Artigo Acesso aberto Revisado por pares

Glycine Transporter Dimers

2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês

10.1074/jbc.m800622200

ISSN

1083-351X

Autores

Ingo Bartholomäus, Laura Milan‐Lobo, Annette Nicke, Sébastien Dutertre, Hanne Hastrup, Alok Jha, Ulrik Gether, Harald H. Sitte, Heinrich Betz, Volker Eulenburg,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Different Na+/Cl--dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuTAa, as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs. Different Na+/Cl--dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuTAa, as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs. After presynaptic release and postsynaptic receptor activation, neurotransmitters have to be rapidly removed from the synaptic cleft in order to allow synaptic transmission to proceed with high spatial and temporal resolution. This is achieved by neurotransmitter transporters located in the plasma membrane of nerve terminals and adjacent glia cells. The family of Na+/Cl--dependent neurotransmitter transporters (SLC6a) includes transporters for γ-aminobutyric acid (GAT1 to -3), 5The abbreviations used are: GAT, γ-aminobutyric acid transporter; CFP, cyan fluorescent protein; CRFR1, corticotropine-releasing factor receptor 1; CuP, copper o-phenanthroline; DAT, dopamine transporter; EL, extracellular loop; FRET, fluorescence resonance energy transfer; FRETc, corrected FRET; GlyT, glycine transporter; HEK 293T cells, human embryonic kidney 293T cells; His, heptahistidyl; NET, norepinephrine transporter; PBS, phosphate-buffered saline; SERT, serotonin transporter; TM, transmembrane domain; YFP, yellow fluorescent protein; WT, wild type; NTA, nitrilotriacetic acid. glycine (GlyT1 and -2), dopamine (DAT), serotonin (SERT), and norephinephrine (NET) (1Torres G.E. Gainetdinov R.R. Caron M.G. Nat. Rev. Neurosci. 2003; 4: 13-25Crossref PubMed Scopus (738) Google Scholar, 2Gether U. Andersen P.H. Larsson O.M. Schousboe A. Trends Pharmacol. Sci. 2006; 27: 375-383Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 3Eulenburg V. Armsen W. Betz H. Gomeza J. Trends Biochem. Sci. 2005; 30: 325-333Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). All of these transporters display significant sequence similarity and share a common membrane topology with 12 transmembrane segments (TMs), a large second extracellular loop (EL2) connecting TM3 and TM4, and cytoplasmic N- and C-terminal regions. Although the functional unit of Na+/Cl--dependent transporters is thought to be a monomer, there is increasing evidence that these transporters form dimers or even higher oligomers within the plasma membrane (4Farhan, H., Freissmuth, M., and Sitte, H. H. (2006) Handb. Exp. Pharmacol. 233-249Google Scholar). Oxidative treatment of the DAT produces dimers and tetramers due to intermolecular disulfide bond formation between two cysteine residues, Cys243 and Cys306, located within TM4 and at the end of EL3, respectively (5Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar, 6Hastrup H. Sen N. Javitch J.A. J. Biol. Chem. 2003; 278: 45045-45048Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, chemical cross-linking and co-isolation of differentially tagged transporter polypeptides has shown that the recombinant SERT forms oligomers in human embryonic kidney (HEK) 293 cells (7Jess U. Betz H. Schloss P. FEBS Lett. 1996; 394: 44-46Crossref PubMed Scopus (54) Google Scholar, 8Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar). Furthermore, for the GAT, DAT, and SERT it has been shown that co-expression of the transporter polypeptides fused to cyan fluorescent protein (CFP) with the respective yellow fluorescent protein (YFP)-tagged protein results in a Förster resonance energy transfer (FRET) signal, suggesting that at least dimers of these transporters exist (9Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 10Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). A possible oligomerization interface has been identified within TM2 of GAT1, where both the disruption of a leucine heptad motif and substitution of polar amino acid residues caused intracellular retention of the transporter and a loss of the FRET signal (11Scholze P. Freissmuth M. Sitte H.H. J. Biol. Chem. 2002; 277: 43682-43690Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12Korkhov V.M. Farhan H. Freissmuth M. Sitte H.H. J. Biol. Chem. 2004; 279: 55728-55736Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Also, x-ray