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• W3122533229 startingPage "43991" @default.
• W3122533229 abstract "The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions. The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions. Withdrawal: Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter: a specific role for sialic acid residuesJournal of Biological ChemistryVol. 294Issue 24PreviewVOLUME 278 (2003) PAGES 43991–44000 Full-Text PDF Open AccessExpression of Concern: Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter: A specific role for sialic acid residues.Journal of Biological ChemistryVol. 294Issue 13PreviewVOLUME 278 (2003) PAGES 43991–44000 Full-Text PDF Open Access The serotonin transporter (SERT) 1The abbreviations used are: SERT, serotonin transporter; rSERT, rat SERT; NET, norepinephrine transporter; DAT, dopamine transporter; CHO, Chinese hamster ovary; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3′-dithiopropionate; PNGase F, peptide N-glycosidase F; MTSEA, (2-aminoethyl)methanethiosulfonate; IP, immunoprecipitation; NEM, N-ethylmaleimide. is a member of the Na+- and Cl--dependent solute carrier family that includes transporters for the biogenic amine neurotransmitters norepinephrine (NET) and dopamine (DAT) (1Blakely R. Berson H. Fremeau Jr., R. Caron M. Peek M. Priace H. Bradley C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (687) Google Scholar, 2Hoffman B. Mezey E. Browstein M. Science. 1991; 254: 579-580Crossref PubMed Scopus (508) Google Scholar, 3Uhl G. Trends Neurosci. 1992; 15: 265-268Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 4Kanner B. J. Exp. Biol. 1994; 196: 237-249Crossref PubMed Google Scholar, 5Amara S. Kuhar M. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 6Ramamoorthy S. Baumen A. Moore K. Han H. Yang-Fen T. Chang A. Ganapathy V. Blakely R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2542-2546Crossref PubMed Scopus (786) Google Scholar, 7Uhl G. Johnson P. J. Exp. Biol. 1995; 196: 229-236Google Scholar, 8Amara S.G. Arriza J.L. Curr. Opin. Neurobiol. 1993; 3: 337-344Crossref PubMed Scopus (127) Google Scholar, 9Patel A. Reith M.E.A. Reith M.E.A. Neurotransmitter Transporters. 2nd Ed. Humana Press Inc., Totowa2002: 355Google Scholar). Following neurotransmission, SERT is responsible for the clearance of serotonin from neurons, platelets, and other cells via a re-uptake mechanism. The transport system for serotonin is the target of many clinically important drugs used in the treatment of a variety of disorders such as cocaine, amphetamines, and antidepressants (1Blakely R. Berson H. Fremeau Jr., R. Caron M. Peek M. Priace H. Bradley C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (687) Google Scholar, 2Hoffman B. Mezey E. Browstein M. Science. 1991; 254: 579-580Crossref PubMed Scopus (508) Google Scholar, 3Uhl G. Trends Neurosci. 1992; 15: 265-268Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 4Kanner B. J. Exp. Biol. 1994; 196: 237-249Crossref PubMed Google Scholar, 5Amara S. Kuhar M. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 6Ramamoorthy S. Baumen A. Moore K. Han H. Yang-Fen T. Chang A. Ganapathy V. Blakely R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2542-2546Crossref PubMed Scopus (786) Google Scholar, 7Uhl G. Johnson P. J. Exp. Biol. 1995; 196: 229-236Google Scholar, 8Amara S.G. Arriza J.L. Curr. Opin. Neurobiol. 1993; 3: 337-344Crossref PubMed Scopus (127) Google Scholar, 9Patel A. Reith M.E.A. Reith M.E.A. Neurotransmitter Transporters. 2nd Ed. Humana Press Inc., Totowa2002: 355Google Scholar, 10Rudnick G. Clark J. Biochim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (370) Google Scholar, 11Blakely R. De Felice L. Hartzell H. J. Exp. Biol. 1994; 196: 263-281Crossref PubMed Google Scholar). Regulation of the transporter function is the key mechanism for the control of neurotransmitter action. Importantly, alterations in SERT and/or its antidepressant-binding activity are reported in patients with major neuropsychiatric disorders, including affective disorders, anxiety disorders, obsessive-compulsive disorders, and autism (12Murphy D.L. Andrews A.M. Wichems C.H. Li Q. Tohda M. Greenberg B. J. Clin. Psychiatry. 