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  • 1 Marmara Univ. School of Medicine Dept. of Biophysics Istanbul Turkey
  • | 2 University of Copenhagen Dept. of Pharmacology, Molecular Neuropharmacology Group, The Panum Institute Copenhagen Denmark
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We have introduced tetracysteine motifs into different positions of the dopamine transporter (DAT) for specific FlAsH labeling. Two of the constructs expressed at the cell surface and were functional as determined by [ 3 H] dopamine uptake experiments. The N-terminally modified transporter showed uptake levels comparable to the wild-type DAT, while the construct with tetracysteine motif at position 511 displayed an uptake level about 1/3 of its wild-type counterpart. In addition, these two transporter constructs were visualized on the cell surface following labeling with a fluorescent cocaine analog. YFP introduced into the same N-terminal position was also shown to have surface staining in agreement with activity tests. We propose that these two sites are suitable targets for tetracysteine labeling to be used in FlAsH staining studies, while p134, p342, p427, p433 and p517 sites are not.

  • Adams, S. R., Campbell, R. E., Gross, L. A., Martin, B. R., Walkup, G. K., Yao, Y., Llopis, J., Tsien, R. Y. (2002) New biarsenical ligands and tetracycteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124 , 6063–6076.

    Tsien R. Y. , 'New biarsenical ligands and tetracycteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications ' (2002 ) 124 J. Am. Chem. Soc. : 6063 -6076.

    • Search Google Scholar
  • Androutsellis-Theotokis, A., Ghassemi, F., Rudnick, G. (2001) A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter. J. Biol. Chem. 276 , 45933–45938.

    Rudnick G. , 'A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter ' (2001 ) 276 J. Biol. Chem. : 45933 -45938.

    • Search Google Scholar
  • Bjerggaard, C., Fog, J. U., Hastrup, H., Madsen, K., Loland, C. J., Javitch, J. A., Gether, U. (2004) Surface targeting of the dopamine transporter involves discrete epitopes in the distal C terminus but does not require canonical PDZ domain interactions. J. Neurosci. 24 , 7024–7036.

    Gether U. , 'Surface targeting of the dopamine transporter involves discrete epitopes in the distal C terminus but does not require canonical PDZ domain interactions ' (2004 ) 24 J. Neurosci. : 7024 -7036.

    • Search Google Scholar
  • Cha, J. H., Zou, M. F., Adkins, E. M., Rasmussen, S. G., Loland, C. J., Schoenenberger, B., Gether, U., Newman, A. H. (2005) Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl) tropane analogues as high-affinity fluorescent probes for the dopamine transporter. J. Med. Chem. 48 , 7513–7516.

    Newman A. H. , 'Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl) tropane analogues as high-affinity fluorescent probes for the dopamine transporter ' (2005 ) 48 J. Med. Chem. : 7513 -7516.

    • Search Google Scholar
  • Ferrer, J. V., Javitch, J. A. (1998) Cocaine alters the accessibility of endogenous cysteines in putative extracellular and intracellular loops of the human dopamine transporter. Proc. Natl. Acad. Sci. USA 95 , 9238–9243.

    Javitch J. A. , 'Cocaine alters the accessibility of endogenous cysteines in putative extracellular and intracellular loops of the human dopamine transporter ' (1998 ) 95 Proc. Natl. Acad. Sci. USA : 9238 -9243.

    • Search Google Scholar
  • Gether, U., Andersen, P. H., Larsson, O. M., Schousboe, A. (2006) Neurotransmitter transporters: molecular function of important drug targets. Trends Pharmacol. Sci. 27 , 375–383.

    Schousboe A. , 'Neurotransmitter transporters: molecular function of important drug targets ' (2006 ) 27 Trends Pharmacol. Sci. : 375 -383.

    • Search Google Scholar
  • Griffin, B. A., Adams, S. R., Jones, J., Tsien, R. Y. (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol. 327 , 565–579.

    Tsien R. Y. , 'Fluorescent labeling of recombinant proteins in living cells with FlAsH ' (2000 ) 327 Methods Enzymol. : 565 -579.

    • Search Google Scholar
  • Hoffmann, C., Gaietta, G., Bünemann, M., Adams, S. R., Oberdorff-Maass, S., Behr, B., Vilardaga, J. P., Tsien, R. Y., Ellisman, M. H., Lohse, M. J. (2005) A FlAsH-based FRET approach to determine G protein coupled receptor activation in living cells. Nature Methods 2 , 171–176.

    Lohse M. J. , 'A FlAsH-based FRET approach to determine G protein coupled receptor activation in living cells ' (2005 ) 2 Nature Methods : 171 -176.

    • Search Google Scholar
  • Kahlig, K. M., Binda, F., Khoshbouei, H., Blakely, R. D., McMahon, D. G., Javitch, J. A., Gali, A. (2005) Amphetamine induces dopamine efflux through a dopamine transporter channel. Proc. Natl. Acad. Sci. USA 102 , 3495–3500.

    Gali A. , 'Amphetamine induces dopamine efflux through a dopamine transporter channel ' (2005 ) 102 Proc. Natl. Acad. Sci. USA : 3495 -3500.

    • Search Google Scholar
  • Koch, H. P., Larsson, H. P. (2005) Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters. J. Neurosci. 25 , 1730–1736.

    Larsson H. P. , 'Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters ' (2005 ) 25 J. Neurosci. : 1730 -1736.

