Characterization of mENT1Delta11, a novel alternative splice variant of the mouse equilibrative nucleoside transporter 1.
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 (mENT1Delta11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Delta11 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 mENT1Delta11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Delta11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Delta11 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 mENT1Delta11 could not be photolabeled with [3H]NBMPR and that mENT1Delta11 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.
Robillard
Kevin R
KR
Dept. of Physiology and Pharmacology, M266 Medical Sciences Building, University of Western Ontario, London, Ontario N6A5C1, Canada.
Bone
Derek B J
DB
Park
Jamie S
JS
Hammond
James R
JR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D033703:Nucleoside Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection
D033721:Equilibrative Nucleoside Transporter 1; D033703:Nucleoside Transport Proteins; D020033:Protein Isoforms; C498476:SLC29A1 protein, mouse