crystallography of a bacterial ortholog of the SLC6a transporter family, LeuTAa, revealed that this transporter forms homodimers (13Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1360) Google Scholar). Together, all of these data support the idea of Na+/Cl--dependent neurotransmitter transporters being oligomeric proteins. There is, however, one reported exception from this rule, the glycine transporters GlyT1 and GlyT2 (14Horiuchi M. Nicke A. Gomeza J. Aschrafi A. Schmalzing G. Betz H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1448-1453Crossref PubMed Scopus (45) Google Scholar, 15Lopez-Corcuera B. Alcantara R. Vazquez J. Aragon C. J. Biol. Chem. 1993; 268: 2239-2243Abstract Full Text PDF PubMed Google Scholar, 16Haugeto O. Ullensvang K. Levy L.M. Chaudhry F.A. Honore T. Nielsen M. Lehre K.P. Danbolt N.C. J. Biol. Chem. 1996; 271: 27715-27722Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). These transporters mediate the uptake of the inhibitory neurotransmitter glycine from the extracellular space into glial cells (GlyT1) and glycinergic neurons (GlyT2), respectively (3Eulenburg V. Armsen W. Betz H. Gomeza J. Trends Biochem. Sci. 2005; 30: 325-333Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Studies on GlyT-deficient mice have shown that, after birth, GlyT1 is essential for removing glycine from postsynaptic glycine receptors (17Gomeza J. Hulsmann S. Ohno K. Eulenburg V. Szoke K. Richter D. Betz H. Neuron. 2003; 40: 785-796Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), whereas GlyT2 is required for the reuptake of glycine into the presynaptic terminal (18Gomeza J. Ohno K. Hulsmann S. Armsen W. Eulenburg V. Richter D.W. Laube B. Betz H. Neuron. 2003; 40: 797-806Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Additionally, GlyT1 has been shown to be involved in the regulation of glutamatergic neurotransmission by controlling the occupancy of the glycine binding site of the N-methyl-d-aspartate subtype of glutamate receptors (19Dingledine R. Kleckner N.W. McBain C.J. Adv. Exp. Med. Biol. 1990; 268: 17-26Crossref PubMed Scopus (40) Google Scholar, 20Gabernet L. Pauly-Evers M. Schwerdel C. Lentz M. Bluethmann H. Vogt K. Alberati D. Mohler H. Boison D. Neurosci. Lett. 2005; 373: 79-84Crossref PubMed Scopus (76) Google Scholar, 21Tsai G. Ralph-Williams R.J. Martina M. Bergeron R. Berger-Sweeney J. Dunham K.S. Jiang Z. Caine S.B. Coyle J.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8485-8490Crossref PubMed Scopus (176) Google Scholar). Evidence for a predominantly monomeric state of these transporters came from two independent studies that failed to detect oligomeric GlyTs within the plasma membrane. Both hydrodynamic analysis of GlyT1 solubilized from rat spinal cord (15Lopez-Corcuera B. Alcantara R. Vazquez J. Aragon C. J. Biol. Chem. 1993; 268: 2239-2243Abstract Full Text PDF PubMed Google Scholar) and affinity purification of surface-labeled recombinant GlyT1 and GlyT2 followed by blue native PAGE (14Horiuchi M. Nicke A. Gomeza J. Aschrafi A. Schmalzing G. Betz H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1448-1453Crossref PubMed Scopus (45) Google Scholar) provided evidence for GlyTs being monomers at the cell surface. Dimers and oligomers were only detected in intracellular membranes and suggested to represent overexpression artifacts (14Horiuchi M. Nicke A. Gomeza J. Aschrafi A. Schmalzing G. Betz H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1448-1453Crossref PubMed Scopus (45) Google Scholar). Here, we used a combination of mutagenesis and cysteine-mediated cross-linking as well as FRET analysis in living cells to reexamine whether GlyTs form oligomers. Our data indicate that these transporters are dimers not only in intracellular compartments but also in the plasma membrane of HEK 293T cells. Homology Modeling—The crystal structure of the bacterial leucine transporter LeuTAa (Protein Data Bank code 2A65) was used as a template to build a homology model of GlyT2. To this end, the amino acid sequence of GlyT2 (accession no. Q761V0) (22Ebihara S. Yamamoto T. Obata K. Yanagawa Y. Biochem. Biophys. Res. Commun. 2004; 317: 857-864Crossref PubMed Scopus (24) Google Scholar) was truncated at its N and C termini (residues 1-192 and 747-799, respectively), and the large EL2 (residues 313-364) was deleted. The remaining sequence was aligned with LeuTAa according to Yamashita et al. (13Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1360) Google Scholar). Three-dimensional models (10 structures) of GlyT1 and GlyT2 were built from the aligned sequences on a Silicon Graphics Octane R12000 work station using the MODELLER program (23Fiser A. Sali A. Methods Enzymol. 