1998; 59: 4-12PubMed Google Scholar, 13Stahl S.M. J. Affect. Disord. 1998; 51: 215-235Crossref PubMed Scopus (403) Google Scholar, 14Jones B.J. Blackburn T.P. Pharmacol. Biochem. Behav. 2002; 71: 555-568Crossref PubMed Scopus (203) Google Scholar, 15Kilic F. Murphy D. Rudnick G. Mol. Pharmacol. 2003; 64: 440-446Crossref PubMed Scopus (127) Google Scholar). SERT, NET, and DAT proteins share extensive sequence homology (5Amara S. Kuhar M. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 9Patel A. Reith M.E.A. Reith M.E.A. Neurotransmitter Transporters. 2nd Ed. Humana Press Inc., Totowa2002: 355Google Scholar, 11Blakely R. De Felice L. Hartzell H. J. Exp. Biol. 1994; 196: 263-281Crossref PubMed Google Scholar, 16Blakely R. Ramamoorthy S. Qian Y. Schroeter S. Bradley C. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 29-72Crossref Google Scholar, 17Barker E. Blakely R. Methods Enzymol. 1998; 296: 475-498Crossref PubMed Scopus (19) Google Scholar) with several common structural characteristics, including homo-oligomeric properties (18Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 19Jess U. Betz H. Schloss P. FEBS Lett. 1996; 23: 44-46Crossref Scopus (54) Google Scholar, 20Schmid J. Scholze P. Kudlacek O. Freissmuth M. Singer E. Sitte H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21Kocabas A. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (50) Google Scholar, 22Hastrup H. Karlin A. Javitch J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar, 23Berger S. Farrell K. Conant D. Kempner E. Paul S. Mol. Pharmacol. 1994; 46: 726-731PubMed Google Scholar, 24Torres G. Gainetdinov R. Caron M. Nature. 2003; 4: 13-25Google Scholar, 25Torres G.E. Carneiro A. Seamans K. Fiorentini C. Sweeney A. Yao W.D. Caron M.G. J. Biol. Chem. 2003; 278: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 61Jess U. Betz H. Schloss P. FEBS Lett. 1996; 394: 44-46Crossref PubMed Scopus (54) Google Scholar) and multiple sites for N-linked glycosylation within a large extracellular loop between transmembrane domains 3 and 4 (9Patel A. Reith M.E.A. Reith M.E.A. Neurotransmitter Transporters. 2nd Ed. Humana Press Inc., Totowa2002: 355Google Scholar). N-Glycosylation of SERT (27Gielen W. Viefhöfer B. Experientia (Basel). 1974; 30: 1177-1178Crossref PubMed Scopus (6) Google Scholar, 28Dette G.A. Wesemann W. Experientia (Basel). 1979; 35: 1152-1153Crossref PubMed Scopus (5) Google Scholar, 29Szabados L. Mester L. Michal F. Born G.V. Biochem. J. 1975; 148: 335-336Crossref PubMed Scopus (5) Google Scholar, 30Launay J. Geoffroy C. Mutel V. Buckle M. Cesura A. Alouf J. Da Prada M. J. Biol. Chem. 1992; 267: 11344-11351Abstract Full Text PDF PubMed Google Scholar, 31Tate C.G. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar, 32Tate C.G. Whiteley E. Betenbaugh M. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 33Chen J.-G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (150) Google Scholar), NET (34Nguyen T.T. Amara S.G. J. Neurochem. 1996; 67: 645-655Crossref PubMed Scopus (64) Google Scholar, 35Melikian H. McDonald J. Gu H. Rudnick G. Moore K. Blakely R. J. Biol. Chem. 1994; 269: 12290-12297Abstract Full Text PDF PubMed Google Scholar, 36Melikian H. Ramamoorthy S. Tate C. Blakely R. Mol. Pharmacol. 1996; 50: 266-276PubMed Google Scholar), and DAT (24Torres G. Gainetdinov R. Caron M. Nature. 2003; 4: 13-25Google Scholar, 25Torres G.E. Carneiro A. Seamans K. Fiorentini C. Sweeney A. Yao W.D. Caron M.G. J. Biol. Chem. 2003; 278: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 26Chen N. Vaughan R. Reith M. J. Neurochem. 2001; 77: 1116-1127Crossref PubMed Scopus (67) Google Scholar) is important for their neurotransmitter uptake functions (9Patel A. Reith M.E.A. Reith M.E.A. Neurotransmitter Transporters. 2nd Ed. Humana Press Inc., Totowa2002: 355Google Scholar); however, neither the mechanism by which N-glycosyl groups contribute to the serotonin uptake function nor the degree of glycosyl modification required to maintain the efficient uptake function of SERT is known. Initially, Gielen and Viefhöfer (27Gielen W. Viefhöfer B. Experientia (Basel). 1974; 30: 1177-1178Crossref PubMed Scopus (6) Google Scholar), Dette and Wesemann (28Dette G.A. Wesemann W. Experientia (Basel). 