Binding of a Photoaffinity Analogue of Glutathione to MRP1 (ABCC1) within Two Cytoplasmic Regions (L0 and L1) as Well as Transmembrane Domains 10-11 and 16-17

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• 作者：Joel Karwatsky ; Roni Daoud ; Jie Cai ; Philippe Gros ; and Elias Georges
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：March 25, 2003
• 年：2003
• 卷：42
• 期：11
• 页码：3286 - 3294
• 全文大小：157K
• 年卷期：v.42,no.11(March 25, 2003)
• ISSN：1520-4995

文摘

MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural productsas well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH isrequired for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating theinfluence of GSH on MRP1 is poorly understood, and the location of GSH binding site(s) within MRP1have yet to be determined. To address these issues, we have synthesized a [¹²⁵I] labeled azido-derivativeof GSH (IAAGSH) to photoaffinity label MRP1. Our results revealed that IAAGSH labeled MRP1 withhigh specificity, and binding was inhibited by MRP1 substrates leukotriene C₄ and MK571. Interestingly,verapamil and vincristine enhanced IAAGSH photolabeling of MRP1, in agreement with observationsthat both drugs enhance GSH transport. We observed GSH to be the best inhibitor of photoaffinity labeling,as compared to oxidized glutathione (GSSG) and four different GSH alkyl derivatives. These observationsindicate that IAAGSH interacted with MRP1 in a similar manner as unmodified GSH. Moreover, usingeight MRP1-HA variants, each containing hemagglutinin A (HA) epitopes inserted at different sites inMRP1, we mapped the GSH binding sites in MRP1. Our GSH analogue photoaffinity labeled four MRP1polypeptides that were located within two cytoplasmic domains in linker sequences (L0 and L1) as wellas transmembrane domains 10-11 and 16-17. The photoaffinity labeling of polypeptides within L0 andL1 domains is further confirmed using two MRP1-specific monoclonal antibodies (MRPr1 and QCRL1)with epitopes within the linker domains. Taken together, this study provides the most precise informationto date on the location of GSH binding sites in MRP1.