lansdell S.J. Gee V.J. Harkness P.C. Doward A.I. Baker E.R. Gibb A.J. Millar N.S. Mol. Pharmacol. 2005; 68: 1431-1438Crossref PubMed Scopus (123) Google Scholar) are beginning to offer insight into protein-specific chaperone activity. Intriguingly, 14-3-3 has been reported to alter the stoichiometry of α4β2 nACh receptors, as a result of the stabilization of α4 subunits (5Exley R. Moroni M. Sasdelli F. Houlihan L.M. Lukas R.J. Sher E. Zwart R. Bermudez I. J. Neurochem. 2006; 98: 876-885Crossref PubMed Scopus (35) Google Scholar). Similarly, RIC-3 has been reported to promote the folding, assembly, and surface expression of some (α7) nACh receptors yet inhibit the expression of others (α3β4 and α4β2) when expressed in Xenopus oocytes (7Halevi 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 (143) Google Scholar). However, when analyzed in mammalian cells, nACh receptor compositions were either promoted (α7, α8, α3β2, α3β4, α4β2, α4β4) or unaffected (α9, α9α10), with no evidence of inhibition (9Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2005; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 12Lansdell S.J. Gee V.J. Harkness P.C. Doward A.I. Baker E.R. Gibb A.J. Millar N.S. Mol. Pharmacol. 2005; 68: 1431-1438Crossref PubMed Scopus (123) Google Scholar). Similarly, RIC-3 has been shown to promote the functional surface expression of 5-HT3A in mammalian cells (10Cheng A. McDonald N.A. Connolly C.N. J. Biol. Chem. 2005; 280: 22502-22507Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The 5-HT3 receptors in the peripheral nervous system are thought to modulate pain and intestinal and cardiovascular functions (13Thompson A.J. Lummis S.C. Curr. Pharm. Des. 2006; 12: 3615-3630Crossref PubMed Scopus (179) Google Scholar). In the central nervous system, 5-HT3 receptors are important targets for the control of emesis induced by chemotherapy/radiotherapy and have been implicated in schizophrenia (13Thompson A.J. Lummis S.C. Curr. Pharm. Des. 2006; 12: 3615-3630Crossref PubMed Scopus (179) Google Scholar). The 5-HT3A receptor is capable of functioning as a homomeric ion channel. However, the conductance of these recombinant receptors is too small to be resolved directly (sub-picosiemens) and the receptors do not resemble many native neuronal 5-HT3 receptors (14Hussy N. Lukas W. Jones K.A. J. Physiol. 1994; 481: 311-323Crossref PubMed Scopus (91) Google Scholar, 15Fletcher S. Barnes N.M. Trends Pharmacol. Sci. 1998; 19: 212-215Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In contrast, 5-HT3B subunits do not function as homomeric channels but are retained in the endoplasmic reticulum (ER) unless assembled into heteromeric (5-HT3A/B) receptors (16Boyd G.W. Low P. Dunlop J.I. Robertson L.A. Vardy A. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (67) Google Scholar, 17Boyd 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). Electrophysiological analysis suggests that both 5-HT3 receptor types may co-exist within the same neuron (14Hussy N. Lukas W. Jones K.A. J. Physiol. 1994; 481: 311-323Crossref PubMed Scopus (91) Google Scholar, 18Dubin A.E. Huvar R. D'Andrea M.R. Pyati J. Zhu J.Y. Joy K.C. Wilson S.J. Galindo J.E. Glass C.A. Luo L. Jackson M.R. Lovenberg T.W. Erlander M.G. J. Biol. Chem. 1999; 274: 30799-30810Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 19Yang J. Mathie A. Hille B. J. Physiol. 1992; 448: 237-256Crossref PubMed Scopus (85) Google Scholar, 20Morales M. McCollum N. Kirkness E.F. J. Comp. Neurol. 2001; 438: 163-172Crossref PubMed Scopus (55) Google Scholar), raising the question of how this may be achieved. In this study we have investigated the role of RIC-3 isoforms (a and d) (7Halevi 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 (143) Google Scholar) on 5-HT3A and 5-HT3A/B receptor expression in mammalian cells. The two RIC-3 isoforms exhibit overlapping, but distinct, localizations between the ER and Golgi. In keeping with a role in receptor biogenesis, these compartments are involved in protein synthesis, assembly, and N-linked glycosylation. Despite differences in RIC-3 isoform localization and the propensity to aggregate, both isoforms promote the surface expression of homomeric receptors and inhibit the formation of heteromeric receptors. Thus, RIC3 can manipulate 5-HT3 receptor composition. Cell Culture and Transfection—Simian COS7 cells (ATCC CRL 1651) cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 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 μF, Bio-Rad Gene Electropulser II). 10 μg of DNA was used per transfection (2 × 106 cells), using equimolar ratios of expression constructs (unless otherwise stated). Cells were analyzed 12 to 48 h after transfection. DNA Constructs—Human 5-HT3A subunit cDNAs were expressed from the mammalian expression vector pGW1 (16Boyd G.W. Low P. Dunlop J.I. Robertson L.A. Vardy A. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (67) Google Scholar). The 5-HT3Amyc and 5-HT3Bmyc have been reported previously (17Boyd 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). 