The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites†
Identifieur interne : 001A05 ( Istex/Corpus ); précédent : 001A04; suivant : 001A06The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites†
Auteurs : Roni Daoud ; Jose Desneves ; Leslie W. Deady ; Leann Tilley ; Rik J. Scheper ; Philippe Gros ; Elias GeorgesSource :
- Biochemistry [ 0006-2960 ] ; 2000.
Abstract
Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has 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 binding and 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 of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as 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, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation 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 (∼111 and ∼85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids ∼900−1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1−900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ∼6 plus 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 the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP−drug interactions.
Url:
DOI: 10.1021/bi9922188
Links to Exploration step
ISTEX:36ABA24296CC0F5175E3849F9CFC34622FCCF8CALe document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites†</title>
<author><name sortKey="Daoud, Roni" sort="Daoud, Roni" uniqKey="Daoud R" first="Roni" last="Daoud">Roni Daoud</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Institute of Parasitology, McGill University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Desneves, Jose" sort="Desneves, Jose" uniqKey="Desneves J" first="Jose" last="Desneves">Jose Desneves</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Deady, Leslie W" sort="Deady, Leslie W" uniqKey="Deady L" first="Leslie W." last="Deady">Leslie W. Deady</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> La Trobe University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Tilley, Leann" sort="Tilley, Leann" uniqKey="Tilley L" first="Leann" last="Tilley">Leann Tilley</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> La Trobe University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Scheper, Rik J" sort="Scheper, Rik J" uniqKey="Scheper R" first="Rik J." last="Scheper">Rik J. Scheper</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Free University Hospital.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Gros, Philippe" sort="Gros, Philippe" uniqKey="Gros P" first="Philippe" last="Gros">Philippe Gros</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Department of Biochemistry, McGill University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Georges, Elias" sort="Georges, Elias" uniqKey="Georges E" first="Elias" last="Georges">Elias Georges</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Institute of Parasitology, McGill University.</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> To whom correspondence should be addressed: Institute ofParasitology, McGill University, 21, 111 Lakeshore Road, Ste-Annede Bellevue, PQ H9X 3V9. Telephone: (514) 398-8137. Fax: (514)398-7857. E-mail: Elias_Georges@maclan.McGill.CA.</mods:affiliation>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">ISTEX</idno>
<idno type="RBID">ISTEX:36ABA24296CC0F5175E3849F9CFC34622FCCF8CA</idno>
<date when="2000" year="2000">2000</date>
<idno type="doi">10.1021/bi9922188</idno>
<idno type="url">https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/fulltext.pdf</idno>
<idno type="wicri:Area/Istex/Corpus">001A05</idno>
<idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001A05</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based
Drug at Multiple Sites<ref type="bib" target="#bi9922188AF2"><hi rend="superscript">†</hi>
</ref>
</title>
<author><name sortKey="Daoud, Roni" sort="Daoud, Roni" uniqKey="Daoud R" first="Roni" last="Daoud">Roni Daoud</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Institute of Parasitology, McGill University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Desneves, Jose" sort="Desneves, Jose" uniqKey="Desneves J" first="Jose" last="Desneves">Jose Desneves</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Deady, Leslie W" sort="Deady, Leslie W" uniqKey="Deady L" first="Leslie W." last="Deady">Leslie W. Deady</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> La Trobe University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Tilley, Leann" sort="Tilley, Leann" uniqKey="Tilley L" first="Leann" last="Tilley">Leann Tilley</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> La Trobe University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Scheper, Rik J" sort="Scheper, Rik J" uniqKey="Scheper R" first="Rik J." last="Scheper">Rik J. Scheper</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Free University Hospital.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Gros, Philippe" sort="Gros, Philippe" uniqKey="Gros P" first="Philippe" last="Gros">Philippe Gros</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Department of Biochemistry, McGill University.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Georges, Elias" sort="Georges, Elias" uniqKey="Georges E" first="Elias" last="Georges">Elias Georges</name>
<affiliation><mods:affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> Institute of Parasitology, McGill University.</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation> To whom correspondence should be addressed: Institute ofParasitology, McGill University, 21, 111 Lakeshore Road, Ste-Annede Bellevue, PQ H9X 3V9. Telephone: (514) 398-8137. Fax: (514)398-7857. E-mail: Elias_Georges@maclan.McGill.CA.</mods:affiliation>
</affiliation>
</author>
</analytic>
<monogr></monogr>
<series><title level="j" type="main">Biochemistry</title>
<title level="j" type="abbrev">Biochemistry</title>
<idno type="ISSN">0006-2960</idno>
<idno type="eISSN">1520-4995</idno>
<imprint><publisher>American Chemical Society</publisher>
<date type="e-published" when="2000-04-26">2000</date>
<date when="2000-05-23">2000</date>
<biblScope unit="vol">39</biblScope>
<biblScope unit="issue">20</biblScope>
<biblScope unit="page" from="6094">6094</biblScope>
<biblScope unit="page" to="6102">6102</biblScope>
</imprint>
<idno type="ISSN">0006-2960</idno>
</series>
</biblStruct>
</sourceDesc>
<seriesStmt><idno type="ISSN">0006-2960</idno>
