The human SLC29 transporter family contains four members, hENT1 (SLC29A1), hENT2 (SLC29A2), hENT3 (SLC29A3), and hENT4 (SLC29A4) that are primarily distinguished from the concentrative nucleoside transporters by their facilitated diffusion transport mechanism. Early research distinguished SLC29 family members by their sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), as either es, equilibrative sensitive, or ei, equilibrative insensitive; however, the usefulness of this terminology for identification has been superceded by the cloning and subsequent characterization of both hENT1 and hENT2, which are responsible for these transport mechanisms (Griffiths and Jarvis, 1996; Kong et al., 2004).
Structurally, SLC29 family members are characterized by 11 putative TMD, with an intracellular amine terminus and extracellular carboxy terminus. The gene encoding hENT1 is localized to chromosome 6p21.1-2 (Coe et al., 1997), and encodes a protein that is 456 amino acids long (Griffiths et al., 1997a). Both the rat and mouse homologues have also been cloned and exhibit approximately 78% identity with hENT1 (Yao et al., 1997; Kiss et al., 2000). Similarly, the genes encoding hENT2 and hENT3 have been identified and are chromosoma-lly localized to 11q13 and 10q22.1, respectively (Griffiths et al., 1997b; Williams et al., 1997; Hyde et al., 2001; Clark et al., 2003). Additionally, hENT2 has been shown to be 456 amino acids long, sharing approximately 46% identity with hENT1 (Griffiths etal., 1997b), while hENT3 is 475 amino acids long and exhibits approximately 30-33% identity with mouse, rat, and human ENT1 and ENT2 isoforms (Hyde et al., 2001). Although it has been confirmed as a nucleoside transporter capable of low affinity adenosine transport, hENT4 is a 530 amino acid protein that shows only 18% identity to hENT1 (Acimovic and Coe, 2002; Baldwin et al., 2004). The gene encoding hENT4 is localized to chromosome 7p22.1 (Acimovic and Coe, 2002; Strausberg et al., 2002; Baldwin et al., 2004). Interestingly, hENT4 has recently been shown to be a low affinity monoamine transporter and renamed plasma membrane monoamine transporter, or PMAT (Engel et al., 2004). As such, ENT4 will not be discussed further.
Both hENT1 and hENT2 exhibit glycosylation sites in the extracellular loops between TMD one and two, on Asn residue 48 (Yao et al., 1997; Crawford et al., 1998; Ward et al., 2003). In the case of hENT1, glycosylation is not required for transport activity, but may affect the binding affinity to transport inhibitors, such as NBMPR (Vickers et al., 1999; Ward et al., 2003). HENT2 also contains an additional glycosylation site on Asn57, which most likely functions to target the protein to the plasma membrane (Ward et al., 2003). Such structural assessments have not yet been conducted for hENT3, although it does differ from both hENT1 and hENT2 in possessing a long, hydrophilic amine terminus region preceding transmembrane domain one that contains dileucine motifs, which are responsible for its intracellular localization (Baldwin et al., 2005).
188.8.131.52 The Substrate Specificities of Nucleoside Transporters CNT (SLC28A)
Vijayalakshmi and Belt (1988) first showed the differing substrate specificities of CNT1 and CNT2 by their observations in mouse intestinal epithelium of two classes of sodium dependent concentrative nucleoside transport, which was dependent on purine/pyrimidine species. It is now known that CNT1 transports primarily pyrimidine nucleosides and the purine adenosine (N2 transport), while CNT2 transports primarily purine nucleosides and uridine (N1 transport) (Huang et al., 1994; Ritzel et al., 1997; Wang et al., 1997). In contrast, CNT3 has been shown to be more broadly selective, transporting both purine and pyrimidine nucleosides (N3 transport) (Wu et al., 1992). Interestingly, while CNT1 is specific for pyrim-idines, it does exhibit specificity for adenosine in a high-affinity, low-capacity manner (Ritzel et al., 2001). Furthermore, while both CNT1 and CNT2 employ a 1:1 Na+ :nucleoside coupling ratio, CNT3 requires a 2:1 ratio (Plagemann and Aran, 1990; Ritzel et al., 2001).
Given their respective substrate specificities, SLC28 family members exhibit transport activity for a wide range of pharmaceutically relevant compounds. CNT1 has been shown to exhibit high transport affinity for the antiviral nucleo-side analogs zidovudine, lamivudine, and zalcitabine, and the cytotoxic cytidine analogs cytarabine and gemcitabine used for treatment of a wide spectrum of tumors, while CNT2 has been shown to transport the antiviral compounds didano-sine (ddI) and ribavirin (Gray et al., 2004). With its broader substrate specificity, CNT3 transports a number of anticancer nucleoside analogs including cladrabine, gemcitabine, 5-fluorouridine, fludarabine, and zebularine (Ritzel et al., 2001).
Substrate and cation recognition sites for CNT transporters are both located extracellularly on the carboxy half of the proteins, on TMD 7, 8, and 9 (Wang and Giacomini, 1999; Loewen et al., 1999). Changing serine 319 of CNT1 to glycine has been shown to alter the substrate selectivity to include purines, while the adjacent glutamine residue was shown to be important in modulating the apparent affinity for nucleosides (Wang and Giacomini, 1999). Moreover, changing serine 353 to threonine changed CNT1 into a transporter that was highly selective for uridine (Loewen et al., 1999). Recently, Lai et al. (2005) have demonstrated that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1 and that F316H mutation confers guanine sensitivity. These researchers speculated that the naturally occurring F316H mutation in hCNT1 is responsible for one of the two CNT activities for which a transporter has not been identified (N4) (Griffiths and Jarvis, 1996; Lai et al., 2005). Although such selectivity studies have not been performed for CNT2 per se, Chang et al. (2004) did explore the structural requirements necessary for purine and pyrimidine transport by hCNT1, hCNT2, and hENT1. Their computer modeling studies, which explore the relationships between hydroxylation position, substrate selectivity, and transporter inhibition, could prove useful for rational drug design of future nucleoside analogs for both cancer and antiviral treatment.
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