Stable Polarized Expression of hCAT-1 in an Epithelial Cell Line |
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Authors: | R Cariappa E Heath-Monnig TC Furesz SG Kamath CH Smith |
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Institution: | Department of Pediatrics, Washington University School of Medicine, and St. Louis Children's Hospital, One Children's Place, St. Louis, MO 63110, USA. |
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Abstract: | Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization. |
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