</seriesStmt>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract">Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has 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 binding and 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 of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as 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, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation 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 (∼111 and ∼85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids ∼900−1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1−900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ∼6 plus 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 the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP−drug interactions.</div>
</front>
</TEI>
<istex><corpusName>acs</corpusName>
<keywords><teeft><json:string>iaci</json:string>
<json:string>photoaffinity</json:string>
<json:string>ltc4</json:string>
<json:string>polypeptide</json:string>
<json:string>digestion</json:string>
<json:string>msd1</json:string>
<json:string>trypsin</json:string>
<json:string>biochemistry</json:string>
<json:string>deeley</json:string>
<json:string>mrpm6</json:string>
<json:string>chem</json:string>
<json:string>msd2</json:string>
<json:string>biol</json:string>
<json:string>mrpr1</json:string>
<json:string>msd3</json:string>
<json:string>molar excess</json:string>
<json:string>photolabeled</json:string>
<json:string>biochem</json:string>
<json:string>transmembrane</json:string>
<json:string>monoclonal</json:string>
<json:string>photolabeling</json:string>
<json:string>iaciphotolabeled</json:string>
<json:string>peptide</json:string>
<json:string>doxorubicin</json:string>
<json:string>drug binding</json:string>
<json:string>sucrose</json:string>
<json:string>nbd1</json:string>
<json:string>photoreactive</json:string>
<json:string>colchicine</json:string>
<json:string>nbd2</json:string>
<json:string>helix</json:string>
<json:string>mabs</json:string>
<json:string>epitope</json:string>
<json:string>protease</json:string>
<json:string>apparent molecular mass</json:string>
<json:string>transmembrane helix</json:string>
<json:string>direct binding</json:string>
<json:string>molar</json:string>
<json:string>drug accumulation</json:string>
<json:string>unmodified</json:string>
<json:string>drug binding site</json:string>
<json:string>mrpm6 mabs</json:string>
<json:string>photoreactive drug</json:string>
<json:string>sodium azide</json:string>
<json:string>photolabeled peptide</json:string>
<json:string>drug sensitive</json:string>
<json:string>resistant cell</json:string>
<json:string>plasma membrane</json:string>
<json:string>mcgill university</json:string>
<json:string>multidrug resistance protein</json:string>
<json:string>protease inhibitor</json:string>
<json:string>monoclonal antibody</json:string>
<json:string>cole</json:string>
<json:string>membrane</json:string>
<json:string>membrane vesicle</json:string>
<json:string>iaci accumulation</json:string>
<json:string>ltc4 transport</json:string>
<json:string>drug binding domain</json:string>
<json:string>exhaustive digestion</json:string>
<json:string>silver staining</json:string>
<json:string>drug transport</json:string>
<json:string>carboxyl third</json:string>
<json:string>result show</json:string>
<json:string>multiple site</json:string>
<json:string>linker domain</json:string>
<json:string>several study</json:string>
</teeft>
</keywords>
<author><json:item><name>DAOUD Roni</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
<json:string>Institute of Parasitology, McGill University.</json:string>
</affiliations>
</json:item>
<json:item><name>DESNEVES Jose</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
</affiliations>
</json:item>
<json:item><name>DEADY Leslie W.</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
<json:string>La Trobe University.</json:string>
</affiliations>
</json:item>
<json:item><name>TILLEY Leann</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
<json:string>La Trobe University.</json:string>
</affiliations>
</json:item>
<json:item><name>SCHEPER Rik J.</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
<json:string>Free University Hospital.</json:string>
</affiliations>
</json:item>
<json:item><name>GROS Philippe</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
<json:string>Department of Biochemistry, McGill University.</json:string>
</affiliations>
</json:item>
<json:item><name>GEORGES Elias</name>
<affiliations><json:string>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</json:string>
<json:string>Institute of Parasitology, McGill University.</json:string>
<json:string>To whom correspondence should be addressed: Institute ofParasitology, McGill University, 21, 111 Lakeshore Road, Ste-Annede Bellevue, PQ H9X 3V9. Telephone: (514) 398-8137. Fax: (514)398-7857. E-mail: Elias_Georges@maclan.McGill.CA.</json:string>
</affiliations>
</json:item>
</author>
<arkIstex>ark:/67375/TPS-J0RG5SH4-V</arkIstex>
<language><json:string>unknown</json:string>
</language>
<originalGenre><json:string>research-article</json:string>
</originalGenre>
<abstract>Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has 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 binding and 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 of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as 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, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation 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 (∼111 and ∼85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids ∼900−1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1−900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ∼6 plus 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 the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP−drug interactions.</abstract>
<qualityIndicators><refBibsNative>true</refBibsNative>
<abstractWordCount>351</abstractWordCount>
<abstractCharCount>2420</abstractCharCount>
<keywordCount>0</keywordCount>
<score>10</score>
<pdfWordCount>6697</pdfWordCount>
<pdfCharCount>39822</pdfCharCount>
<pdfVersion>1.2</pdfVersion>
<pdfPageCount>9</pdfPageCount>
<pdfPageSize>612 x 792 pts (letter)</pdfPageSize>
<pdfWordsPerPage>744</pdfWordsPerPage>
<pdfText>true</pdfText>
</qualityIndicators>
<title>The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites†</title>
<genre><json:string>research-article</json:string>
</genre>
<host><title>Biochemistry</title>
<language><json:string>unknown</json:string>
</language>
<issn><json:string>0006-2960</json:string>
</issn>
<eissn><json:string>1520-4995</json:string>
</eissn>
<volume>39</volume>
<issue>20</issue>
<pages><first>6094</first>
<last>6102</last>
</pages>
<genre><json:string>journal</json:string>
</genre>
</host>
<ark><json:string>ark:/67375/TPS-J0RG5SH4-V</json:string>
</ark>