5-HT3A-YFP was generated as reported previously (21Ilegems 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). 5-HT3B-CFP was generated by PCR to position CFP immediately downstream of the hemagglutinin epitope (YPYDVPDYA/SR/MVSK... DELYK/IE/QDSAL). Underlined are residues introduced as the result of the incorporation of restriction sites for cloning purposes. Both 5-HT3A-YFP and 5-HT3B-CFP were capable of assembling into cell surface-expressed receptors as wild-type receptors. Human RIC-3a was a kind gift from M. Treinin (Hebrew University) and used as previously described (10Cheng A. McDonald N.A. Connolly C.N. J. Biol. Chem. 2005; 280: 22502-22507Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). RIC-3 isoform d and tRIC3 were generated by PCR to generate RIC-3 variants terminating with the sequence... YILFKLSKGK (tRIC-3) or... YILFKVSRIILTILHQ (isoform d). RIC-3-N and RIC-3-C possessing artificial N-glycosylation sites, AFAKANGSGGGAGGGG (K80N for RIC-3-N) and LRKRNGSGLE (P364G, Q365S for RIC-3-C), were generated by site-directed mutagenesis. RIC-3-SS-DsRed was generated by replacing the amino-terminal methionine of DsRed with the amino-terminal 33 residues of RIC-3 isoform a. RIC-3-YFP, RIC-3-pHluorin, and RIC-3-DsRed were generated by PCR, cloning the relevant fluorescent protein between residues 30 and 31 of RIC-3. The fidelity of all constructs was verified by DNA sequencing. The addition of these fluorescent probes to the amino terminus of RIC-3 did not alter their subcellular distribution when compared with untagged RIC-3. Furthermore, these chimeras exhibited the same modulatory behavior on 5-HT3 receptors. ER-Red and Golgi-YFP were purchased from Clontech. Antibodies and Reagents—Rabbit anti-Myc antibodies (Santa Cruz Biotechnology) were used as directed. Antisera (sheep) to RIC-3 were generated as previously reported (10Cheng A. McDonald N.A. Connolly C.N. J. Biol. Chem. 2005; 280: 22502-22507Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and used at 0.5 μg/ml for Western blotting. The secondary horseradish peroxidase-conjugated antibodies were purchased from Perbio and used at 1/5000. Lysotracker-Red (Molecular Probes) and monodansylcadaverine (Sigma) were used as recommended by the supplier. Quantification of Cell Surface Expression—Transfected COS7 cells were plated into 24-well dishes. Cells were fixed in 3% paraformaldehyde (in phosphate-buffered saline) 12–24 h post-transfection. Cell surface detection was performed in the absence of detergent, and total expression levels were determined following Triton X-100 (0.5%, 15 min) treatment. Briefly, following permeabilization (if required) cells were washed twice in 50 mm NH4Cl (in phosphate-buffered saline) and blocked (5% Marvel, 0.5% bovine serum albumin in phosphate-buffered saline) for 1 h. Receptor expression was detected via the extracellular Myc epitope using rabbit anti-Myc for 1 h, followed by five washes in block, and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody for 1 h. Excess antibody was removed by five further washes in block. Horseradish peroxidase levels were assayed using 10 μm Amplex Red (Invitrogen) as the substrate (plus 0.003% H2O2 in sodium phosphate buffer) with excitation at 560 nm and detection at 590 nm after 30 min using a Spectramax Gemini EM plate reader (Molecular Devices). Immunoprecipitation—COS7 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. 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). Triton X-114-soluble and -insoluble fractions were produced using 30 °C, 15 min, to precipitate the detergent/membranes and centrifuged at 15,000 × g. The insoluble pellet was washed twice (washed at 4 °C and then precipitated at 30 °C) prior to returning to 4 °C for immunoprecipitation. Immunoprecipitations were performed as described previously (16Boyd G.W. Low P. Dunlop J.I. Robertson L.A. Vardy A. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (67) Google Scholar) and analyzed by SDS-polyacrylamide gel electrophoresis, followed by autoradiography. Radioligand Binding—[3H]GR65630 binding was performed on intact and transiently transfected COS7 cells cultured in 24-well plates and analyzed 1 day post-transfection. Mock-transfected cells were used as controls. A saturating concentration (3 nm) of [3H]GR65630 was incubated in sextuplicate for 2 h in binding buffer (135 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm D(+)glucose, pH 7.4). Following five washes in binding buffer, cells were solubilized with 0.5% Triton X-100 and counted in a scintillation counter. Photobleaching Studies—Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching in COS7 cells were carried out using a Zeiss LSM510 confocal imaging system incorporating a heated chamber (35 °C). Optimal conditions for photobleaching were determined using a 488-nm argon laser line (100% power, 20 