Abstract
The ATP-binding cassette multidrug resistance protein 1 MRP1 (ABCC1) mediates the cellular efflux of organic anions including conjugated metabolites, chemotherapeutic agents, and toxicants. We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C4 (LTC4) transport but increased 17β-estradiol 17β-d-glucuronide (E217βG) and methotrexate (MTX) transport. Vanadate-induced trapping of [α-32P]8N3ADP by the Pro1150Ala mutant in the absence of substrate was also greatly reduced compared with wild-type MRP1 suggesting an uncoupling of ATP hydrolysis and transport activity. To determine whether the functional importance of MRP1-Pro1150 is conserved, the analogous Pro1158 and Pro1147 residues in the MRP2 and MRP3 transporters, respectively, were mutated to Ala. Expression levels of the three mutants were unaffected; however, the vesicular transport activity of at least one organic anion substrate was significantly altered. As observed for MRP1-Pro1150Ala, LTC4 transport by MRP2-Pro1158Ala was decreased. However, E217βG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala. In the case of MRP3-Pro1147Ala, LTC4 transport was increased, whereas E217βG transport was unaffected. MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala. In contrast, all three mutants showed a marked reduction in levels of vanadate-induced trapped [α-32P]8N3ADP. We conclude that MRP1-Pro1150, MRP2-Pro1158, and MRP3-Pro1147 in CL7 differ in their influence on substrate specificity but share a common role in the nucleotide interactions of these transporters.
Footnotes
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This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) (MOP-10519). I.J.L. is the recipient of a CIHR Doctoral Award and A.J.S. is the recipient of a Queen's University Robert John Wilson Fellowship. S.P.C.C. is the recipient of a Canada Research Chair in Cancer Biology and is the Queen's University Bracken Chair in Genetics and Molecular Medicine.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.015479.
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ABBREVIATIONS: MRP, multidrug resistance protein; ABC, ATP-binding cassette; TM, transmembrane; NBD, nucleotide binding domain; GSH, glutathione; CL, cytoplasmic loop; LTC4, leukotriene C4;E217βG, 17β-estradiol 17β-d-glucuronide; MTX, methotrexate; kb, kilobase; HEK, human embryonic kidney; mAb, monoclonal antibody; TSB, Tris-sucrose buffer; PAGE, polyacrylamide gel electrophoresis.
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↵1 Current affiliation: St. Jude Children's Research Hospital, Department of Pharmaceutical Sciences, Memphis, Tennessee.
- Received February 28, 2007.
- Accepted May 8, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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