<categories><wos><json:string>1 - science</json:string>
<json:string>2 - biochemistry & molecular biology</json:string>
</wos>
<scienceMetrix><json:string>1 - health sciences</json:string>
<json:string>2 - biomedical research</json:string>
<json:string>3 - biochemistry & molecular biology</json:string>
</scienceMetrix>
<scopus><json:string>1 - Life Sciences</json:string>
<json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string>
<json:string>3 - Biochemistry</json:string>
</scopus>
<inist><json:string>1 - sciences appliquees, technologies et medecines</json:string>
<json:string>2 - sciences biologiques et medicales</json:string>
<json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string>
</inist>
</categories>
<publicationDate>2000</publicationDate>
<copyrightDate>2000</copyrightDate>
<doi><json:string>10.1021/bi9922188</json:string>
</doi>
<id>36ABA24296CC0F5175E3849F9CFC34622FCCF8CA</id>
<score>1</score>
<fulltext><json:item><extension>pdf</extension>
<original>true</original>
<mimetype>application/pdf</mimetype>
<uri>https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/fulltext.pdf</uri>
</json:item>
<json:item><extension>zip</extension>
<original>false</original>
<mimetype>application/zip</mimetype>
<uri>https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/bundle.zip</uri>
</json:item>
<json:item><extension>txt</extension>
<original>false</original>
<mimetype>text/plain</mimetype>
<uri>https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/fulltext.txt</uri>
</json:item>
<istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based
Drug at Multiple Sites†</title>
</titleStmt>
<publicationStmt><authority>ISTEX</authority>
<publisher>American Chemical Society</publisher>
<availability><licence>Copyright © 2000 American Chemical Society</licence>
<p>American Chemical Society</p>
</availability>
<date type="e-published" when="2000-04-26">2000</date>
<date when="2000-05-23">2000</date>
<date type="Copyright" when="2000">2000</date>
</publicationStmt>
<notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note>
<note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note>
</notesStmt>
<sourceDesc><biblStruct type="article"><analytic><title level="a" type="main" xml:lang="en">The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based
Drug at Multiple Sites<ref type="bib" target="#bi9922188AF2"><hi rend="superscript">†</hi>
</ref>
</title>
<author xml:id="author-0000"><persName><surname>Daoud</surname>
<forename type="first">Roni</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
<note place="foot"><ref>‡</ref>
<p>
Institute of Parasitology, McGill University.</p>
</note>
</author>
<author xml:id="author-0001"><persName><surname>Desneves</surname>
<forename type="first">Jose</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
</author>
<author xml:id="author-0002"><persName><surname>Deady</surname>
<forename type="first">Leslie W.</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
<note place="foot"><ref>§</ref>
<p>
La Trobe University.</p>
</note>
</author>
<author xml:id="author-0003"><persName><surname>Tilley</surname>
<forename type="first">Leann</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
<note place="foot"><ref>§</ref>
<p>
La Trobe University.</p>
</note>
</author>
<author xml:id="author-0004"><persName><surname>Scheper</surname>
<forename type="first">Rik J.</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
<note place="foot"><ref>‖</ref>
<p>
Free University Hospital.</p>
</note>
</author>
<author xml:id="author-0005"><persName><surname>Gros</surname>
<forename type="first">Philippe</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
<note place="foot"><ref>⊥</ref>
<p>
Department of Biochemistry, McGill University.</p>
</note>
</author>
<author xml:id="author-0006" role="corresp"><persName><surname>Georges</surname>
<forename type="first">Elias</forename>
</persName>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</affiliation>
<note place="foot"><ref>‡</ref>
<p>
Institute of Parasitology, McGill University.</p>
</note>
<affiliation role="corresp"> To whom correspondence should be addressed: Institute of Parasitology, McGill University, 21, 111 Lakeshore Road, Ste-Anne de Bellevue, PQ H9X 3V9. Telephone: (514) 398-8137. Fax: (514) 398-7857. E-mail: Elias_Georges@maclan.McGill.CA.</affiliation>
</author>
<idno type="istex">36ABA24296CC0F5175E3849F9CFC34622FCCF8CA</idno>
<idno type="ark">ark:/67375/TPS-J0RG5SH4-V</idno>
<idno type="DOI">10.1021/bi9922188</idno>
</analytic>
<monogr><title level="j" type="main">Biochemistry</title>
<title level="j" type="abbrev">Biochemistry</title>
<idno type="acspubs">bi</idno>
<idno type="coden">bichaw</idno>
<idno type="pISSN">0006-2960</idno>
<idno type="eISSN">1520-4995</idno>
<imprint><publisher>American Chemical Society</publisher>
<date type="e-published" when="2000-04-26">2000</date>
<date when="2000-05-23">2000</date>
<biblScope unit="vol">39</biblScope>
<biblScope unit="issue">20</biblScope>
<biblScope unit="page" from="6094">6094</biblScope>
<biblScope unit="page" to="6102">6102</biblScope>
</imprint>
</monogr>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><abstract><p>Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both
transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP
has 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 binding
and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug,
<hi rend="italic">N</hi>
-(hydrocinchonidin-8‘-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of
the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling
of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein
as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore,
a molar excess of leukotriene C<hi rend="subscript">4</hi>
, doxorubicin, colchicine, and other quinoline-based drugs, including
MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI
accumulation 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 (∼111
and ∼85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody
epitopes corresponds to the C-terminal half of the MRP (amino acids ∼900−1531) containing the third
multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide
which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the
MRP sequence (amino acids 1−900) containing the MSD1 and MSD2 plus the first nucleotide binding
domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ∼6
plus 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 the
gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to
the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize
to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying
MRP−drug interactions.