2003; 374: 461-491Crossref PubMed Scopus (1372) Google Scholar). The models resulting in the lowest root mean square deviation as compared with the original LeuTAa structure were retained for analysis without further refinement. Dimers of GlyT2 were created by juxtaposing two transporter molecules using Thr464 as an anchoring point. Figures were generated using PyMOL software (Delano Scientific, Palo Alto, CA). cDNA Constructs and Heterologous Expression—An expression construct for the human GlyT1c was kindly provided by Dr. Katherine Fisher (Groton Laboratories, Pfizer, NY). The GlyT2 cDNA was isolated from mouse brain stem mRNA using standard cloning techniques. N-terminal heptahistidyl (His), FLAG, and Myc tags were added by PCR-based mutagenesis. After subcloning into the pcDNA3.1+ vector (Invitrogen), the respective substitutions were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). For fluorescence analysis, the coding regions of GlyT1 and GlyT2 were subcloned by PCR into pECFP-C1 or pEYFP-C1 (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France) to create CFP- or YFP-tagged GlyT1 or GlyT2, respectively. All constructs were verified by sequencing, and all surface-expressed transporters were shown to be functional upon heterologous expression in HEK 293T cells as revealed by [3H]glycine uptake measurements (data not shown). An expression construct for the human DAT (24Giros B. el Mestikawy S. Godinot N. Zheng K. Han H. Yang-Feng T. Caron M.G. Mol. Pharmacol. 1992; 42: 383-390PubMed Google Scholar) was kindly provided by Dr. Marc G. Caron (Duke University, Durham, NC), and a membrane-bound form of YFP (25Hein P. Frank M. Hoffmann C. Lohse M.J. Bunemann M. EMBO J. 2005; 24: 4106-4114Crossref PubMed Scopus (180) Google Scholar) was kindly provided by Viacheslav Nikolaev (University of Würzburg, Germany). HEK 293T cells were grown in modified Eagle's medium supplemented with glutamine (2 mm), 10% (v/v) fetal calf serum, penicillin (50 units/ml), streptomycin (50 μg/ml), and 50 μm β-mercaptoethanol (all reagents from Invitrogen) at 37 °C in a humidified 5% CO2 atmosphere. Cells were seeded on the day prior to transfection into 6-well plates, and transfection was performed at 70-85% confluence using the Polyfect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For GlyT1 expression, GripTite 293 cell lines (Invitrogen) stably expressing GlyT1, GlyT1L343C, and GlyT1C116A;L343C were generated and seeded 48 h before the experiment. Oxidative Cross-linking—Cross-linking experiments were performed 48-72 h after transfection. Adherent cells were washed twice with phosphate-buffered saline (PBS) or HEPES buffered salt solution (130 mm NaCl, 1.3 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 2 mm CaCl2, 10 mm glucose, 10 mm NaOH/HEPES, pH 7.4) and incubated with 0.1-0.5 mm CuSO4 and 0.8-2 mm o-phenanthroline (CuP) in PBS or HEPES buffered salt solution for 10 min at room temperature. The reaction was stopped by removal of the reagent and incubation of the cells with 10 mm N-ethylmaleimide in PBS for 15-20 min. Western Blotting—Cells were lysed at 4 °C in lysis buffer containing 150 mm NaCl, 5 mm EDTA, 1% (v/v) Triton X-100, 0.25% (w/v) desoxycholate, 0.1% (w/v) SDS, 1 mm Pefabloc SC (Roche Applied Science), and 50 mm HEPES/Tris, pH 7.4. After centrifugation for 15 min at 16,000 × g, the protein content in the supernatant was determined using the Bio-Rad Protein assay (Bio-Rad, Munich, Germany), and 10 μg of supernatant protein were mixed with the appropriate volume of 4× nonreducing loading buffer (Invitrogen) prior to separation by SDS-PAGE on 3-8% Tris acetate gels (Invitrogen). Proteins were transferred to either nitrocellulose (Whatman, Dassel, Germany) or polyvinylidene fluoride (Amersham Biosciences) membranes. The membranes were blocked in Tris-buffered saline supplemented with 0.1% (w/v) Tween 20 and 5% (w/v) nonfat milk powder for at least 30 min prior to a ≥30-min incubation with primary antibodies directed against either the N terminus of GlyT2 (polyclonal rabbit) (18Gomeza J. Ohno K. Hulsmann S. Armsen W. Eulenburg V. Richter D.W. Laube B. Betz H. Neuron. 