scans). Post-photobleach imaging was performed using minimal laser excitation to reduce indirect bleaching. Much controversy exists over whether the first hydrophobic domain in RIC-3 represents a signal sequence (10Cheng A. McDonald N.A. Connolly C.N. J. Biol. Chem. 2005; 280: 22502-22507Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) or a transmembrane domain (6Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar, 7Halevi 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 (143) Google Scholar, 8Ben-Ami H.C. Yassin L. Farah H. Michaeli A. Eshel M. Treinin M. J. Biol. Chem. 2005; 280: 28053-28060Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Although the original Caenorhabditis elegans RIC-3 clone is not predicted to possess an amino-terminal signal sequence (6Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar), human (NP_078833) and mouse (NP_001033713) RIC-3 do not share homology with the first 48 residues of the C. elegans clone (NP_501299). Thus, it is possible that C. elegans and the mammalian form of RIC-3 are distinct at their amino terminus and may differ with respect to the existence of a cleavable signal sequence or a transmembrane domain. To address this issue, we performed a SignalP3.0 analysis (22Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5634) Google Scholar) on human RIC-3 and determined that a cleavable signal sequence is predicted between amino acids 28 and 29 (AFL-SR) with an S-mean of 0.862 and D-score of 0.601 (Fig. 1A). This compares with that of a well characterized signal sequence from human growth hormone (23Connolly C.N. Futter C.E. Gibson A. Hopkins C.R. Cutler D.F. J. Cell Biol. 1994; 127: 641-652Crossref PubMed Scopus (130) Google Scholar) (AAA72555) that has an S-mean of 0.860 and D-score of 0.807. However, unlike the majority of amino-terminal signal sequences, the amino-terminal 8 amino acids of RIC-3 are not consistent with a hydrophobic signal sequence. Therefore, to determine experimentally whether the predicted signal sequence region is capable of directing protein import into the ER and undergoing cleavage, we generated a chimera incorporating the first 33 amino acids of RIC-3 followed by the sequence of DsRed monomer in which the initial methionine had been removed (RIC-3-SS-DsRed). As expected, the RIC-3-SS-DsRed is localized to the ER, as evidenced by the reticular staining pattern (Fig. 1B). To test whether the signal sequence is cleaved, we probed for the secretion of DsRed into the culture medium. RIC-3-SS-DsRed-transfected cells were pulse-labeled with [35S]methionine and chased overnight in the absence of radiolabel. DsRed was immunoprecipitated from the medium and cell lysates. To distinguish soluble and membrane-bound DsRed within cells, cells were lysed using Triton X-114. Lysates were separated into soluble and insoluble (membrane) fractions prior to immunoprecipitation. DsRed was detected in both the medium and the soluble cellular fraction, confirming that the signal sequence is cleaved (Fig. 1C). It is not clear why two molecular mass forms (∼38- and ∼28-kDa bands) exist, when only one of ∼28 kDa is expected, but this finding is reproducible (n = 3). It is not likely to be due to the presence/absence of the signal sequence as both forms are detected in the medium and soluble fractions. No sites for N-glycosylation exist within DsRed, ruling out different glycosylation. Regardless, these results indicate that the amino-terminal region of RIC-3 is capable of functioning as a cleavable signal sequence. As stated above, RIC-3 has no predicted sites for N-glycosylation with which to probe the membrane topology of RIC-3. Therefore, we introduced two novel sites by site-directed mutagenesis at positions 80 or 363. These are predicted to reside extracellularly (RIC3-N) or intracellularly (RIC3-C), respectively. A comparison of the molecular mass of each protein, in the presence or absence of tunicamycin (to inhibit N-glycosylation) reveals that the molecular mass of RIC3-N is increased by ∼2–3 kDa, consistent with N-glycosylation at this engineered site (Fig. 1D, +). Furthermore, this increase in size is lost in the presence of tunicamycin (Fig. 1D, –). In contrast, no changes in the molecular mass of RIC3-C were evident. These findings support the predicted extracellular location of the pre-TMDI region (downstream from the signal sequence) and the cytoplasmic localization of the carboxyl terminus of RIC-3. Controversy also exists with respect to the subcellular localization of RIC-3. The consensus of opinion supports an endoplasmic reticulum localization of RIC-3 isoform a (RIC-3a) (6Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar, 7Halevi 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 (143) Google Scholar, 10Cheng A. McDonald N.A. Connolly C.N. J. Biol. Chem. 2005; 280: 22502-22507Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Mol. Neurosci. 