title: Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2.
abstract: Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.
authors.affiliation: Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Surtaj H
authors.initials: SH
authors.lastname: Iram
authors.forename: Susan P C
authors.initials: SP
authors.lastname: Cole
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001386:Azides / Q000493:pharmacokinetics; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D003461:Crystallography; D004958:Estradiol / Q000493:pharmacokinetics; D057809:HEK293 Cells; D006801:Humans; D017997:Leukotriene C4 / Q000493:pharmacokinetics; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D009711:Nucleotides / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D014316:Tritium
chemical_list: D000255:Adenosine Triphosphate; D000596:Amino Acids; D001386:Azides; D004958:Estradiol; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D014316:Tritium; C017040:8-azidoadenosine 5'-triphosphate; C421109:multidrug resistance-associated protein 1