Characterization of drug transport by the human multidrug resistance protein 3 (ABCC3)

We have characterized the substrate specificity and mechanism of transport of the human multidrug resistance-associated protein 3 (MRP3). A murine fibroblast-like cell line generated from the kidneys of mice that lack Mdr1a/b and Mrp1 was retrovirally transduced with MRP3 cDNA. Stable clones overproducing MRP3 were resistant to the epipodophyllotoxins etoposide and teniposide but not to vincristine, doxorubicin, and cisplatin, drugs suggested to be MRP3 substrates by others. The resistance to etoposide was associated with reduced cellular accumulation and enhanced efflux of this drug and was not affected by depleting cells of glutathione but was inhibited by several common organic anion transport inhibitors. Membrane vesicles from infected insect cells expressing MRP3 mediated ATP-dependent transport of estradiol 17-beta-D-glucuronide, leukotriene C(4), dinitrophenyl S-glutathione but not glutathione itself, and etoposide glucuronide, a major metabolite of etoposide in vivo. The transport of estradiol 17-beta-D-glucuronide by MRP3 was inhibited in a concentration-dependent manner by both etoposide and methotrexate. Even though etoposide glucuronide is an excellent substrate for MRP3, this compound is not involved in the etoposide resistance of our MRP3 cells, as these cells extrude unmodified etoposide rather than etoposide glucuronide.     

Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.     

Negative regulation of human insulin gene expression by the 5'-flanking region in non-pancreatic cells

We have examined the effect of the 5'-flanking region of the human insulin gene on its expression in non-pancreatic cells. The presence of the region containing the insulin gene enhancer (-339 to -169 bp) markedly repressed the promoter activity of the insulin gene. This suppressive phenomenon was restored by the addition of forskolin or dibutyryl cAMP, suggesting that this region alone is not sufficient to repress completely insulin gene expression in the presence of extracellular stimuli which increase the intracellular cAMP level. The hypervariable region (HVR) located at -365 bp also repressed the promoter activity. These results show negative regulation of human insulin gene expression in non-pancreatic cells by these regions.     

Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr³²⁴ in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.     

Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1)

The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.