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• W2084361108 abstract "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 inXenopus oocytes and used native and engineeredN-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. 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 inXenopus oocytes and used native and engineeredN-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. concentrative nucleoside transporter equilibrative nucleoside transporter nitrobenzylthioinosine p-chloromercuriphenylsulfonate polymerase chain reaction enzyme-linked immunosorbent assay bovine serum albumin phosphate-buffered saline Nucleoside transporters play key roles in physiology and pharmacology (1Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Uptake of exogenous nucleosides, for example, is a critical first step of nucleotide synthesis in tissues such as bone marrow and intestinal epithelium and certain parasitic organisms that lack de novo pathways for purine biosynthesis (2Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Barrett K.E. Donowitz M. Current Topics in Membranes. 50. Academic Press, San Diego, CA2000: 329-378Google Scholar, 3Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). The same transport mechanisms function as drug transporters and mediate uptake of many synthetic nucleoside analogs used in cancer (and viral) chemotherapy (2Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Barrett K.E. Donowitz M. Current Topics in Membranes. 50. Academic Press, San Diego, CA2000: 329-378Google Scholar). Nucleoside transporters also control the extracellular concentration of adenosine in the vicinity of its cell surface receptors and regulate processes such as neurotransmission and cardiovascular activity (1Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 2Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Barrett K.E. Donowitz M. Current Topics in Membranes. 50. Academic Press, San Diego, CA2000: 329-378Google Scholar, 3Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). Adenosine itself is used clinically to treat cardiac arrhythmias, and nucleoside transport inhibitors such as dipyridamole, dilazep, and draflazine function as coronary vasodilators. In mammals, plasma membrane transport of nucleosides is brought about by members of the concentrative, Na+-dependent (CNT)1 and equilibrative, Na+-independent (ENT) nucleoside transporter families (1Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 2Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Barrett K.E. Donowitz M. Current Topics in Membranes. 50. Academic Press, San Diego, CA2000: 329-378Google Scholar, 3Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). CNTs are expressed in a tissue-specific fashion; ENTs are present in most, possibly all, cell types. Two ENT isoforms have been identified in human and rat tissues (4Griffiths M. Beaumont N. Yao S.Y.M. Sundaram M. Boumah C.E. Davies A. Kwong F.Y.P. Coe I.R. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (358) Google Scholar, 5Yao S.Y.M. Ng A.M.L. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 6Griffiths M. Yao S.Y.M. Abidi F. Phillips S.E.V. Cass C.E. Young J.D. Baldwin S.A. Biochem. J. 1997; 328: 739-743Crossref PubMed Scopus (226) Google Scholar, 7Crawford C.R. Patel D.H. Naeve C. Belt J.A. J. Biol. Chem. 1998; 273: 5288-5293Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Human (h) and rat (r) ENT1 and ENT2 (456–457 amino acid residues) transport both purine and pyrimidine nucleosides, including adenosine, and are distinguished functionally by a difference in sensitivity to inhibition by NBMPR: hENT1 and rENT1 are potently inhibited by NBMPR (Kd 1–10 nm) and have the functional designation equilibrative-sensitive (es), while hENT2 and rENT2 are unaffected by micromolar concentrations of NBMPR and have the functional designationequilibrative-insensitive (ei) (4Griffiths M. Beaumont N. Yao S.Y.M. Sundaram M. Boumah C.E. Davies A. Kwong F.Y.P. Coe I.R. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (358) Google Scholar, 5Yao S.Y.M. Ng A.M.L. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 6Griffiths M. Yao S.Y.M. Abidi F. Phillips S.E.V. Cass C.E. Young J.D. Baldwin S.A. Biochem. J. 1997; 328: 739-743Crossref PubMed Scopus (226) Google Scholar, 7Crawford C.R. Patel D.H. Naeve C. Belt J.A. J. Biol. Chem. 1998; 273: 5288-5293Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). They also differ in sensitivity to inhibition by vasodilator drugs (hENT1 > hENT2 > rENT1 = rENT2) and by the ability of hENT2 and rENT2 to transport nucleobases as well as nucleosides (1Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 3Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar, 7Crawford C.R. Patel D.H. Naeve C. Belt J.A. J. Biol. Chem. 1998; 273: 5288-5293Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 8Osses N. Pearson J.D. Yudilevich D.L. Jarvis S.M. Biochem. J. 1996; 317: 843-848Crossref PubMed Scopus (43) Google Scholar). ENTs are widely distributed in other eukaryotes, including insects, nematodes, protozoa, yeast, and plants and do not appear to be present in prokaryotes (3Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). The predicted membrane topology of hENT1, the first identified member of the ENT family, contains 11 putative transmembrane helices (4Griffiths M. Beaumont N. Yao S.Y.M. Sundaram M. Boumah C.E. Davies A. Kwong F.Y.P. Coe I.R. