Mutational analysis of a highly conserved proline residue in MRP1, MRP2, and MRP3 reveals a partially conserved function

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 C-4 (LTC4) transport but increased 17 beta-estradiol 17 beta-D-glucuronide (E(2)17 beta G) and methotrexate (MTX) transport. Vanadate-induced trapping of [alpha-P-32]8N(3)ADP 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-Pro(1150) is conserved, the analogous Pro(1158) and Pro(1147) 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, E(2)17 beta G 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 E(2)17 beta G 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 [alpha-P-32]8N(3)ADP. We conclude that MRP1-Pro(1150), MRP2-Pro(1158), and MRP3-Pro(1147) in CL7 differ in their influence on substrate specificity but share a common role in the nucleotide interactions of these transporters.