Nucleoside transporters are important in transporting synthetic nucleoside drugs into their target cells where these drugs are metabolized and become cytotoxic. Nucleoside drugs are used in chemotherapy (eg. Ara C in leukemia) and as antiviral agents (eg. AZT in the treatment of AIDS). Pharmacologically, the Na independent nucleoside transport systems can be separated into the nitrobenzylthioinosine (NBMPR) sensitive (ES) and the NBMPR insensitive (EI) systems. NBMPR is an inhibitor of nucleoside transport. Recently, our laboratory cloned the human Na-independent Equilibrative Nucleoside Transporters (ENT1(encoding ES) and ENT2 (encoding EI)) and developed a Nucleoside Transporter Deficient cell line, PK15NTD cells. This will allow us to study the structure/function relationship of the nucleoside transporters and the structural determinants of nucleosides and nucleoside drugs as substrates of the nucleoside transporters. Through the knowledge of nucleoside transport at the molecular level, a long term goal is to facilitate the design of nucleoside analogue drugs for the treatment of diseases. In this application, we will characterize functionally and pharmacologically the cloned human ENT1 and ENT2 stably expressed in PK15NTD cells (Aim 1). Results obtained from the cloned proteins will be compared with the endogenous ES and EI transporters in human colonic cancer cell line, T84. Using kinetic studies, we will identify structural determinants of nucleosides for the transport by ENT1 and ENT2.
In Aim 2, we will study the structure/function relationship of ENT1 and ENT2. We will define the role of glycosylation in the function of ENT1 and ENT2 since glycosylation is important for nucleoside transport. Nucleoside transporter function is thiol-sensitive and the thiol sensitive group is exofacial in ENT2 but is cytoplasmic in ENT1. We propose to identify cysteine residues that confer thiol sensitivity of ENT1 and ENT2. ENT1 and ENT2 have a 3000 fold difference in sensitivity to NBMPR and an 8-10 fold difference in the affinity for guanosine and cytidine transport, ENT1/ENT2 chimeras will be constructed to identify the binding domain and substrate recognition domain in ENT1 and ENT2. Protein kinase C (PKC) inhibits the cloned ENT1 and ENT2 and endogenous ES and EI transporters in T84 cells. Experiments are proposed in the third aim to study the molecular mechanisms of how PKC inhibits ENT1 and ENT2. These proposed studies should provide insights into the molecular mechanisms and kinase regulation of nucleoside transport, and the structural determinants of nucleosides/nucleoside drugs for transport by the nucleoside transporters.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA085428-05
Application #
6750672
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Lees, Robert G
Project Start
2000-06-01
Project End
2007-05-31
Budget Start
2004-06-01
Budget End
2007-05-31
Support Year
5
Fiscal Year
2004
Total Cost
$257,513
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Hoque, Kazi Mirajul; Chen, Linxi; Leung, George P H et al. (2008) A purine-selective nucleobase/nucleoside transporter in PK15NTD cells. Am J Physiol Regul Integr Comp Physiol 294:R1988-95
Leung, George P H; Man, Ricky Y K; Tse, Chung-Ming (2005) D-Glucose upregulates adenosine transport in cultured human aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 288:H2756-62
Donowitz, Mark; Cha, Boyoung; Zachos, Nicholas C et al. (2005) NHERF family and NHE3 regulation. J Physiol 567:3-11
Cha, Boyoung; Kim, Jae Ho; Hut, Hans et al. (2005) cGMP inhibition of Na+/H+ antiporter 3 (NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein. J Biol Chem 280:16642-50
Zachos, Nicholas C; Tse, Ming; Donowitz, Mark (2005) Molecular physiology of intestinal Na+/H+ exchange. Annu Rev Physiol 67:411-43
Leung, George P H; Man, Ricky Y K; Tse, Chung-Ming (2005) Effect of thiazolidinediones on equilibrative nucleoside transporter-1 in human aortic smooth muscle cells. Biochem Pharmacol 70:355-62
Ward, Jeffrey L; Leung, George P H; Toan, Shuy-Vang et al. (2003) Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2. Arch Biochem Biophys 411:19-26
Toan, Shuy-Vang; To, Kenneth K W; Leung, George P H et al. (2003) Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells. Pflugers Arch 447:195-204
Jobbagy, Zsolt; Ward, Jeffrey L; Toan, Shuy-Vang et al. (2002) One-step unidirectional cloning of tandem repeats of DNA fragments: an application for fusion protein production. Anal Biochem 303:104-7
Leung, G P; Ward, J L; Wong, P Y et al. (2001) Characterization of nucleoside transport systems in cultured rat epididymal epithelium. Am J Physiol Cell Physiol 280:C1076-82

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