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Abstract
Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C(4) and the cholestatic estrogen, 17beta-estradiol 17(beta-D-glucuronide) (E(2)17betaG). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E(2)17betaG. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E(2)17betaG transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments of the COOH-terminal third of the mouse protein by replacement of either amino acids 959-1187 or 1188-1531 with those of MRP produced proteins capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C(4) with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E(2)17betaG with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E(2)17betaG.
Highlights
Multidrug resistance protein (MRP)1 and P-glycoprotein (Pgp) are very distantly related members of the superfamily of
The levels of vector-encoded protein were severalfold lower in the mrp transfectant populations used for the original study than in the MRP transfectants used for comparison
Transport of LTC4 and E217G by Hybrid Proteins—We examined the ability of the hybrid proteins to transport two well characterized potential physiological substrates, LTC4 and E217G
Summary
Materials—Doxorubicin HCl, vincristine sulfate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Sigma. The resulting insert was excised using HindIII, which cut in the polylinker region 5Ј to the insert and at the 3Ј end of the insert at the HindIII site introduced by PCR This fragment was ligated to an 11.5-kb HindIII fragment containing nucleotides 2875– 4823 of MRP attached to the pCEBV7 expression vector to generate construct pCEBV7-mrp/ MRP959 –1531 [7]. The vector encoding mrp/MRP959–1187 was generated by ligating a HindIII-EcoRI fragment encompassing nucleotides 2875–3880 of MRP into HindIII-EcoRI digested pBluescript II KSϩ This construct was digested with StuI at nucleotide 3551 of the insert and with SpeI at a site in the polylinker region 3Ј to the insert to generate a 3.7-kb StuI-SpeI fragment containing the vector attached to nucleotides 2875– 3554 of MRP. Uptake was terminated at various times by removing aliquots (20 l) and samples processed as described above
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