In Vitro Transport Activity and Trafficking of MRP2/ABCC2 Polymorphic Variants

Abstract

Purpose Multidrug resistance-associated protein 2 (MRP2/ABCC2) is an efflux pump that removes drugs and chemicals from cells. We sought to characterize the expression, trafficking and in vitro activity of seven single nucleotide polymorphisms (SNPs) in the ABCC2 gene. Methods ABCC2 SNPs were generated using site-directed mutagenesis and stably expressed in Flp-In HEK293 cells, which allows targeted insertion of transgenes within the genome. Total and cell surface expression of MRP2 as well as accumulation of substrates (calcein AM and 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate, CDCF) were quantified in cells or inverted membrane vesicles expressing wild-type (WT) or variant forms. Results The cell surface expression of the C-24T-, G1249A-, G3542T-, T3563A-, C3972T- and G4544A-MRP2 variants was similar to WT-MRP2. While expression was similar, transport of calcein AM was enhanced in cells expressing the G3542T-, T3563A-, C3972T-, and G4544A-MRP2 variants. By comparison, cells expressing the C2366T-MRP2 variant had 40-50% lower surface expression, which increased the accumulation of calcein AM up to 3-fold. Accumulation of CDCF in inverted membrane vesicles expressing the C2366T-MRP2 variant was also reduced by 50%. In addition, the G1249A-MRP2 variant had decreased transport of CDCF. Conclusions Taken together, these data demonstrate that genetic variability in the ABCC2 gene influences the in vitro expression, trafficking, and transport activity of MRP2.

Pharmacogenetics of toxicity of 5-fluorouracil, doxorubicin and cyclophosphamide chemotherapy in breast cancer patients

“…ABCC2 transporter is crucial for glutathione, glucuronide and sulfate conjugates of many drugs, thus it mediates their intracellular concentration. Variant p.Val417Ile (rs2273697) has been shown in in vitro models to decrease the apparent affinity for substrates conjugated with glutathione and glucuronide, and to decrease the transport activity [ 105 , 106 ]. Opposite findings came from the work of Haenisch and colleagues on bioavailability of talinolol, who reported that p.Val417Ile variant was associated with higher activity of intestinal ABCC2 [ 107 ].…”

A novel ABCC2 p.G693R mutation resulting in loss of function of MRP2: a recurrent cause of hyperbilirubinemia in Dubin-Johnson syndrome in China

“…In the present study, the p.G693R mutation was observed in 2 out of 7 patients, indicating that the p.G693R mutation might be a potential hotspot mutation in Chinese DJS patients. ABCC2 p.G693R is a missense mutation located in the first ATP-binding domain of MRP2 protein [17]. We demonstrated that while wild-type MRP2 predominantly localized to the cell surface and cytoplasm, the MRP2 p.G693R mutant was predominantly retained in the cytoplasm rather than the cell surface.…”

A recurrent ABCC2 p.G693R mutation resulting in loss of function of MRP2 and hyperbilirubinemia in Dubin-Johnson syndrome in China

“…In the present study, the p.G693R mutation was observed in 2 out of 7 patients, indicating that the p.G693R mutation might be a potential hotspot mutation in Chinese DJS patients. ABCC2 p.G693R is a missense mutation located in the first ATP-binding domain of MRP2 protein [25]. We demonstrated that while wild-type MRP2 predominantly localized to the cell surface and cytoplasm, the MRP2 p.G693R mutant was predominantly retained in the cytoplasm rather than the cell surface.…”