1979; 35: 1152-1153Crossref PubMed Scopus (5) Google Scholar), and Szabados et al. (29Szabados L. Mester L. Michal F. Born G.V. Biochem. J. 1975; 148: 335-336Crossref PubMed Scopus (5) Google Scholar) reported that, at nerve ending membranes, sialic acid-containing structures involved in the serotonin uptake process and sialic acid moieties contribute to the transport function of SERT. Later, during the purification of SERT from human platelets, Launay et al. (30Launay J. Geoffroy C. Mutel V. Buckle M. Cesura A. Alouf J. Da Prada M. J. Biol. Chem. 1992; 267: 11344-11351Abstract Full Text PDF PubMed Google Scholar) demonstrated that SERT has two sialic acid residues on each complex N-linked oligosaccharide molecule. In general, N-glycosylation has an important role in the quality control pathway that ensures correct folding and processing of membrane proteins (37Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar, 38Helenius A. Tatu T. Marquardt T. Braakman I. Rupp R.G. Oka M.S. Cell Biology and Biotechnology. Springer-Verlag, Berlin1992Google Scholar, 39Hurtley S. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (779) Google Scholar, 40Braakman I. Hoover-Litty H. Wagner K. Helenius A. J. Cell Biol. 1991; 114 (401): 401Crossref PubMed Scopus (252) Google Scholar, 41Hurtley S. Bole D. Hoover-Litty H. Helenius A. Copeland C. J. Cell Biol. 1989; 108: 2117-2126Crossref PubMed Scopus (224) Google Scholar). Defects in the glycosylation pathway retain the glycoproteins in the endoplasmic reticulum (42Hammond C. Helenius A. Curr. Biol. 1995; 7: 523-529Crossref Scopus (589) Google Scholar, 43Klausner R. New Biol. 1989; 1: 3-8PubMed Google Scholar). However, removal of glycosylation sites neither keeps SERT in the endoplasmic reticulum nor impairs its expression on the plasma membrane, but reduces its uptake function (31Tate C.G. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar, 32Tate C.G. Whiteley E. Betenbaugh M. J. Biol. Chem. 1999; 274: 17551-17558Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 33Chen J.-G. Liu-Chen S. Rudnick G. Biochemistry. 1997; 36: 1479-1486Crossref PubMed Scopus (150) Google Scholar). In this study, using recombinant SERT overexpressed in Lec4 cells (44Stanley P. Vivona G. Atkinson P. Arch. Biochem. Biophys. 1984; 230: 363-374Crossref PubMed Scopus (15) Google Scholar, 45Chaney W. Sundaram S. Friedman N. Stanley P. J. Cell Biol. 1989; 109: 2089-2096Crossref PubMed Scopus (46) Google Scholar, 46Stanley P. Annu. Rev. Genet. 1984; 18: 525-552Crossref PubMed Scopus (167) Google Scholar, 47Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar) and N-glycosylation site mutants of SERT, QQ (31Tate C.G. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar), overexpressed in Chinese hamster ovary (CHO) cells, we investigated the roles of sialylated N-glycans in SERT function. Lec4 cells are N-acetylglucosamine transferase V-defective, and Lec2 cells are nucleotide cytidine monophosphosialic acid (CMP-sialic acid) transferase-defective CHO cells. Glycoproteins and glycolipids synthesized in Lec2 and -4 mutants have defects in sialic acid content (44Stanley P. Vivona G. Atkinson P. Arch. Biochem. Biophys. 1984; 230: 363-374Crossref PubMed Scopus (15) Google Scholar, 45Chaney W. Sundaram S. Friedman N. Stanley P. J. Cell Biol. 1989; 109: 2089-2096Crossref PubMed Scopus (46) Google Scholar, 46Stanley P. Annu. Rev. Genet. 1984; 18: 525-552Crossref PubMed Scopus (167) Google Scholar, 47Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar). Our overall findings indicate that (i) functional oligomerization requires sialylated N-glycans; (ii) sialylated glycans are not required for normal cell-surface expression; and (iii) sialylated glycans are also required for normal cytoskeletal associations of SERT with myosin IIA. Based on these findings, we propose a structural role for sialylated N-glycans in conferring an optimum conformation to SERT that facilitates homo-oligomerization upon biosynthesis and, in turn, exposes the domain(s) for myosin cytoskeletal associations. To explore if the impaired oligomeric properties of SERT were due to the lack of sialylation or branching, we also used Lec2 cells and found the same result with those using Lec4 cells. Utilizing the Lec mutant cells in this study was a novel approach to elucidate the significance of sialic acid residues in SERT function. Materials—The mutant form of rat SERT (rSERT; Asn208 and Asn217 of the N-linked glycosylation consensus sequences replaced with glutamine residues by site-directed mutagenesis), the QQ construct in plasmid pCGT148 (31Tate C.G. