Different N-Terminal Motifs Determine Plasma Membrane Targeting of the Human Concentrative Nucleoside Transporter 3 in Polarized and Nonpolarized Cells

Abstract

Human concentrative nucleoside transporter 3 (hCNT3) is a broad-selectivity, high-affinity protein implicated in the uptake of most nucleoside-derived anticancer and antiviral drugs. Regulated trafficking of hCNT3 has been recently postulated as a suitable way to improve nucleoside-based therapies. Moreover, the recent identification of a putative novel hCNT3-type transporter lacking the first 69 amino acids and retained at the endoplasmic reticulum anticipated that the N terminus of hCNT3 contains critical motifs implicated in trafficking. In the current study, we have addressed this issue by using deletions and site-directed mutagenesis and plasma membrane expression and nucleoside uptake kinetic analysis. Data reveal that 1) a segment between amino acids 50 and 62 contains plasma membrane-sorting determinants in nonpolarized cells; 2) in particular, the Val 57 -Thr 58 -Val 59 tripeptide seems to be the core of the export signal, whereas acidic motifs upstream and downstream of it seem to be important for the kinetics of the process; and 3) in polarized epithelia, the ␤-turn-forming motif 17 VGFQ 20 is necessary for proper apical expression of the protein.

Structural determinants for rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

“…Previous studies in our laboratory suggested that sorting signals implicated in the membrane insertion of CNTs are localized in the intracellular N-terminal tail of these proteins [13,16,26]. This is based on the evidence that an alternative splicing variant of hCNT3, which is truncated in its N-terminus tail is retained in the ER [16].…”

“…This is based on the evidence that an alternative splicing variant of hCNT3, which is truncated in its N-terminus tail is retained in the ER [16]. Moreover, a β-turn domain in this segment of the hCNT3 protein has been shown to be essential for proper sorting of the transporter to the apical plasma membrane in polarized epithelia [26].…”

“…On the basis of previous observations on hCNT3 N-terminal tail which associated this intracellular domain with sorting regulation [16,26], the rCNT2 intracellular N-terminal tail was considered to be a candidate to regulate the sorting of the transporter to the plasma membrane. To elucidate the possible sequences contained in this domain of rCNT2 responsible for this process, sequential deletions of the N-terminal tail were performed ( Figure 1A).…”

Identification of a Membrane-targeting Domain of the Transient Receptor Potential Canonical (TRPC)4 Channel Unrelated to Its Formation of a Tetrameric Structure

“…Finally, the Val 57 -Thr 58 -Val 59 tripeptide at the N terminus of human concentrative nucleoside transporter 3 (hCNT3) appears to be the core of the endoplasmic reticulum export signal (47). Our preceding results have demonstrated that the 23 VRAETEL 29 aa residues require TRPC4 expression at the cell surface.…”

Nucleoside transporters and human organic cation transporter 1 determine the cellular handling of DNA‐methyltransferase inhibitors

“…MDCK cells were plated on 12 mm diameter, 0.3 μm pore Transwell plates (Corning Incorporated, Corning, NY, USA) and transfected as described previously (Errasti‐Murugarren et al ., ). Proper expression and insertion of transporter proteins at the plasma membrane has been verified using YFP‐tagged transporters, as previously reported (Errasti‐Murugarren et al ., ; 2010a,b), and transport assays, as detailed below, using model substrates of hCNT‐ and hOCT‐type proteins (uridine and cytidine for hCNT1 and hCNT3, and MPP + for hOCT1). Uptake rates were determined as a control of transporter expression levels at the plasma membrane.…”