A functional role of intracellular loops of human multidrug resistance protein 1.
Multidrug resistance protein 1 (MRP1) is a human ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance to tumor cells by actively effluxing intracellular drugs. To examine the functional significance of intracellular loops (ICLs) in MRP1, we determined the effect of mutation of the amino acid sequence EXXXG, which is conserved in ICL5 and ICL7 of human MRP1, 2 and 3, sulfonylurea receptor (SUR) 1 and 2, and mouse MRP1 and 2. E and G in the ICLs of human MRP1 were mutated to L and P, respectively, and the N-terminal (including ICL5) and C-terminal (including ICL7) wild type or mutant halves of MRP1 were co-expressed in insect cells. The mutation of either ICL5 or ICL7 considerably decreased ATP-dependent LTC4 uptake into vesicles of insect cells expressing mutated MRP1. GSH-dependent photolabeling of MRP1 with an 125I-labeled photoaffinity analog of azido agosterol A (azido AG-A) was abolished by the mutations in ICL5 and ICL7. Mutations in ICL5 of MRP1 almost completely inhibited the labeling of NBD2, but not NBD1, by 8-azido-alpha-[32P]ATP. In contrast, mutations in ICL7 of MRP1 abolished the labeling of both NBDs. Mutation of either ICL5 or ICL7 of MRP1 almost completely inhibited vanadate trapping with 8-azido-alpha-[32P]ATP by both NBD1 and NBD2 domains. These findings indicate that the intramolecular signaling between NBD and ICLs in MRP1 is vital for MRP1 function.
Ren
Xiao-Qin
XQ
Department of Molecular Oncology and Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520.
Furukawa
Tatsuhiko
T
Yamamoto
Masatatsu
M
Aoki
Shunji
S
Kobayashi
Motomasa
M
Nakagawa
Masayuki
M
Akiyama
Shin-Ichi
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers; D006801:Humans; D017997:Leukotriene C4 / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels; D017434:Protein Structure, Tertiary; D013045:Species Specificity; D022161:Transport Vesicles / Q000378:metabolism; D014316:Tritium
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate; D017931:DNA Primers; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019920:Photoaffinity Labels; D014316:Tritium