Premium
Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells
Author(s) -
Arlanov Rudolf,
Porter Andrew,
Strand Dennis,
Brough Rachel,
Karpova Darja,
Kerb Reinhold,
Wojnowski Leszek,
Schwab Matthias,
Lang Thomas
Publication year - 2012
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22041
Subject(s) - multidrug resistance associated protein 2 , biology , multidrug resistance associated proteins , ht1080 , efflux , transport protein , transporter , glutathione , endoplasmic reticulum , microbiology and biotechnology , atp binding cassette transporter , genetics , in vitro , biochemistry , gene , enzyme
The multidrug resistance‐associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel “ Sc reen and In sert” (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30–65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum‐like distribution. Variants N1244K and R1174H decreased transport of glutathione–methylfluorescein (GS–MF) and glutathione–monochlorobimane (GS–MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS–MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS–MF and GS–MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation. Hum Mutat 33:750–762, 2012. © 2012 Wiley Periodicals, Inc.