</p>
</abstract>
<textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term>
</keywords>
</textClass>
<langUsage><language ident="zxx"></language>
</langUsage>
</profileDesc>
</teiHeader>
</istex:fulltextTEI>
</fulltext>
<metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration>
<istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id>
<journal-id journal-id-type="coden">bichaw</journal-id>
<journal-title-group><journal-title>Biochemistry</journal-title>
<abbrev-journal-title>Biochemistry</abbrev-journal-title>
</journal-title-group>
<issn pub-type="ppub">0006-2960</issn>
<issn pub-type="epub">1520-4995</issn>
<publisher><publisher-name>American Chemical Society</publisher-name>
</publisher>
<self-uri>pubs.acs.org/biochemistry</self-uri>
</journal-meta>
<article-meta><article-id pub-id-type="doi">10.1021/bi9922188</article-id>
<article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based
Drug at Multiple Sites<xref rid="bi9922188AF2"><sup>†</sup>
</xref>
</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Daoud</surname>
<given-names>Roni</given-names>
</name>
<xref rid="bi9922188AF3"><sup>‡</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Desneves</surname>
<given-names>Jose</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Deady</surname>
<given-names>Leslie W.</given-names>
</name>
<xref rid="bi9922188AF4"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Tilley</surname>
<given-names>Leann</given-names>
</name>
<xref rid="bi9922188AF4"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Scheper</surname>
<given-names>Rik J.</given-names>
</name>
<xref rid="bi9922188AF5"><sup>‖</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Gros</surname>
<given-names>Philippe</given-names>
</name>
<xref rid="bi9922188AF6"><sup>⊥</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Georges</surname>
<given-names>Elias</given-names>
</name>
<xref rid="bi9922188AF1">*</xref>
<xref rid="bi9922188AF3"><sup>‡</sup>
</xref>
</contrib>
<aff>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,
Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,
and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
</aff>
</contrib-group>
<author-notes><fn id="bi9922188AF3"><label>‡</label>
<p>
Institute of Parasitology, McGill University.</p>
</fn>
<fn id="bi9922188AF4"><label>§</label>
<p>
La Trobe University.</p>
</fn>
<fn id="bi9922188AF5"><label>‖</label>
<p>
Free University Hospital.</p>
</fn>
<fn id="bi9922188AF6"><label>⊥</label>
<p>
Department of Biochemistry, McGill University.</p>
</fn>
<corresp id="bi9922188AF1">
To whom correspondence should be addressed: Institute of
Parasitology, McGill University, 21, 111 Lakeshore Road, Ste-Anne
de Bellevue, PQ H9X 3V9. Telephone: (514) 398-8137. Fax: (514)
398-7857. E-mail: Elias_Georges@maclan.McGill.CA.</corresp>
</author-notes>
<pub-date pub-type="epub"><day>26</day>
<month>04</month>
<year>2000</year>
</pub-date>
<pub-date pub-type="ppub"><day>23</day>
<month>05</month>
<year>2000</year>
</pub-date>
<volume>39</volume>
<issue>20</issue>
<fpage>6094</fpage>
<lpage>6102</lpage>
<history><date date-type="received"><day>23</day>
<month>09</month>
<year>1999</year>
</date>
<date date-type="rev-recd"><day>28</day>
<month>12</month>
<year>1999</year>
</date>
<date date-type="asap"><day>26</day>
<month>04</month>
<year>2000</year>
</date>
<date date-type="issue-pub"><day>23</day>
<month>05</month>
<year>2000</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2000 American Chemical Society</copyright-statement>
<copyright-year>2000</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
</permissions>
<abstract><p>Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both
transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP
has 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 binding
and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug,
<italic toggle="yes">N</italic>
-(hydrocinchonidin-8‘-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of
the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling
of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein
as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore,
a molar excess of leukotriene C<sub>4</sub>
, doxorubicin, colchicine, and other quinoline-based drugs, including
MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI
accumulation 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 (∼111
and ∼85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody
epitopes corresponds to the C-terminal half of the MRP (amino acids ∼900−1531) containing the third
multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide
which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the
MRP sequence (amino acids 1−900) containing the MSD1 and MSD2 plus the first nucleotide binding
domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ∼6
plus 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 the
gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to
the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize
to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying
MRP−drug interactions.
</p>
</abstract>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>bi9922188</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
<notes id="bi9922188AF2"><label>†</label>
<p>
This work was supported by grants from the Natural Sciences and
Engineering Research Council of Canada to E.G. Research at the
Institute of Parasitology is partially supported by a grant from the FCAR
pour l'aide à la recherche.</p>
</notes>
</front>
<body><sec id="d7e196"><title></title>
<p>Treatment of cancer patients with chemotherapeutic drugs
is often unsuccessful due to the emergence of drug resistant
tumors. Similarly, tumor cell lines selected in vitro with
anticancer drugs become resistant to multiple drugs with
concurrent overexpression of either or both transmembrane
proteins, the P-glycoprotein (P-gp1)<sup>1</sup>
and the multidrug
resistance protein (MRP) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00001" ref-type="bibr"></xref>
−<xref rid="bi9922188b00002" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00003" ref-type="bibr"></xref>
</named-content>
</italic>
). Gene transfer studies with
human P-gp1 or MRP cDNA were shown to confer resistance
to similar anticancer drugs onto previously drug susceptible
cells (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00004" ref-type="bibr"></xref>
, <xref rid="bi9922188b00005" ref-type="bibr"></xref>
</named-content>
</italic>
). Furthermore, disruption of P-gp1 or MRP genes
in mice led to increased sensitivity to natural product toxins
and elevated glutathione levels in MRP-expressing tissues
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00006" ref-type="bibr"></xref>
−<xref rid="bi9922188b00007" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00008" ref-type="bibr"></xref>
</named-content>
</italic>
). Although both proteins are likely to mediate several
physiological functions, P-gp1 appears to function as a
nonspecific efflux pump at the blood−brain barrier (<italic toggle="yes"><xref rid="bi9922188b00006" ref-type="bibr"></xref>
</italic>
), while
MRP functions include mediation of inflammatory responses
(<italic toggle="yes"><xref rid="bi9922188b00008" ref-type="bibr"></xref>
</italic>
). Moreover, both proteins have been shown to function
as “flipases” of short chain lipids (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00009" ref-type="bibr"></xref>
, <xref rid="bi9922188b00010" ref-type="bibr"></xref>
</named-content>
</italic>
) and to mediate the
transport of normal cell metabolites and xenobiotics (<italic toggle="yes">5</italic>
,<italic toggle="yes"> 11−13</italic>
).
