Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity

Abstract

Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17␤-estradiol 17-(␤-D-glucuronide) (E 2 17␤G), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu 1089 of MRP1 as being critical for anthracycline resistance. However, Glu 1089 mutations had no effect on E 2 17␤G transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala 1239 in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E 2 17␤G transport 3-fold. Any mutation of mrp1 Ala 1239 , including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr 1242 for E 2 17␤G transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E 2 17␤G transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C 4 transport ϳ2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/ T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C 4 and E 2 17␤G. These results demonstrate the important role of Thr 1242 for E 2 17␤G transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.Human multidrug resistance protein 1 (MRP1), 1 a 190-kDa glycoprotein, is a member of the ATP-binding cassette (ABC) transporter superfamily (1-3). The predicted structure of MRP1 differs from that of a typical eukaryotic ABC transporter such as P-glycoprotein (P-gp). It contains a P-gp-like core region, which consists of two membrane-spanning domains (MSDs), each containing six transmembrane (TM) ␣-helices, and two nucleotide binding domains. However, MRP1 contains a third MSD predicted to consist of five TM ␣-helices with an extracellular NH 2 terminus and a cytoplasmic linker connecting the additional MSD with the core region (3-7). Like P-gp, MRP1 confers resistance to many commonly used, structurally diverse natural product chemotherapeutic agents including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (7-10). However, several lines of evidence suggest that MRP1 and P-gp confer resistance to these drugs by different mechanisms. In vitro studies using P-gp-enriched membrane vesicles or purifi...

The MRP family of drug efflux pumps

“…In addition, agosterol A, a polyhydroxylated sterol acetate that is a potent glutathione-dependent inhibitor, was also reported to crosslink TM17 (Ren et al, 2001(Ren et al, , 2002. The results of site-directed mutagenesis studies support the involvement of TM14 and TM17 in MRP1 activity, and have also implicated residues in TM6 (Ito et al, 2001b;Zhang et al, 2001aZhang et al, , b, 2002Haimeur et al, 2002;Ren et al, 2002).…”

Structural and Functional Consequences of Mutating Cysteine Residues in the Amino Terminus of Human Multidrug Resistance-associated Protein 1

“…Yet, these mutations had no effect on transport of conjugated organic anions such as LTC 4 . Thr 1242 in the transmembrane segment 17 of human MRP1 was also found to be important for MRP1 function (26). Mutation of Thr 1242 reduced MRP1-mediated transport of LTC 4 and E 2 17␤G and drug resistance.…”

Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione

“…The transfected cell lines that have been described previously include: human MRP1 and murine mrp1 (43), chimeric mrp1/MRP1s (44), and mutant MRP1 and mrp1 (28,30). MRP1 half molecules (amino acids 1-932 and 932-1531) were also cloned into the mammalian dual expression vector pBudCE4.1 (Invitrogen).…”

“…Recent studies indicate that a number of amino acids in predicted TM helix 17 and at least one residue in TM helix 14 are important in determining substrate specificity (28 -30). Furthermore, it is possible to make compensatory mutations in these two helices, suggesting that they may be in spatial proximity to one another in the native protein (30).…”

“…3 We have shown that sequence variations in the COOH-proximal third of the two proteins play a major role in these functional differences (28,30,44). Because this region of the protein contains the site that is strongly labeled by LY475776, we compared the relative ability of LY475776 to inhibit transport of LTC 4 by MRP1 and murine mrp1 (Fig.…”