The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites

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• 作者：Roni Daoud ; Jose Desneves ; Leslie W. Deady ; Leann Tilley ; Rik J. Scheper ; Philippe Gros ; and Elias Georges
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：May 23, 2000
• 年：2000
• 卷：39
• 期：20
• 页码：6094 - 6102
• 全文大小：217K
• 年卷期：v.39,no.20(May 23, 2000)
• ISSN：1520-4995

文摘

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or bothtransmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRPhas been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-,glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug bindingand transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug,N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling ofthe 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labelingof the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled proteinas the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore,a molar excess of leukotriene C₄, doxorubicin, colchicine, and other quinoline-based drugs, includingMK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACIaccumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells.Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides (~111and ~85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibodyepitopes corresponds to the C-terminal half of the MRP (amino acids ~900-1531) containing the thirdmultiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptidewhich contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of theMRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide bindingdomain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ~6plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in thegradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI tothe MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localizeto the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studyingMRP-drug interactions.