2003; 40: 797-806Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), DAT (polyclonal rabbit, Chemicon, Temecula, CA), the Myc epitope (polyclonal rabbit, Abcam, Cambridge, UK) (all diluted 1:2000 in blocking buffer), or the FLAG epitope (monoclonal mouse; Sigma; 4 μg/ml). After washing with Tris-buffered saline containing 0.5% (w/v) Tween 20, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies (Invitrogen) and detected with the SuperSignal kit (Pierce). Affinity Purification of His-tagged GlyT2—HEK 293T cells were transfected with the indicated constructs and, 48 h later, lysed in 100 mm sodium phosphate buffer, pH 8.0, containing 0.2% (w/v) dodecylmaltoside and 1 mm Pefabloc SC. The lysates were centrifuged at 16,000 × g for 15 min, and 190 μg of supernatant protein were incubated under continuous agitation with Ni2+-NTA-agarose beads (Qiagen, Hilden, Germany) in the aforementioned lysis buffer supplemented with 10 mm imidazole for 1 h at 4 °C. After washing with lysis buffer containing 30 mm imidazole and 0.075% (w/v) dodecylmaltoside, agarose-bound proteins were eluted with 2× SDS loading buffer (Invitrogen) for 10 min at 25 °C. Functional Characterization of GlyT2—His-GlyT2WT or His-GlyT2T464C was expressed in Xenopus laevis oocytes as described previously (14Horiuchi M. Nicke A. Gomeza J. Aschrafi A. Schmalzing G. Betz H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1448-1453Crossref PubMed Scopus (45) Google Scholar). Glycine (30 μm)-induced currents were monitored using the two-electrode voltage clamp technique as described previously (26Dutertre S. Nicke A. Lewis R.J. J. Biol. Chem. 2005; 280: 30460-30468Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The oocytes were treated with 5 mm CuP for 15 min, and glycine-induced currents were measured again. To analyze the efficacy of CuP-induced cross-linking, His-tagged proteins were isolated from detergent extracts of these oocytes as described (14Horiuchi M. Nicke A. Gomeza J. Aschrafi A. Schmalzing G. Betz H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1448-1453Crossref PubMed Scopus (45) Google Scholar) and subjected to Western blot analysis. Dimers of 200 kDa were only seen after CuP treatment of His-GlyT2T464C but not of untreated or His-GlyT2WT-expressing cells (data not shown). Surface Biotinylation—Surface biotinylation of transfected HEK 293T cells with 1 mm NHS-SS-biotin (Pierce) was performed essentially as described (27Eulenburg V. Becker K. Gomeza J. Schmitt B. Becker C.M. Betz H. Biochem. Biophys. Res. Commun. 2006; 348: 400-405Crossref PubMed Scopus (58) Google Scholar). After biotinylation, cell lysates (50 μg of protein) were incubated with streptavidin-agarose beads (60 μl; Pierce) for 3 h at 4 °C. The beads were then washed three times with lysis buffer, and bound biotinylated proteins were eluted by a 30-min incubation at 4 °C with 30 μl of 1× loading buffer (Invitrogen) supplemented with 0.6 mm dithiothreitol. Aliquots of the lysates (15 μg of protein) and biotinylated fractions (30 μl) were then analyzed by SDS-PAGE and Western blotting. Fluorescence Resonance Energy Transfer—FRET signals (28Schmid J.A. Sitte H.H. Curr. Opin. Oncol. 2003; 15: 55-64Crossref PubMed Scopus (45) Google Scholar) were measured with an epifluorescence microscope (Carl Zeiss Axiovert 200) using the "three-filter method" according to Xia and Liu (29Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Images were taken from HEK 293 cells maintained and transfected as described previously (30Seidel S. Singer E.A. Just H. Farhan H. Scholze P. Kudlacek O. Holy M. Koppatz K. Krivanek P. Freissmuth M. Sitte H.H. Mol. Pharmacol. 2005; 67: 140-151Crossref PubMed Scopus (103) Google Scholar) using a ×63 oil objective and a LUDL filter wheel that allows for rapid exchange of filters. The system was equipped with the following fluorescence filters: CFP filter (ICFP; excitation = 436 nm, dichroism = 455 nm, emission = 480 nm), YFP filter (IYFP; excitation = 500 nm, dichroism = 515 nm, emission = 535 nm), and FRET filter (IFRET; excitation = 436 nm, dichroism = 455 nm, emission = 535 nm). The acquisition of the images was done with MetaMorph version 4.6. (Molecular Devices Corp., Downingtown, PA). Background fluorescence was subtracted from all images, and fluorescence intensity was measured at the plasma membrane and in cytosolic regions in all images. To calculate a normalized FRET signal (NFRET), we used the following equation, NFRET=IFRET−a×IYFP−b×ICFPIYFP×ICFP(Eq. 1) where a and b represent the bleed-through values for YFP and CFP. All NFRET values are expressed as means ± S.E. Corrected