Journal of Biochemistry Advance Access originally published online on July 21, 2006
Journal of Biochemistry 2006 140(3):313-318; doi:10.1093/jb/mvj155
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© 2006 The Japanese Biochemical Society.
ARTICLE |
A Functional Role of Intracellular Loops of Human Multidrug Resistance Protein 1
1 Department of Molecular Oncology and 2 Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520; and 3 Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871
To whom correspondence should be addressed. Phone: +81-99-275-5490, Fax: +81-99-265-9687, E-mail: akiyamas{at}m3.kufm.kagoshima-u.ac.jp
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-
-[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--[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.
* Present address: Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, 2020 Gravier Street, New Orleans, LA, 70112, USA
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