</p>
<p>P-gp1 and the MRP are members of a large family of
membrane-trafficking proteins that couple ATP hydrolysis
to ligand transport across the cell membrane (<italic toggle="yes"><xref rid="bi9922188b00014" ref-type="bibr"></xref>
</italic>
); however,
the amino acid sequences of the two proteins are 15%
identical (<italic toggle="yes"><xref rid="bi9922188b00015" ref-type="bibr"></xref>
</italic>
). MRP primary structure encodes an MDR-like
core of two membrane-spanning domains (MSD2 and
MSD3) and two nucleotide-binding domains (NBD1 and
NBD2), in addition to a 220-amino acid N-terminal membrane-spanning domain (MSD1) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00016" ref-type="bibr"></xref>
−<xref rid="bi9922188b00017" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
). Although the role of
MSD1 remains to be clarified, it is thought to contain five
transmembrane helices with an extracytosolic N-terminus
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00017" ref-type="bibr"></xref>
, <xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
). Furthermore, deletion of the first transmembrane
helix from MSD1 or the entire MSD1 plus the linker
sequence between MDS1 and MDS2 was shown to inhibit
MRP-mediated transport of LTC<sub>4</sub>
(<italic toggle="yes"><xref rid="bi9922188b00019" ref-type="bibr"></xref>
</italic>
). More recently, Bakos
et al. (<italic toggle="yes"><xref rid="bi9922188b00020" ref-type="bibr"></xref>
</italic>
) showed that the deletion of all transmembrane
helices of MSD1 had no effect on MRP-mediated LTC<sub>4</sub>
transport. Interestingly, deletion of the linker domain between
MSD1 and MSD2 abolished LTC<sub>4</sub>
transport (<italic toggle="yes"><xref rid="bi9922188b00020" ref-type="bibr"></xref>
</italic>
).
</p>
<p>Several studies have shown MRP-mediated transport of
glutathione-, glucuronidate-, and sulfate-conjugated drugs
(<italic toggle="yes">11</italic>
,<italic toggle="yes"> 21−23</italic>
). The glutathione-conjugated eicosanoid, leukotriene C4 (LTC<sub>4</sub>
), is the highest-affinity substrate for MRP
transporter (<italic toggle="yes">11</italic>
,<italic toggle="yes"> 21</italic>
,<italic toggle="yes"> 22</italic>
). Indeed, the MRP appears to function
as a cotransporter of glutathione; hence, MRP-like proteins
are known as GS-X pumps or multispecific organic anion
transporters (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00024" ref-type="bibr"></xref>
, <xref rid="bi9922188b00025" ref-type="bibr"></xref>
</named-content>
</italic>
). The ability of MRP to bind and
transport unmodified drugs remains to be resolved. The MRP
was shown to transport several unmodified drugs and natural
products (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00026" ref-type="bibr"></xref>
, <xref rid="bi9922188b00027" ref-type="bibr"></xref>
</named-content>
</italic>
). For example, direct binding and transport
of unmodified quinoline-based drugs in MRP-expressing
MDR cells has been previously described (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00027" ref-type="bibr"></xref>
−<xref rid="bi9922188b00028" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00029" ref-type="bibr"></xref>
</named-content>
</italic>
). However,
Loe et al. (<italic toggle="yes"><xref rid="bi9922188b00030" ref-type="bibr"></xref>
</italic>
) found MRP-mediated active transport of
unmodified vincristine only in the presence of GSH.
</p>
<p>Photoreactive drug analogues have been previously employed to study protein receptor interactions with small ligand
molecules (<italic toggle="yes"><xref rid="bi9922188b00031" ref-type="bibr"></xref>
</italic>
). The use of photoreactive drug analogues that
photoaffinity label P-gp has demonstrated the presence of
at least two drug binding sites which map to sequences in
transmembranes 5 and 6, and 11 and 12 (<italic toggle="yes"><xref rid="bi9922188b00032" ref-type="bibr"></xref>
</italic>
). The drug
binding domains identified by photoaffinity labeling were
later confirmed by mutational analyses of P-gp1 TMs
sequences (<italic toggle="yes"><xref rid="bi9922188b00033" ref-type="bibr"></xref>
</italic>
). Several attempts to photoaffinity label the
MRP with commercially available P-gp-specific photoreactive drugs have not been successful (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00028" ref-type="bibr"></xref>
, <xref rid="bi9922188b00034" ref-type="bibr"></xref>
</named-content>
</italic>
). Leier et al.