FRET (FRETc) images were obtained according to Ref. 31Sorkin A. McClure M. Huang F. Carter R. Curr. Biol. 2000; 10: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar. Briefly, after background subtraction from all three images, CFP and YFP images were multiplied with their corresponding bleed-through value. The following equation was used for the calculation of FRETc images, FRETc = FRET - (b × CFP) - (a × YFP). Confocal Microscopy—GlyT cell surface expression was visualized by confocal microscopy using a Zeiss Axiovert 200-LSM 510 confocal microscope (argon laser, 30 milliwatts; helium/neon laser, 1 milliwatt) equipped with an oil immersion objective (Zeiss Plan-Neofluar ×40/1.3). In brief, HEK 293 cells transfected with the indicated construct were seeded onto glass coverslips and examined 1 day later. In co-expression experiments, fluorescent protein-tagged constructs were detected with a band pass filter (475-525 nm) using the 458-nm (for CFP, at 30-45% input power) or 488 nm (for YFP, at 8-10% input power) laser lines. Plasma membranes were visualized after the addition of 20 μl of trypan blue (0.05% (w/v) in PBS) at an excitation wavelength of 543 nm with a long pass filter (585 nm). CFP and trypan blue images were captured sequentially, and overlay images were produced with Zeiss imaging software as described (32Korkhov V.M. Holy M. Freissmuth M. Sitte H.H. J. Biol. Chem. 2006; 281: 13439-13448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). For the DAT, residue Cys306, located at the end of EL3 between TM5 and TM6, has been shown to be essential for intermolecular disulfide bond formation upon oxidative treatment (5Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar). Sequence comparison revealed that in the GlyTs, leucine and threonine residues are found at the homologous positions (Leu343 in GlyT1c and Thr464 in GlyT2; Fig. 1A). To assess whether these residues reside at the surfaces of the respective transporter proteins and might be close to a possible dimerization interface, we generated a model of GlyT2 by using the crystal structure of LeuTAa as a template (13Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1360) Google Scholar). Regions that did not display significant homology to the LeuTAa protein, like the intracellular N and C termini and the large EL2 between TM3 and TM4, were deleted from the GlyT2 sequence to optimize sequence alignment. The resulting GlyT2 model showed a root mean square deviation of 3.62 Å from the LeuTAa structure when 498 of 504 pairwise Cα atoms were aligned (root mean square deviation of 1.157 Å for 398 Cα atoms). In this model, the side chain of Thr464 was located on the surface of the transporter above the helix formed by TM11 (Fig. 1, B and C). Thus, Thr464 might be close to a possible dimerization interface. Apposition of two monomers at this interface created a GlyT2 dimer, in which the two Thr464 residues are in close proximity (Fig. 1, B-E). This dimeric model suggests that, in addition to interactions between the EL3 regions of the monomers, side chains of TM11 could contribute to dimer stabilization. Like-wise, in a similar model of GlyT1, Leu343, the amino acid residue corresponding to Thr464 in GlyT2 and Cys306 in DAT, is predicted to be localized at the surface of the transporter protein (data not shown). To establish whether Thr464 is indeed located at a dimerization interface of GlyT2, we replaced this residue by a cysteine in order to examine whether GlyT2 oligomers can be stabilized by intermolecular disulfide cross-linking, as reported for the DAT (5Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar). As controls, we generated two GlyT2 expression constructs, in which residues Lys462 and Thr557 were substituted by cysteines. According to our model, both residues are unlikely to face the proposed dimerization interface (Fig. 1, B-E). HEK 293T cells were transfected with these three cysteine-substituted GlyT2 constructs and analyzed for [3H]glycine uptake activity and subcellular localization of GlyT2 immunoreactivity. This did not reveal any significant differences as compared with wild-type GlyT2 (GlyT2WT; data not shown). Western blot analysis of detergent extracts prepared from the transfected cells confirmed that the mutant transporters were expressed in amounts comparable with that obtained with the GlyT2WT cDNA (Fig. 2C). Two bands of 65 and 90 kDa, which represent the core-glycosylated and fully glycosylated transporter forms (33Nunez