Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells

Abstract

Human CNT3 encodes the concentrative nucleoside transport N3 system. Previous expression studies in oocytes showed that the Km values for nucleosides of the cloned hCNT3 were 7- to 25-fold lower than the endogenous N3 transporter in HL60 cells. Therefore, in the present study we re-examined the kinetic properties of the cloned hCNT3 using mammalian cell expression systems by transient expression in Cos7L cells and stably expression in nucleoside transporter deficient PK15NTD cells. We demonstrated that hCNT3 is a Na-dependent, broadly-selective nucleoside transporter with affinities (<11 microM) for nucleosides closely resembling the endogenous N3 transporter. Pharmacological studies showed that phloridzin is a mixed-type inhibitor of hCNT3 (Ki=15 microM), and the dideoxyuridine analogs are poor substrates. By epitope-tagging, we further demonstrated that hCNT3 is N-glycosylated as PNGase F and Endo H deglycosylated hCNT3 from 67 kDa to 58 kDa. Searching the human genome database, we identified the genomic organization of hCNT3. This gene contains 19 exons and its exon-intron boundaries within the coding sequence exactly match with those of hCNT1 and hCNT2 with one additional exon in the N-terminus. Our data suggest that hCNT3 gene is evolutionarily conserved with hCNT1 and hCNT2. Physiologically, hCNT3 is a glycoprotein, which transports purine and pyrimidine nucleosides in a Na-dependent manner with high affinities.

Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3)

“…The primer sequences can be found at http://www.pharmgkb.org. The genomic structure of SLC28A3 was based on a recent analysis by Toan et al 25 Variant positions are relative to the ATG start site and are based on the reference cDNA sequence of SLC28A3.…”

Structure-Activity Relationship and Mechanistic Studies on the Chemopreventive Activity of Dipyridamole and Its Analogues

“…In this group, the 4-, 8-position tertiary amine was replaced by a secondary amine to explore the SAR effects of the resulting free hydrogen (Figure 4.5). Substitutions ranged from monoalkyl (128)(129)(130)(131)(132)(133)(134)(135)(136)(137)(138)(139)(140)(141)(142)(143)(144)(145)(146), cycloalkyl (147)(148)(149)(150)(151)(152)(153), to aromatic rings (154)(155)(156)(157). These compounds showed a wide range of activities from 12 -100 % inhibition of colony formation.…”

“…Inhibitors of the sodium dependent concentrative nucleoside transporters are less characterized. The few concentrative transport inhibitors that are known currently are phloridzin, 5'-position modified nucleoside derivatives, benzimidazole ribofuranosides, and 8-position modified purine nucleoside derivatives (147)(148)(149)(150).…”

Flavonoids and Related Compounds as Nucleoside Transporter Inhibitors

“…• Phloridzin, 48 • 5′-position modified nucleoside derivatives of adenosine, 49 • Benzimidazole ribofuranosides,…”

“…It inhibits CNT1 with an IC 50 of 250 μM, CNT2 with an IC 50 of 100 μM and CNT3 with a K i of 15 μM at CNT3. 48 An addition, phloridzin has also been shown to inhibit concentrative nucleoside transport in rat intestinal epithelium with an IC 50 of 700 μM and in renal brush border membranes with an IC 50 of 60 μM. …”