(<italic toggle="yes"><xref rid="bi9922188b00011" ref-type="bibr"></xref>
</italic>
) demonstrated the photoaffinity labeling of MRP with
[<sup>3</sup>
H]LTC<sub>4</sub>
. The specificity of LTC<sub>4</sub>
photoaffinity labeling was
confirmed by competition experiments with nonradiolabeled
LTC<sub>4</sub>
and MK571, a quinoline-based antagonist of the LTD<sub>4</sub>
receptor which reverses MRP-mediated MDR (<italic toggle="yes"><xref rid="bi9922188b00035" ref-type="bibr"></xref>
</italic>
). However,
photoaffinity labeling of MRP by [<sup>3</sup>
H]LTC<sub>4</sub>
suffers from a
weak photolabeling efficiency which limits its usefulness in
studying MRP−drug interactions. Furthermore, with respect
to MRP binding to anticancer drugs, it is not known if LTC<sub>4</sub>
and unmodified anticancer drugs bind to the same sites. In
this report, we demonstrate specific photoaffinity labeling
of the MRP by a quinoline-based photoreactive drug.
Moreover, our results show for the first time photoaffinity
labeling of three sites in the N- and C-halves of the MRP.
</p>
</sec>
<sec id="d7e381"><title>Materials and Methods</title>
<p><italic toggle="yes">Materials.</italic>
Iodine-125 (100.7 mCi/mL) was purchased
from Amersham Biochemical Inc. (Mississauga, ON). Protein
A-coupled Sepharose was purchased from Pharmacia Inc.
(Montreal, PQ). The LTD<sub>4</sub>
receptor antagonist MK571 was
kindly provided by A. W. Ford-Hutchinson (Merck-Frost
Centre for Therapeutic Research, Point Claire-Dorval, PQ;
<italic toggle="yes">36</italic>
). Leukotriene C<sub>4</sub>
(LTC<sub>4</sub>
) was purchased from Cayman
Chemical Co. (Ann Arbor, MI). The small cell lung cancer
cells (H69 and H69/AR) and the MRP-specific monoclonal
antibody (QCRL-1) were kind gifts from S. P. C. Cole
(Cancer Research Laboratories, Queen's University, Kingston, ON). All other chemicals were of the highest commercial grade available.
</p>
<p><italic toggle="yes">Cell Culture and Plasma Membrane Preparations.</italic>
Drug
sensitive (H69) and resistant (H69/AR) cells were grown in
RPMI 1640 medium containing 4 mM glutamine and 5%
fetal calf serum (Hyclone). Resistant cells were cultured
continuously in the presence of 0.8 μM doxorubicin;
however, cells used for drug transport studies were grown
in drug-free medium for 10 days prior to the date of the
experiment. Plasma membranes from H69 and H69/AR cells
were prepared as described by Lin et al. (<italic toggle="yes"><xref rid="bi9922188b00037" ref-type="bibr"></xref>
</italic>
). In brief, cells
were collected by low-speed centrifugation and washed three
times with ice-cold phosphate-buffered saline (PBS) (pH 7.4).
Cells were homogenized in 50 mM mannitol, 5 mM Hepes,
and 10 mM Tris-HCl (pH 7.4) (containing 2 mM PMSF and
3 μg/mL leupeptin) in a Dounce glass homogenizer. A
calcium chloride solution was then added to the homogenate
to a final concentration of 10 mM and the solution mixed
by stirring to ensure even distribution of the cation. The
slightly turbid supernatant solution that contains plasmalemma vesicles was pelleted by high-speed centrifugation at
100000<italic toggle="yes">g</italic>
for 1 h at 4 °C using a Beckman SW28 rotor. For
sucrose gradient purification, the membrane suspension was
adjusted to a final sucrose concentration of 45% with the
addition of sucrose powder. The gradient was set up in 14
mL polycarbonate tubes using 1 mL of 60% sucrose, 5 mL
of membranes in 45% sucrose, 2.5 mL of 35% sucrose, and
2.5 mL of 30% sucrose. Samples were spun for 3 h at 35 000
rpm and 4 °C. Membranes floating at the 30, 35, and 45%
interfaces were harvested and washed with 10 mM Tris-HCl
(pH 7.4). The enriched plasma membrane fraction was
resuspended in the same buffer containing 250 mM sucrose.
Membranes were stored at −80 °C if not immediately used.
Protein concentrations were determined by the Lowry method
(<italic toggle="yes"><xref rid="bi9922188b00038" ref-type="bibr"></xref>
</italic>
).
</p>
<p><italic toggle="yes">Radioiodination and Photoaffinity Labeling.</italic>
The coupling
method used in the synthesis of photoreactive drugs (IACI)
has been previously described elsewhere (<italic toggle="yes"><xref rid="bi9922188b00039" ref-type="bibr"></xref>
</italic>
). Details of the
synthesis will be described elsewhere. Iodination of IACI
was carried out in the dark. Briefly, IACI (10 nmol) was
dissolved in 20 μL of dimethyl sulfoxide (DMSO) and mixed
with 10 μL of carrier-free Na<sup>125</sup>
I (1 mCi, 0.5 nmol) and 10
μL of chloramine T (10 nmol) in 1 M K<sub>2</sub>
HPO<sub>4</sub>
(pH 7.4).
The reaction was allowed to continue for 5 min and was
stopped by the addition of sodium metabisulfite [50 μL of a
5% (w/v) solution]. The reaction mixture was loaded onto a
C<sub>18</sub>
cartridge (Sep-Pak, Waters-Millipore) prewashed with
10 mM K<sub>2</sub>
HPO<sub>4</sub>
(pH 7.4). The column was washed with 5
mL aliquots of 10 mM K<sub>2</sub>
HPO<sub>4</sub>
(pH 7.4) containing 10%
(v/v) methanol until no significant radiolabel was detected.
IACI was eluted with 2.5 mL of methanol and vacuum-dried
in the dark. The dried residue was resuspended in DMSO
and the concentration of the radioactive, photoactive drug
determined by HPLC.
