title: Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells.
abstract: 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 "Screen and Insert" (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.
authors.affiliation: Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Arlanov
authors.forename: Andrew
authors.initials: A
authors.lastname: Porter
authors.forename: Dennis
authors.initials: D
authors.lastname: Strand
authors.forename: Rachel
authors.initials: R
authors.lastname: Brough
authors.forename: Darja
authors.initials: D
authors.lastname: Karpova
authors.forename: Reinhold
authors.initials: R
authors.lastname: Kerb
authors.forename: Leszek
authors.initials: L
authors.lastname: Wojnowski
authors.forename: Matthias
authors.initials: M
authors.lastname: Schwab
authors.forename: Thomas
authors.initials: T
authors.lastname: Lang
mesh_terms: D001741:Black or African American / Q000235:genetics; D001213:Asian / Q000235:genetics; D045744:Cell Line, Tumor; D002700:Chloramines / Q000378:metabolism; D005354:Fibrosarcoma / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D014644:Genetic Variation; D057809:HEK293 Cells; D006239:Haplotypes; D006801:Humans; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism*; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D002700:Chloramines; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; D013752:Tetracycline; C000715087:ABCC2 protein, human; C030816:chloramine