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Mutational Analysis of a Highly Conserved Proline Residue in MRP1, MRP2, and MRP3 Reveals a Partially Conserved Function

Departments of Pharmacology and Toxicology (I.J.L., S.P.C.C.) and Pathology and Molecular Medicine (A.J.S., R.G.D., S.P.C.C.) and Division of Cancer Biology and Genetics, Cancer Research Institute (I.J.L., A.J.S., R.G.D., S.P.C.C.), Queen's University, Kingston, Ontario, Canada

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 17ß-estradiol 17ß-D-glucuronide (E₂17ßG) and methotrexate (MTX) transport. Vanadate-induced trapping of [-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, E₂17ßG 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 E₂17ßG 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 [-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.

This article has been cited by other articles:

				A. J. Slot, D. D. Wise, R. G. Deeley, T. J. Monks, and S. P. C. Cole Modulation of Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP2 (ABCC2) Transport Activities by Endogenous and Exogenous Glutathione-Conjugated Catechol Metabolites Drug Metab. Dispos., March 1, 2008; 36(3): 552 - 560. [Abstract] [Full Text] [PDF]