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar), was a generous gift from Dr. C. G. Tate (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK). Lec2 was purchased from American Type Culture Collection (ATCC, CRL 1736) (Manassas, VA). Lec4 cells were a generous gift from Dr. P. Stanley (Albert Einstein College of Medicine, Bronx, NY). Myosin IIA cDNA was kindly provided by Dr. R. Adelstein (NHLBI, NIH, Bethesda, MD). Restriction endonucleases and ligases were from New England Biolabs Inc. (Beverly, MA). Expression vectors, cell culture materials, Lipofectin, and LipofectAMINE 2000 were from Invitrogen. The micro BCA protein assay kit, the enhanced chemiluminescence (ECL) Western blotting system, streptavidin-agarose beads, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3′-dithiopropionate (NHS-SS-biotin), and immunopure horseradish peroxidase-conjugated streptavidin were from Pierce. Mouse monoclonal anti-Myc antibody was from Chemicon International, Inc. (Temecula, CA). Mouse monoclonal anti-FLAG, rabbit polyclonal anti-FLAG, and biotinylated anti-FLAG antibodies and peptide N-glycosidase F (PNGase F) peroxidase-conjugated donkey anti-mouse and anti-rabbit secondary antibodies were from Sigma. Rabbit polyclonal anti-myosin IIA antibody was supplied by Covance Research Products (Denver, PA). CHO and JAR cells were provided by American Type Culture Collection (Manassas, VA). Protein A-Sepharose beads and nonimmune rabbit serum were purchased from Zymed Laboratories Inc. Co. (South San Francisco, CA). [3H]Serotonin was purchased from PerkinElmer Life Sciences. (2-Aminoethyl)methanethiosulfonate (MTSEA) was from Toronto Research Chemicals, Inc. (North York, Ontario, Canada). Plasmids, Constructs, and Cell Line Expression Systems—QQ and rSERT were subcloned into the EcoRV/XbaI and KpnI/NotI sites, respectively, of the pcDNA3 expression vector, placing rSERT expression under the dual control of the cytomegalovirus and T7 RNA polymerase promoters, suitable for expression in CHO cells (52Blakely R. Clark J. Rudnick G. Amara S. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar). A Myc or FLAG epitope was tagged to the N and C termini of rSERT and QQ, respectively. Res-FLAG-QQ (where “Res” is resistant to MTSEA inactivation) and Sens-Myc-QQ (where “Sens” is sensitive to MTSEA inactivation) were prepared with two altered glycosylation sites (i.e. Asn208 and Asn217 to Gln) by site-directed mutagenesis using oligonucleotides 5′-CTCCTGGAACACTG GCCAATGCACCAACTACTTCGCC-3′ and 5′-GCACCAACTACTTCGCCCAGGACCAAATCACCTGGAC-3′. Mutant transporters with a single glycosylation site, Q1 (N208Q) or Q2 (N217Q), were prepared by site-directed mutagenesis with PCR using oligonucleotides 5′-CTCCTGGAACACTGGCCAATGCACCAACTACTTCGCC-3′ and 5′-GCACCAACTACTTCGCCCAGGACCAAATCACCTGGAC-3′, respectively. We confirmed the subcloning processes by sequencing the genes at the University of Arkansas for Medical Sciences DNA Sequencing Facility. Lec and CHO cells were maintained in α-minimal essential medium, and JAR cells in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Neither CHO nor Lec cells endogenously express rSERT proteins. To measure whole cell and surface expression and protein-protein interactions, these cells were transiently transfected with rSERT constructs using a 1:2 ratio of LipofectAMINE 2000 reagent in Opti-MEM. Cells were used in biotinylation, immunoblotting, and immunoprecipitation (IP) assays 24 h post-transfection. Anti-SERT Antibody—Blakely and co-workers (53Qian Y. Melikian H.E. Rye D.B. Levey A.I. Blakely R.D. J. Neurosci. 