PMID- 10455109
OWN - NLM
STAT- MEDLINE
DCOM- 19990930
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 35
DP  - 1999 Aug 27
TI  - Identification of amino acid residues responsible for the pyrimidine and purine
      nucleoside specificities of human concentrative Na(+) nucleoside cotransporters
      hCNT1 and hCNT2.
PG  - 24475-84
AB  - hCNT1 and hCNT2 mediate concentrative (Na(+)-linked) cellular uptake of
      nucleosides and nucleoside drugs by human cells and tissues. The two proteins
      (650 and 658 residues, 71 kDa) are 72% identical in sequence and contain 13
      putative transmembrane helices (TMs). When produced in Xenopus oocytes,
      recombinant hCNT1 is selective for pyrimidine nucleosides (system cit), whereas
      hCNT2 is selective for purine nucleosides (system cif). Both transport uridine.
      We have used (i) chimeric constructs between hCNT1 and hCNT2, (ii) sequence
      comparisons with a newly identified broad specificity concentrative nucleoside
      transporter (system cib) from Eptatretus stouti, the Pacific hagfish (hfCNT), and
      (iii) site-directed mutagenesis of hCNT1 to identify two sets of adjacent
      residues in TMs 7 and 8 of hCNT1 (Ser(319)/Gln(320) and Ser(353)/Leu(354)) that, 
      when converted to the corresponding residues in hCNT2 (Gly(313)/Met(314) and
      Thr(347)/Val(348)), changed the specificity of the transporter from cit to cif.
      Mutation of Ser(319) in TM 7 of hCNT1 to Gly enabled transport of purine
      nucleosides, whereas concurrent mutation of Gln(320) to Met (which had no effect 
      on its own) augmented this transport. The additional mutation of Ser(353) to Thr 
      in TM 8 converted hCNT1/S319G/Q320M, from cib to cif, but with relatively low
      adenosine transport activity. Additional mutation of Leu(354) to Val (which had
      no effect on its own) increased the adenosine transport capability of
      hCNT1/S319G/Q320M/S353T, producing a full cif-type transporter phenotype. On its 
      own, the S353T mutation converted hCNT1 into a transporter with novel
      uridine-selective transport properties. Helix modeling of hCNT1 placed Ser(319)
      (TM 7) and Ser(353) (TM 8) within the putative substrate translocation channel,
      whereas Gln(320) (TM 7) and Leu(354) (TM 8) may exert their effects through
      altered helix packing.
FAU - Loewen, S K
AU  - Loewen SK
AD  - Membrane Transport Research Group, University of Alberta, Edmonton, Alberta T6G
      2H7, Canada.
FAU - Ng, A M
AU  - Ng AM
FAU - Yao, S Y
AU  - Yao SY
FAU - Cass, C E
AU  - Cass CE
FAU - Baldwin, S A
AU  - Baldwin SA
FAU - Young, J D
AU  - Young JD
LA  - eng
SI  - GENBANK/AF132298
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Carrier Proteins)
RN  - 0 (Membrane Transport Proteins)
RN  - 0 (Purine Nucleosides)
RN  - 0 (Pyrimidine Nucleosides)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (cif nucleoside transporter)
RN  - K72T3FS567 (Adenosine)
SB  - IM
MH  - Adenosine/metabolism
MH  - Amino Acid Sequence
MH  - Animals
MH  - Carrier Proteins/*genetics/metabolism
MH  - Hagfishes
MH  - Humans
MH  - Kinetics
MH  - *Membrane Transport Proteins
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutation
MH  - Oocytes
MH  - Purine Nucleosides/chemistry/metabolism
MH  - Pyrimidine Nucleosides/chemistry/metabolism
MH  - Recombinant Fusion Proteins/metabolism
MH  - Sequence Alignment
MH  - Substrate Specificity
MH  - Xenopus
EDAT- 1999/08/24 00:00
MHDA- 1999/08/24 00:01
CRDT- 1999/08/24 00:00
PHST- 1999/08/24 00:00 [pubmed]
PHST- 1999/08/24 00:01 [medline]
PHST- 1999/08/24 00:00 [entrez]
AID - 10.1074/jbc.274.35.24475 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Aug 27;274(35):24475-84. doi: 10.1074/jbc.274.35.24475.