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (358) Google Scholar). Binding domains for NBMPR and vasodilator drugs, which compete with permeant for the substrate-binding site at the extracellular surface, comprise a region of the h/rENT1 protein (amino acid residues 100–231) encompassing putative transmembrane helices 3–6 (9Sundaram M. Yao S.Y.M. Ng A.M.L. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1998; 273: 21519-21525Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 10Sundaram M. Yao S.Y.M. Ng A.M.L. Cass C.E. Baldwin S.A. Young J.D. Biochemistry. 2001; 40: 8146-8151Crossref PubMed Scopus (50) Google Scholar). A residue in the same structural domain of rENT2 (Cys140) is responsible for substrate-protectable inhibition of the transporter by PCMBS (11Yao S.Y.M. Sundaram M. Chomey E.G. Cass C.E. Baldwin S.A. Young J.D. Biochem. J. 2001; 353: 387-393Crossref PubMed Google Scholar). In protozoa, mutations of Gly183 in transmembrane helix 5 of Leishmania donovani LdNT1.1 result in altered substrate specificity and drug resistance to tubercidin (12Vasudevan G. Ullman B. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6092-6097Crossref PubMed Scopus (60) Google Scholar). A mutant form of TbAT1 from Trypanosoma brucei brucei that confers resistance to melaminophenyl arsenicals contains six amino acid substitutions in different transmembrane helices and loops of its sequence (13Maser P. Sutterlin C. Kralli A. Kaminsky R. Science. 1999; 285: 242-244Crossref PubMed Scopus (222) Google Scholar). While these studies have been successful in identifying functionally important roles for transmembrane helices 3–6 and other regions, there is currently no experimentally based model of ENT topology. Such information is essential to provide a structural basis for further molecular and mechanistic studies of ENT transporter function. One approach to test the two-dimensional orientation of integral membrane proteins is to identify sites of N-glycosylation. During protein synthesis, attachment of N-linked oligosaccharides to nascent polypeptide occurs on Asn residues in the motif Asn-X-Ser/Thr, where X can be any amino acid except Pro (14Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar, 15Shakin-Eshleman S.H. Spitalnik S.L. Kasturi L. J. Biol. Chem. 1996; 271: 6363-6366Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Due to strict compartmentalization of enzymes, N-glycosylation is carried out exclusively on the luminal side of the endoplasmic reticulum, which is topologically equivalent to the extracellular side of the protein. Thus, it is possible to identify exofacial and endofacial segments of the protein from the N-glycosylation profile. Here, we have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological studies of native h/rENT1 in erythrocyes and ventricular myocytes to experimentally define the topology of this prototypical nucleoside transporter protein. When combined with computer-based sequence analyses, the results provide a unified model of membrane architecture for all eukaryotic ENT nucleoside transporters. The locations of transmembrane helices in hENT1 were predicted by analysis of the amino acid sequence using the hidden Markov model procedure of Sonnhammeret al. (16Sonnhammer E.L.L. von Heijne G. Krogh A. Glasgow J. Littlejohn T. Major F. Lathrop R. Sankoff D. Sensen C. Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology. AAAI Press, Menlo Park, CA1998: 175-182Google Scholar) as implemented in the computer program TMHMM (version 2.0). In addition, multiple sequence alignments were analyzed for putative transmembrane helices using the TMAP procedure of Persson and Argos (17Persson B. Argos P. J. Mol. Biol. 1994; 237: 182-192Crossref PubMed Scopus (424) Google Scholar) and the neural network approach (PHDhtm) of Rostet al. (18Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (533) Google Scholar). Analyses were performed on the 34 members of the ENT protein family listed in Table I of Ref. 3Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar. The single mutants N48Q, N277Q, and N288Q (putative loops A andH in Fig. 1) were generated in plasmid pKS-hENT1 (4Griffiths M. Beaumont N. Yao S.Y.M. Sundaram M. Boumah C.E. Davies A. Kwong F.Y.P. Coe I.R. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (358) Google Scholar, 9Sundaram M. Yao S.Y.M. Ng A.M.L. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1998; 273: 21519-21525Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) by the PCR-based megaprimer method (19Sivaprasadarao A. Findlay J.B.C. Biochem. J. 1994; 300: 437-442Crossref PubMed Scopus (43) Google Scholar) using Pfu DNA polymerase." @default.
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• W2084361108 title "Topology of a Human Equilibrative, Nitrobenzylthioinosine (NBMPR)-sensitive Nucleoside Transporter (hENT1) Implicated in the Cellular Uptake of Adenosine and Anti-cancer Drugs" @default.
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