1995; 15: 1261-1274Crossref PubMed Google Scholar) successfully raised an antibody (CT2) against the C-terminal 34 amino acids of SERT. Using this approach as a guide, Proteintech Group, Inc. (Chicago, IL) synthesized a peptide corresponding to the last 26 amino acids of the SERT C terminus (positions 586–630) and generated a polyclonal antibody against this synthetic peptide. This is a portion of the protein not affected by glycosylation mutation and thus should recognize wild-type and QQ transporters equally well. The synthetic peptide sequence (positions 586–630) matches the C terminus of rSERT, a highly conserved region across different SERT species, but divergent from other gene family members (5Amara S. Kuhar M. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar). We first purified this antibody by standard affinity purification via peptide-Sepharose procedures (54Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) and then biotinylated the purified antibody following the manufacturer's instruction (Amersham Biosciences). Briefly, the synthetic peptide corresponding to the last 26 amino acids of the C terminus of SERT was dissolved in coupling buffer (0.1 m NaHCO3 and 0.5 m NaCl (pH 8.0)) and bound to CNBr-activated Sepharose 4B beads (Amersham Biosciences) that were previously washed with 1 mm HCl by overnight incubation at 4 °C. First, the column was packed, and crude antibody was run through the column. After washing the beads with 10 column volumes of phosphate-buffered saline, the antibody was eluted sequentially with 0.1 m glycine (pH 3.0) to tubes containing 50 ml of 1 m Tris (pH 8.9). The protein concentrations of each fraction were obtained using the BCA protein assay kit, and protein-containing fractions were stored at -80 °C. Immunoblot Analysis—Following transfection, cells were solubilized in phosphate-buffered saline containing 0.44% SDS, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture. The protease inhibitor mixture contained 5 mg/ml pepstatin and 5 mg/ml leupeptin, and 5 mg/ml aprotinin was included in each lysis buffer (18Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar), which also contained the alkylating agent N-ethylmaleimide (NEM) at a final concentration of 5 mm to prevent oxidation and formation of nonspecific disulfide bonds during lysis and to retain the native monomeric structures in the gel (21Kocabas A. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (50) Google Scholar). Samples were analyzed by 10% SDS-PAGE (55Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and transferred to nitrocellulose membrane (56Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). Immunoblot analysis was performed first with anti-Myc (diluted 1:2500), biotinylated anti-FLAG (diluted 1:1000), or biotinylated anti-SERT (diluted 1:1000) antibody and then with horseradish peroxidase-conjugated streptavidin or horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (diluted 1:10,000), respectively. The signals were visualized using the ECL Western blotting detection system. Blots were visualized using a VersaDoc 1000 gel visualization and analysis system (Bio-Rad). Co-immunoprecipitation—Protein-protein transporter interaction was demonstrated by coexpressing two different protein forms in CHO or Lec cells at a 1:1 ratio. Following transfection, CHO, Lec, or JAR cells were treated with 10 mm NEM for 30 min. Cells were lysed in IP buffer (55 mm triethylamine (pH 7.5), 111 mm NaCl, 2.2 mm EDTA, and 0.44% SDS + 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture) (18Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar) containing 5 mm NEM. Cell lysates were first precleared by incubation with nonimmune rabbit serum and protein A for 1 h and then centrifuged. The precleared lysate was combined with an equal volume of a 1:1 slurry of rabbit anti-mouse protein A-Sepharose beads as described previously for primary IP with the antibody (21Kocabas A. