Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs.
The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.
Sundaram
M
M
Membrane Transport Research Group, Departments of Physiology and Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada.
Yao
S Y
SY
Ingram
J C
JC
Berry
Z A
ZA
Abidi
F
F
Cass
C E
CE
Baldwin
S A
SA
Young
J D
JD
D000241:Adenosine / Q000493:pharmacokinetics*; D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D033721:Equilibrative Nucleoside Transporter 1; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D026901:Membrane Transport Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012984:Software; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D014981:Xenopus / Q000378:metabolism
D000596:Amino Acids; D000970:Antineoplastic Agents; D033721:Equilibrative Nucleoside Transporter 1; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C438649:SLC29A1 protein, human; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine