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J. Biol. Chem., Vol. 281, Issue 36, 26675-26682, September 8, 2006

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Molecular Determinants of Specificity for Synthetic Nucleoside Analogs in the Concentrative Nucleoside Transporter, CNT2^*

From the Department of Biopharmaceutical Sciences, The University of California, San Francisco, San Francisco, California 94158

Members of the concentrative nucleoside transporter (CNT) family (SLC28) mediate the transport of naturally-occurring nucleosides, and nucleoside analog drugs across the plasma membrane of epithelial cells. Each of the three CNT family members has a distinct specificity for naturally occurring nucleosides, and residues that contribute to the specificity of each transporter have been identified. In contrast, the molecular determinants of specificity for synthetic nucleoside analogs are not known. In this study, we take advantage of the large species difference that exists between human and rat CNT2 (hCNT2 and rCNT2) in their ability to transport the nucleoside analog drug cladribine, 2CdA, (rCNT2 > > > hCNT2) to identify the critical domains and amino acid residues that contribute to the observed difference in specificity between CNT2 orthologs. Using chimeric proteins of human and rat CNT2, we determined that the C-terminal half of CNT2 contained the determinants of 2CdA selectivity. We replaced key residues in the C terminus of hCNT2 with the equivalent residue in rCNT2. One residue in the C-terminal portion of CNT2 was found to significantly contribute to 2CdA selectivity: hCNT2-S354A. This mutant caused an increase of 5-6-fold over hCNT2. The 2-chloro pharmacophore, rather than the 2'-deoxyribose was responsible for the reduced 2CdA uptake by hCNT2. Our data are consistent with a model in which an increased capability for hydrogen bonding in critical amino acids that reside in the C terminus of rCNT2 contributes to its enhanced selectivity for 2CdA.

Received for publication, December 16, 2005 , and in revised form, June 27, 2006.

^* This work was supported by Grants GM42230, GM36780, and GM61390 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biopharmaceutical Sciences, UCSF. 1550 4th St. San Francisco, CA. Tel.: 415-476-1936; Fax: 415-502-4322; E-mail: kmg{at}itsa.ucsf.edu.

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