E. Aragon C. J. Biol. Chem. 1994; 269: 16920-16924Abstract Full Text PDF PubMed Google Scholar), were seen with all three mutant proteins. An additional band at about 155 kDa present in variable amounts presumably corresponded to SDS-resistant dimers of immature transporter polypeptides (14Horiuchi M. Nicke A. Gomeza J. Aschrafi A. Schmalzing G. Betz H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1448-1453Crossref PubMed Scopus (45) Google Scholar). Together, these data indicate that none of the cysteine substitutions introduced affected the expression, function, or plasma membrane insertion of GlyT2 in HEK 293T cells. The GlyT2T464C Mutant Efficiently Forms Dimers—To determine whether position Thr464 is close to a potential dimerization interface of GlyT2, HEK 293T cells transiently expressing GlyT2WT, the GlyT2T464C mutant, or, as a control, DATWT were treated with the oxidizing agent CuP, followed by detergent extraction, nonreducing SDS-PAGE, and Western blot analysis. With DATWT-expressing cells, CuP treatment resulted in the appearance of a distinct band at 170 kDa in addition to immunoreactive bands at 85 and 57 kDa (Fig. 2A), which represent the mature and immature monomeric forms of this transporter (5Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar). The 170 kDa band corresponds in size to the previously described DATWT dimer (5Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar), thereby confirming a close proximity of the Cys306 residues at the dimer interface. Consistent with previous findings (5Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar), the formation of DAT dimers by CuP was completely abolished when Cys306 was replaced by an alanine (DATC306A; Fig. 2A). In contrast to these results obtained with DATWT, CuP treatment of cells expressing GlyT2WT did not result in the formation of SDS-resistant GlyT2 oligomers (Fig. 2B, left). However, CuP treatment of GlyT2T464C-expressing cells generated a major GlyT2-immunoreactive adduct of 200 kDa. This was accompanied by a strong decrease in the intensity of the 90 kDa band, which represents the fully glycosylated GlyT2 monomer (Fig. 2B, left). These results are consistent with GlyT2T464C forming a dimer that is cross-linked upon oxidative treatment. To exclude the possibility that this 200-kDa adduct represents an unspecific aggregate induced by CuP treatment, detergent extracts from CuP-treated cells expressing GlyT2T464C were supplemented with β-mercaptoethanol. This reducing treatment resulted in a strong reduction of the 200 kDa band and an increase in the intensity of the mature monomer band at 90 kDa indicative of cleavage of the disulfide cross-link (Fig. 2B, right). Notably, in contrast to what was found with GlyT2T464C, CuP treatment of GlyT2T557C- or GlyT2K462C-expressing cells failed to produce the 200-kDa GlyT2 immunoreactive adduct (Fig. 2C). Thus, disulfide-mediated cross-linking of the GlyT2T464C mutant is position-specific. The functional consequences of CuP cross-linking were analyzed by measuring glycine-induced currents by two-electrode voltage clamp in Xenopus oocytes expressing His-GlyT2WT or His-GlyT2T464C before and after treatment with CuP. Application of 30 μm glycine induced currents of 76 ± 8 pA for GlyT2WT and 39 ± 5 pA for GlyT2T464C (n = 6). The smaller current monitored for the GlyT2T464C mutant most likely reflects a slightly reduced expression also seen in Western blots prepared from detergent extracts from the oocytes (data not shown). After treatment with CuP, the currents recorded from the same oocytes were not significantly reduced as compared with the currents measured before cross-linking (reduction of 18 ± 13% for His-GlyT2WT and of 15 ± 12% for His-GlyT2T464C, as compared with the untreated controls; p ≥ 0.5). Similar to what was seen in HEK 293T cells, treatment of His-GlyT2T464C but not His-GlyT2WT-expressing oocytes with CuP resulted in the appearance of a prominent GlyT2-immunoreactive band at 200 kDa (data not shown). Together, these results are consistent with cross-linking of GlyT2 dimers by CuP treatment not interfering with transporter function. Very similar findings were also obtained with GlyT1. CuP treatment of GlyT1WT stably expressing cells did not result in the appearance of a immunoreactive band of higher molecular weight. However, treatment of a GlyT1L343C-expressing cell line produced a prominent immunoreactive band at 190

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