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (50) Google Scholar) and then mixed overnight at 4 °C. The lysate immune complexes were recovered by brief centrifugation in a bench-top micro-centrifuge (Beckman), washed several times with high- and low-salt IP buffers, and eluted in Laemmli sample buffer (50 mm Tris-HCl (pH 6.8), 2% SDS, 0.1% bromphenol blue, 10% glycerol, and 1% β-mercaptoethanol). For the nonreducing condition, 5 mm NEM was included in Laemmli sample buffer containing no β-mercaptoethanol. Samples were separated on a 10% SDS-polyacrylamide gel (55Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). After electrophoresis, gels were analyzed by immunoblotting with either biotinylated monoclonal anti-FLAG antibody (diluted 1:1500) or polyclonal anti-myosin IIA antibody (diluted 1:1000) to demonstrate self-association or myosin IIA association, respectively. The signals were developed with the ECL detection system. Cell-surface Biotinylation—Following transfection, cells were biotinylated with the membrane-impermeant biotinylating reagent NHS-SS-biotin as described previously (18Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 21Kocabas A. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (50) Google Scholar). This reagent selectively modifies only the external lysine residues on membrane proteins. All SERT proteins used in this study, with or without epitope tagging, contained the same number of external lysine residues and were expected to react equally with NHS-SS-biotin. Following biotinylation, cells were treated with 100 mm glycine to complete quenching of the unreacted NHS-SS-biotin and lysed in Tris-buffered saline containing 1% SDS, 1% Triton X-100, and protease inhibitor mixture/phenylmethylsulfonyl fluoride. The biotinylated proteins were recovered with streptavidin-agarose beads during overnight incubation. Biotinylated proteins were eluted and separated on SDS-polyacrylamide gel. Immunoblot analysis was performed using anti-SERT, anti-Myc, or anti-FLAG antibody. The primary antibody was detected using horseradish peroxidase-conjugated secondary antibodies and the ECL detection system. PNGase F Treatment—CHO and Lec cells transiently transfected with transporters were first lysed in IP buffer containing NEM at a final concentration of 5 mm. The cell lysate was then treated with rabbit anti-mouse protein A-Sepharose beads coated with biotinylated anti-SERT antibody. The lysate immune complexes were recovered, washed, and then eluted in sample buffer supplemented with 0.4 units/ml PNGase F. After a 3-h incubation at 37 °C (34Nguyen T.T. Amara S.G. J. Neurochem. 1996; 67: 645-655Crossref PubMed Scopus (64) Google Scholar, 35Melikian H. McDonald J. Gu H. Rudnick G. Moore K. Blakely R. J. Biol. Chem. 1994; 269: 12290-12297Abstract Full Text PDF PubMed Google Scholar), the reaction mixture was separated by SDS-PAGE, and immunoblot analysis was performed using horseradish peroxidase-conjugated streptavidin and the ECL blotting system. Transport Assay—CHO and Lec cells in 24-well culture plates were infected with recombinant vTF-7 vaccinia virus, which works with T7 RNA polymerase (52Blakely R. Clark J. Rudnick G. Amara S. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar), and transfected with plasmids bearing mutant SERT cDNA under the control of the T7 promoter as described previously (18Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar). Transfected cells were incubated for 16–20 h at 37 °C and then used to assay serotonin uptake by incubation with 20.5 nm [1,2-3H]serotonin (3400 cpm/pmol) in phosphate-buffered saline containing 0.1 mm CaCl2 and 1 mm MgCl2 for 10 min. The intact cells were washed quickly with ice-cold phosphate-buffered saline, lysed in SDS, transferred to scintillation vials, and counted (18Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar). Cells with no plasmid DNA (mock-transfected) were negative controls. Data Analysis—Nonlinear regression fits of experimental and calculated data were performed with Origin (MicroCal Software, Northampton, MA), which uses the Marquardt-Levenberg nonlinear least-squares curve-fitting algorithm. Each figure shows a representative experiment that was performed at least twice. The statistical analysis given under “Results” is from multiple experiments. Data with error bars represent means ± S.D. of triplicate samples. Glycosylation Pattern of SERT—We first evaluated expression of SERT proteins and their delivery to the cell surface in both model systems. After transfection, expression of wild-type" @default.
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• W3122533229 title "Glycosyl Modification Facilitates Homo- and Hetero-oligomerization of the Serotonin Transporter" @default.
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