    • Search Google Scholar
  • Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy . 2 nd edn, Kluwer Academic/Plenum Publishers, New York, London, Moscow, Dordrecht.

    Lakowicz J. R. , '', in Principles of Fluorescence Spectroscopy , (1999 ) -.

  • Langhorst, M. F., Genisyurek, S., Stuermer, C. A. (2006) Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by b-mercaptoethanol for specific staining of tetracysteine-tagged proteins. Histochem. Cell. Biol. 125 , 743–747.

    Stuermer C. A. , 'Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by b-mercaptoethanol for specific staining of tetracysteine-tagged proteins ' (2006 ) 125 Histochem. Cell. Biol. : 743 -747.

    • Search Google Scholar
  • Loland, C. J., Granas, C., Javitch, J. A., Gether, U. (2004) Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding. J. Biol. Chem. 279 , 3228–3238.

    Gether U. , 'Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding ' (2004 ) 279 J. Biol. Chem. : 3228 -3238.

    • Search Google Scholar
  • Martin, B. R., Giepmans, B. N., Adams, S. R., Tsien, R. Y. (2005) Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23 , 1309–1314.

    Tsien R. Y. , 'Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity ' (2005 ) 23 Nat. Biotechnol. : 1309 -1314.

    • Search Google Scholar
  • Nobles, M., Benians, A., Tinker, A. (2005) Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc. Natl. Acad. Sci. USA 102 , 18706–18711.

    Tinker A. , 'Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells ' (2005 ) 102 Proc. Natl. Acad. Sci. USA : 18706 -18711.

    • Search Google Scholar
  • Reith, M. E., Berfield, J. L., Wang, L. C., Ferrer, J. V., Javitch, J. A. (2001) The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter. J. Biol. Chem. 276 , 29012–29018.

    Javitch J. A. , 'The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter ' (2001 ) 276 J. Biol. Chem. : 29012 -29018.

    • Search Google Scholar
  • Rudnick, G. (1998) Bioenergetics of neurotransmitter transport. J. Bioenergetics and Biomembranes 30 , 173–185.

    Rudnick G. , 'Bioenergetics of neurotransmitter transport ' (1998 ) 30 J. Bioenergetics and Biomembranes : 173 -185.

    • Search Google Scholar
  • Siegel, R. M., Chan, F. K., Zacharias, D. A., Swofford, R., Holmes, K. L., Tsien, R. Y., Lenardo, M. J. (2000) Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein. Sci. STKE. 2000 , 2000, PL1.

    Lenardo M. J. , 'Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein ' (2000 ) 2000 Sci. STKE. : PL1 -.

    • Search Google Scholar
  • Stroffekova, K., Proenza, C., Beam, K. G. (2001) The protein-labeling reagent FLASH-EDT 2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins. Pflugers Arch. 442 , 859–866.

    Beam K. G. , 'The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins ' (2001 ) 442 Pflugers Arch. : 859 -866.

    • Search Google Scholar
  • Torres, G. E., Amara, S. G. (2007) Glutamate and monoamine transporters: new visions of form and function. Curr. Opin. Neurobiol. 17 , 304–312.

    Amara S. G. , 'Glutamate and monoamine transporters: new visions of form and function ' (2007 ) 17 Curr. Opin. Neurobiol. : 304 -312.

    • Search Google Scholar
  • Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., Gouaux, E. (2005) Crystal structure of a bacterial homologue of Na + /Cl -dependent neurotransmitter transporters. Nature 437 , 215–223.

    Gouaux E. , 'Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters ' (2005 ) 437 Nature : 215 -223.

    • Search Google Scholar

Editorial Board

    1. Csányi, Vilmos (Göd)
    1. Dudits, Dénes (Szeged)
    1. Falus, András (Budapest)
    1. Fischer, Ernő (Pécs)
    1. Gábriel, Róbert (Pécs)
    1. Gulya, Károly (Szeged)
    1. Gulyás, Balázs (Stockholm)
    1. Hajós, Ferenc (Budapest)
    1. Hámori, József (Budapest)
    1. Heszky, László (Gödöllő)
    1. Hideg, Éva (Szeged)
    1. E. Ito (Sanuki)
    1. Janda, Tibor (Martonvásár)
    1. Kavanaugh, Michael P. (Missoula)
    1. Kása, Péter (Szeged)
    1. Klein, Éva (Stockholm)
    1. Kovács, János (Budapest)
    1. Brigitte Mauch-Mani (Neuchâtel)
    1. Nässel, Dick R. (Stockholm)
    1. Nemcsók, János (Szeged)
    1. Péczely, Péter (Gödöllő)
    1. Roberts, D. F. (Newcastle-upon-Tyne)
    1. Sakharov, Dimitri A. (Moscow)
    1. Singh, Meharvan (Fort Worth)
    1. Sipiczky, Mátyás (Debrecen)
    1. Szeberényi, József (Pécs)
    1. Székely, György (Debrecen)
    1. Tari, Irma (Szeged)
    1. Vágvölgyi, Csaba (Szeged),
    1. L. Zaborszky (Newark)

 

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Acta Biologica Hungarica
Language English
Size  
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Foundation
1950
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changed title
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Founder Magyar Tudományos Akadémia
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H-1051 Budapest, Hungary, Széchenyi István tér 9.
Publisher Akadémiai Kiadó
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Chief Executive Officer, Akadémiai Kiadó
ISSN 0236-5383 (Print)
ISSN 1588-256X (Online)