2006; 30: 153-156Crossref PubMed Scopus (19) Google Scholar). However, RIC-3 has been reported in the Golgi (11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), within intracellular aggregates (11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Mol. Neurosci. 2006; 30: 153-156Crossref PubMed Scopus (19) Google Scholar), and on the cell surface (9Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. J. Biol. Chem. 2005; 280: 1257-1263Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To address this issue, COS7 cells were co-transfected with organelle markers, ER-Red combined with RIC-3-YFP or Golgi-YFP combined with RIC-3-Red (monomeric DsRed). Clearly, RIC-3a is localized predominantly to the ER (Fig. 2A, left panels). Although RIC-3a is not concentrated in the Golgi, it may be present at low levels (11Castillo M. Mulet J. Gutierrez L.M. Ortiz J.A. Castelan F. Gerber S. Sala S. Sala F. Criado M. J. Biol. Chem. 2005; 280: 27062-27068Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Interestingly, in other cells RIC-3a is also present within anomalous structures that are not observed in the absence of RIC-3 expression (Fig. 2A, right panels). Two distinct types of structures can be observed: A diffuse slick of low fluorescent intensity (Fig. 2B, thin arrows) and an intensely fluorescent aggregate (Fig. 2B, wide arrows) that appears hollow when imaged using lower exposures (Fig. 2, A and C, and supplemental Fig. S1). Both these structures occur adjacent to the ER (Fig. 2B and supplemental Fig. S2). Similar structures are evident upon the expression of untagged RIC-3a (not shown), eliminating the possibility that protein aggregation is being induced by YFP or DsRed dimerization. When RIC-3a-YFP is co-expressed with 5-HT3A-CFP, both proteins are observed to co-localize within the ER (not shown) and within both aggregate structures, depending on which structures are evident within the cell (Fig. 2C and supplemental Fig. S3). Similarly, when 5-HT3A-CFP, 5-HT3B-YFP, and RIC-3a-Red are co-expressed in the same cell, all three proteins are co-localized in the ER and aggregates (Fig. 2C and supplemental Fig. S3). That the 5-HT3 receptors do not accumulate in these structures when expressed in the absence of RIC-3a, combined with the fact that the aggregates are induced by RIC-3a expression, suggests that RIC-3a is able to direct 5-HT3 receptors into these structures. To determine specificity, we investigated the co-expression of the γ-aminobutyric acid type A receptor subunit, γ2Long, which remains unassembled within the ER in the absence of α and β subunits (26Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). In contrast to the results observed for 5-HT3 receptors, the γ2Long-Red protein did not co-localize to these RIC-3a-YFP-containing structures (Fig. 2C and supplemental Fig. S3). To address the question of possible cell surface expression of RIC-3a, a fusion incorporating PHluorin, a pH-sensitive variant of GFP that can report on surface labeling when expressed extracellularly was constructed (27Ashby M.C. Ibaraki K. Henley J.M. Trends Neurosci. 2004; 27: 257-261Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The pHluorin moiety is located within the extracellular (as determined by the introduction of an N-glycosylation site) (Fig. 1D) domain of RIC-3. As such, the pHluorin would be sensitive to pH changes in the extracellular environment but only if RIC-3 were expressed on the cell surface. At an extracellular pH of 8, RIC-3a-pHluorin fluorescence is observed with the ER (∼pH 7) and aggregates, with no evidence of surface fluorescence (Fig. 2D, left panels). Given that the aggregates remain visible, it is unlikely that these represent acidic compartments such as endosomes (∼pH 5.5) or lysosomes (<pH 5). Lowering the extracellular pH to 5.5 did not reduce the fluorescence signal, suggesting that little (if any) RIC-3a is present on the cell surface of COS7 cells. Moreover, the subsequent addition of NH4Cl to quench endosomal pH did not reveal any previously masked fluorescence residing within acidic compartments. Identical results were observed when RIC-3a-pHluorin was expressed in primary hippocampal neurons (Fig. 2D, right panels). Thus, when expressed recombinantly, RIC3a exists predominantly within the ER and ER-associated structures, with no evidence of surface or endosomal expression. These experiments were performed rapidly (<2 min), eliminating any significant effects of the treatments on protein trafficking. Given the pleomorphic nature of the RIC-3a aggregates, we investigated the potential for dynamic morphological changes of RIC-3a-YFP and RIC-3a-Red by time-lapse fluorescence microscopy. In the majority of cells possessing the bright aggre" @default.
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• W2051003806 title "Differential Subcellular Localization of RIC-3 Isoforms and Their Role in Determining 5-HT3 Receptor Composition" @default.
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