</p>
<p>Either plasma membranes (10−20 μg) or intact cells (5
× 10<sup>6</sup>
cells) were photoaffinity labeled with IACI. Briefly,
membranes or cells were photoaffinity labeled by IACI
(0.20−1.0 μM) in the absence or the presence of a molar
excess of colchicine, chloroquine, doxorubicin, LTC<sub>4</sub>
, or
MK571. Membranes or cells were incubated at room
temperature in the dark for 30 min and then transferred to
ice for 10 min. Following the latter incubation, cells were
irradiated for 10 min on ice with a UV source at 254 nm
(Stratagene UV cross-linker, Stratagene, La Jolla, CA). The
free photoactive drug was removed by centrifugation, and
cells were lysed in 20 μL of 50 mM Tris (pH 7.4) containing
1% Nonidet P-40 (NP40), 5 mM MgCl<sub>2</sub>
, and protease
inhibitors (3 μg/mL leupeptin and 2 mM PMSF). Photoaffinity-labeled proteins were isolated by brief centrifugation
at 4 °C and resolved on SDS−PAGE. It should be mentioned
that incubation of cells or membranes with IACI but without
UV irradiation did not result in the photoaffinity labeling of
proteins (data not shown).
</p>
<p><italic toggle="yes">Immunoprecipitation and SDS Gel Electrophoresis.</italic>
IACI
photoaffinity-labeled cells were lysed in 50 mM Tris-HCl
(pH 7.4) containing 0.5% CHAPS, 0.5% sodium deoxycholate, 150 mM NaCl, and protease inhibitors (3 μg/mL
leupeptin and 2 mM PMSF). The cell lysates were clarified
by centrifugation at 12000<italic toggle="yes">g</italic>
and 4 °C. Equal amounts of cell
lysate proteins were separately incubated overnight at 4 °C
with 10 μg of QCRL-1, MRPr1, or MRPm6 monoclonal
antibodies (Mabs) or an irrelevant IgG<sub>2a</sub>
. Protein A-coupled
Sepharose was added to cell lysates and the mixture allowed
to incubate for 1 h at room temperature. After several washes
in lysis buffer, proteins were released from the Sepharose
beads with buffer I [10 mM Tris-HCl (pH 8.0) containing
2% SDS, 50 mM dithiothreitol (DTT), and 1 mM ethylenediaminetetraacetate (EDTA)] and buffer II (2× buffer I and
9 M urea). Immunopurified proteins were eluted and resolved
by SDS−PAGE using the Laemmli or Fairbanks gel system
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00040" ref-type="bibr"></xref>
, <xref rid="bi9922188b00041" ref-type="bibr"></xref>
</named-content>
</italic>
). Gel slabs containing the immunoprecipitated
proteins were fixed in 50% methanol, dried, and exposed to
XAR Kodak film at −70 °C for 2−12 h. Alternatively,
proteins were visualized by silver staining, using the NOVEX
SilverXpress Silver Staining Kit.
</p>
<p><italic toggle="yes">Proteolytic Digestion and HPLC.</italic>
Immunopurified photoaffinity-labeled MRP bands were cut out of dried SDS−PAGE gels and digested with increasing concentrations of
<italic toggle="yes">Staphylococcus aureus</italic>
V8 protease (1−20 μg/gel slice) in
the well of a 15% Laemmli gel (<italic toggle="yes"><xref rid="bi9922188b00041" ref-type="bibr"></xref>
</italic>
) according to the method
of Cleveland et al. (<italic toggle="yes"><xref rid="bi9922188b00042" ref-type="bibr"></xref>
</italic>
). For partial digestion of the IACI
photoaffinity-labeled MRP, 100 μg of H69 and H69/AR
plasma membrane samples was photolabeled with 0.2 μM
IACI and immunoprecipitated overnight with QCRL-1 Mab
as previously described (<italic toggle="yes"><xref rid="bi9922188b00043" ref-type="bibr"></xref>
</italic>
). Protein A-coupled Sepharose
beads were washed in buffer A [0.1% TX-100, 0.03% SDS,
0.05 M Tris-HCl (pH 7.4), 5 mg/mL fraction V bovine serum
albumin (BSA), and 150 mM NaCl] containing protease
inhibitors (0.1 mM PMSF, 3 μg/mL leupeptin, pepstatin A,
and aprotinin) followed by several washes without protease
inhibitors. Mild trypsin digestion was carried out in the
presence of 8 or 16 ng of trypsin at 37 °C for 5 min.
Digestion was stopped with 10 μg/mL leupeptin, pepstatin
A, aprotinin, and 1 mM PMSF followed by incubation for 5
min at 65 °C in SDS−PAGE sample buffer. Samples were
resolved on Fairbanks gels, transferred to nitrocellulose
membranes, and probed with QCRL-1, MRPm6, and MRPr1
monoclonal antibodies. Alternatively, partially digested fragments were immunoprecipitated separately with MRPr1 or
MRPm6. Following an overnight immunoprecipitation, protein A-coupled Sepharose beads with MRP halves (85 and
111 kDa polypeptides) were washed and incubated with 40
μg of V8 protease in 50 mM Na<sub>2</sub>
HPO<sub>4</sub>
(pH 7.4). Digestion
was allowed to proceed for 16 h at 37 °C, and samples were
then resolved on Fairbanks gels (<italic toggle="yes"><xref rid="bi9922188b00041" ref-type="bibr"></xref>
</italic>
).
