Abstract
Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1Δ11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Δ11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1Δ11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Δ11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Δ11 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT1Δ11 could not be photolabeled with [3H]NBMPR and that mENT1Δ11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.
Footnotes
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This study was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (to J.R.H.). K.R. gratefully acknowledges the financial support provided by the Schulich School of Medicine and Dentistry, University of Western Ontario, during the course of his graduate studies. D.B.J.B. is the recipient of an Ontario Graduate Scholarship in Science and Technology.
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A preliminary report of this work was presented at the XVth World Congress of Pharmacology; 2006 July 2-7; Beijing, China.
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ABBREVIATIONS: ENT, equilibrative nucleoside transporter; NBMPR, nitrobenzylmercaptopurine riboside; TM, transmembrane; CK2, casein kinase II; bp, base pair(s); RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; NBTGR, nitrobenzylthioguanosine riboside; CMV, cytomegalovirus; BGS, bovine growth serum; PVDF, polyvinylidene difluoride; TBS-T, Tris-buffered saline/Tween 20; NMG, N-methyl-glucamine; PAGE, polyacrylamide gel electrophoresis; PK15-NTD, pig kidney epithelial cells 15-nucleoside transport deficient; U2-OS, U2-osteosarcoma.
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↵1 Current affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
- Received September 14, 2007.
- Accepted April 14, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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