xCt cystine transporter expression in HEK293 cells: pharmacology and localization.
xCT, the core subunit of the system x(c)(-) high affinity cystine transporter, belongs to a superfamily of glycoprotein-associated amino acid transporters. Although xCT was shown to promote cystine transport in Xenopus oocytes, little work has been done with mammalian cells (Sato, H., Tamba, M., Ishii, T., and Bannai, S. J. Biol. Chem. 274, 11455-11458, 1999). Therefore, we have constructed mammalian expression vectors for murine xCT and its accessory subunit 4F2hc and transfected them into HEK293 cells. We report that this transporter binds cystine with high affinity (81 microM) and displays a pharmacological profile expected for system x(c)(-). Surprisingly, xCT transport activity in HEK293 cells is not dependent on the co-expression of the exogenous 4F2hc. Expression of GFP-tagged xCT indicated a highly clustered plasma membrane and intracellular distribution suggesting the presence of subcellular domains associated with combating oxidative stress. Our results indicate that HEK293 cells transfected with the xCT subunit would be a useful vehicle for future structure-function and pharmacology experiments involving system x(c)(-).
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Department of Psychiatry, University of British Columbia, 4N1-2255 Wesbrook Mall, Vancouver, British Columbia, V6T 1Z3, Canada. thmurphy@interchange.ubc.edu
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D027182:Amino Acid Transport System y+; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003553:Cystine / Q000378:metabolism; D027261:Fusion Regulatory Protein-1; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000493:pharmacokinetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018384:Oxidative Stress / Q000502:physiology; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D027182:Amino Acid Transport System y+; D015703:Antigens, CD; D002352:Carrier Proteins; D027261:Fusion Regulatory Protein-1; D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C118895:SLC7A11 protein, human; C492255:Slc7a11 protein, mouse; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D003553:Cystine