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.
Nies
Anne T
AT
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany. a.nies@dkfz.de
König
Jörg
J
Cui
Yunhai
Y
Brom
Manuela
M
Spring
Herbert
H
Keppler
Dietrich
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D004285:Dogs; D018432:Drug Resistance, Multiple; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011993:Recombinant Fusion Proteins; C000715087:ABCC2 protein, human; C421109:multidrug resistance-associated protein 1