Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2)

The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C‐proximal MRP2 and N‐proximal MRP1 localized to the apical membrane of polarized Madin–Darby canine kidney cells (MDCKII) indicating involvement of the carboxy‐proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven‐amino‐acid extension at its C‐terminus with the last three amino acids (TKF) comprising a PDZ‐interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C‐termini. These constructs were fused via their N‐termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver‐derived human HepG2 cells. Quantitative analysis showed that full‐length GFP–MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C‐terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP–MRP2 was C‐terminally truncated by 15 or more amino acids. Thus, neither the PDZ‐interacting TKF motif nor the full seven‐amino‐acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C‐terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells.