Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues.
Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.
Yao
S Y
SY
Membrane Transport Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Ng
A M
AM
Muzyka
W R
WR
Griffiths
M
M
Cass
C E
CE
Baldwin
S A
SA
Young
J D
JD
D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004109:Dilazep / Q000494:pharmacology; D004176:Dipyridamole / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D033705:Equilibrative Nucleoside Transport Proteins; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D033703:Nucleoside Transport Proteins; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology; D014981:Xenopus
D000345:Affinity Labels; D002352:Carrier Proteins; D033705:Equilibrative Nucleoside Transport Proteins; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D008565:Membrane Proteins; D009687:Nuclear Proteins; D033703:Nucleoside Transport Proteins; C438649:SLC29A1 protein, human; C438654:SLC29A2 protein, human; C438652:Slc29a1 protein, rat; C438655:Slc29a2 protein, rat; D014665:Vasodilator Agents; D013868:Thioinosine; D004176:Dipyridamole; D004109:Dilazep; C001789:4-nitrobenzylthioinosine