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J. Biol. Chem., Vol. 283, Issue 16, 10978-10991, April 18, 2008

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Glycine Transporter Dimers

EVIDENCE FOR OCCURRENCE IN THE PLASMA MEMBRANE^*

Ingo Bartholomäus¹, Laura Milan-Lobo, Annette Nicke, Sébastien Dutertre², Hanne Hastrup^¶, Alok Jha, Ulrik Gether^¶, Harald H. Sitte³, Heinrich Betz, and Volker Eulenburg⁴

From the Department of Neurochemistry, Max Planck Institute for Brain Research, Deutschordenstrasse 46, 60529 Frankfurt, Germany, Center for Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Medical University Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria, and ^¶Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark

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 LeuT_Aa, 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.

Received for publication, January 24, 2008

^* This work was supported by the Max-Planck-Gesellschaft, Deutsche Forschungsgemeinschaft (DFG) Grants SPP1172 and NI 592/3-2, Fonds der Chemischen Industrie, the Cluster of Excellence "Macromolecular Complexes" at the Goethe University Frankfurt (DFG Project EXC 115), and Austrian Science Fund Grants 17076 and 18076. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Present address: Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany.

² Recipient of a European Molecular Biology Organization postdoctoral fellowship.

³ To whom correspondence may be addressed. Tel.: 43-1-4277-64123; Fax: 43-1-4277-9641; E-mail: harald.sitte{at}meduniwien.ac.at.

⁴ To whom correspondence may be addressed. Tel.: 49-69-96769-317; Fax: 49-69-96769-441; E-mail: eulenburg{at}mpih-frankfurt.mpg.de.

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