CLONING OF THE HUMAN EQUILIBRATIVE, NITROBENZYLMERCAPTOPURINE RIBOSIDE (NBMPR)-INSENSITIVE NUCLEOSIDE TRANSPORTER EI BY FUNCTIONAL EXPRESSION IN A TRANSPORT-DEFICIENT CELL-LINE

Mammalian cells obtain nucleic acid precursors through the de novo syn thesis of nucleotides and the salvage of exogenous nucleobases and nuc leosides. The first step in the salvage pathway is transport across th e plasma membrane, Several transport activities, including equilibrati ve and concentrative mechanisms, have been identified by their functio nal properties, We report here the functional cloning of a 2.6-kilobas e pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBM PR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells, Expression of this cDNA conferred an NBMPR-insensitiv e, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characterist ic of the ei transporter, The cDNA contained a single open reading fra me that encoded a 456-residue protein with 11 potential membrane-spann ing regions and two consensus sites for N-glycosylation in the first p redicted extracellular loop, The predicted protein was 50% identical t o the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carbo xyl-terminal portion of the ENT2 protein was nearly identical to a sma ller protein in the GenBank(TM) data base (human HNP36, 326 residues) that has been identified as a growth factor-induced delayed early resp onse gene of unknown function. Comparison of the ENT2 and HNP36 nucleo tide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame, Transient expression studie s with the full-length ENT2 and a 5'-truncated construct that lacks th e first start codon (predicted protein 99% identical to HNP36) demonst rated that only the full-length construct conferred uridine transport activity to the cells, These data suggest that the delayed early respo nse gene HNP36 is a truncated form of ENT2 and that the full-length op en reading frame of ENT2 is required for production of a functional pl asma membrane ei transporter.