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) (LTC(4)) transport but increased 17beta-estradiol 17beta-d-glucuronide (E(2)17betaG) and methotrexate (MTX) transport. Vanadate-induced trapping of [alpha-(32)P]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, LTC(4) transport by MRP2-Pro1158Ala was decreased. However, E(2)17betaG 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, LTC(4) transport was increased, whereas E(2)17betaG 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-(32)P]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.
Létourneau
Isabelle J
IJ
Department of Pharmacology and Toxicology, Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada.
Slot
Andrew J
AJ
Deeley
Roger G
RG
Cole
Susan P C
SP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D017997:Leukotriene C4 / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010777:Photochemistry; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014162:Transfection; D014638:Vanadates / Q000737:chemistry
D000255:Adenosine Triphosphate; D001386:Azides; D002413:Cations, Divalent; D004958:Estradiol; D017997:Leukotriene C4; D026901:Membrane Transport Proteins; D008727:Methotrexate; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011392:Proline; D014638:Vanadates; C000715087:ABCC2 protein, human; C025483:estradiol-17 beta-glucuronide; C114462:multidrug resistance-associated protein 3; C017040:8-azidoadenosine 5'-triphosphate; C421109:multidrug resistance-associated protein 1