</p>
<p>For a complete trypsin digestion, gel slices were rehydrated
for 5 min in water prior to the elution of the IACI
photoaffinity-labeled MRP, using the GE200 SixPacGel
Eluter (Hoefer Scientific Instruments). The buffer of the
eluted protein was changed to 50 mM ammonium bicarbonate (pH 8.0) by repeated washing using the Spin-X UF
concentrators with a 100 000 kDa cutoff. The digestion
commenced at 37 °C with the addition of 2 and 1 μg of
trypsin for 14 and 4 h, respectively. The digested sample
was vacuum-dried, resuspended in 250 μL of 1% trifluoroacetic acid in water, and resolved by reverse phase HPLC
(Vydac 201HS54 C<sub>18</sub>
RP column). The chromatographic
procedure consisted of an 80 min gradient of 0 to 100%
acetonitrile with 1% trifluoroacetic acid at a flow rate of 1
mL/min. Fractions were collected and checked for radioactivity.
</p>
<p><italic toggle="yes">Drug Accumulation.</italic>
Drug sensitive and resistant cells (1
× 10<sup>6</sup>
cells) were washed three times with PBS (pH 7.4)
and incubated at 37 °C with 10 mM <sc>d</sc>
-glucose or 10 mM
2-deoxyglucose and 100 nM sodium azide. One micromole
of IACI was added, and drug accumulation in cells was
assessed in 0 and 60 min incubations as previously described
(<italic toggle="yes"><xref rid="bi9922188b00044" ref-type="bibr"></xref>
</italic>
).
</p>
</sec>
<sec id="d7e522"><title>Results</title>
<p><italic toggle="yes">Photoaffinity Labeling of the MRP with IACI.</italic>
The MRP
has been shown to mediate the transport of conjugated cell
metabolites and natural product toxins (<italic toggle="yes">22</italic>
, <italic toggle="yes">23</italic>
,<italic toggle="yes"> 45</italic>
). Several
studies have now demonstrated a direct binding between one
such glutathione-containing compound, cysteinyl leukotriene
(LTC<sub>4</sub>
), and the MRP (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00011" ref-type="bibr"></xref>
, <xref rid="bi9922188b00046" ref-type="bibr"></xref>
</named-content>
</italic>
). However, MRP interaction
with unmodified compounds remains unclear. In this study,
we examined the photoaffinity labeling of plasma membrane
proteins from a MRP-expressing cell line (H69/AR) by a
photoactive quinoline-derived drug (IACI; Figure <xref rid="bi9922188f00001"></xref>
). To
determine if IACI binds directly to the MRP, plasma
membranes from drug sensitive (H69) and resistant (H69/AR) SCLC cells were incubated in the presence of 0.20 μM
IACI and UV irradiated (see Materials and Methods). The
results in Figure <xref rid="bi9922188f00002"></xref>
A show a 190 kDa protein photolabeled
by IACI in H69/AR but not in H69 membranes. When
similar membrane samples were photoaffinity labeled by
[<sup>125</sup>
I]iodoarylazidoprazosin or [<sup>3</sup>
H]azidopine, shown previously to photolabel P-glycoprotein (<italic toggle="yes"><xref rid="bi9922188b00047" ref-type="bibr"></xref>
</italic>
), no 190 kDa protein
was photoaffinity labeled (results not shown and ref <italic toggle="yes">28</italic>
).
These results suggest that neither iodoarylazidoprazosin nor
azidopine photoaffinity labels the 190 kDa protein in H69/AR membranes. The identity of the 190 kDa protein as the
MRP was confirmed by immunoprecipitation of IACI-photolabeled plasma membranes from H69/AR cells with
the MRP-specific monoclonal antibody, QCRL-1. Figure <xref rid="bi9922188f00002"></xref>
A
shows that the IACI-photolabeled 190 kDa protein can
specifically immunoprecipitate with QCRL-1 from H69/AR
but not from H69 membranes. Moreover, no IACI photoaffinity-labeled 190 kDa protein was immunoprecipitated
with an irrelevant antibody (Figure <xref rid="bi9922188f00002"></xref>
A).
<fig id="bi9922188f00001" position="float" orientation="portrait"><label>1</label>
<caption><p>Organic structure of<italic toggle="yes">N</italic>
-(hydrocinchonidin-8‘-yl)-4-azido-2-hydroxybenzamide (IACI).</p>
</caption>
<graphic xlink:href="bi9922188f00001.gif" position="float" orientation="portrait"></graphic>
</fig>
<fig id="bi9922188f00002" position="float" orientation="portrait"><label>2</label>
<caption><p>Photoaffinity labeling of MRP by IACI. Plasma membranes from drug sensitive (H69) and resistant (H69/AR) cells were photoaffinity labeled with 0.20 μM IACI and resolved on SDS−PAGE (A). Panel A also shows IACI photoaffinity-labeled membranes from H69 and H69/AR cells immunoprecipitated with MRP-specific Mab (QCRL-1) or an irrelevant IgG<sub>2a</sub>
. Panel B shows total membranes
from H69 and H69/AR cells after silver staining. Panel C shows the photoaffinity labeling of H69/AR membranes with increasing
concentrations of IACI (from 0 to 4.0 μM). The inset of panel C shows the increase in the intensity of 190 kDa photolabeled protein which
was excised and the radiolabel quantified. Panel D shows the photoaffinity-labeled proteins from H69 or H69/AR cells incubated in the
absence or presence of excess (0.1−100 μM) noniodinated IACI.</p>
</caption>
<graphic xlink:href="bi9922188f00002.gif" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>To determine if photolabeling of the 190 kDa protein (or
MRP) in membranes from H69/AR cells is due to nonspecific
binding to an abundant protein, total membrane proteins from
H69 and H69/AR cells were resolved by SDS−PAGE on a
6% Laemmli gel (<italic toggle="yes"><xref rid="bi9922188b00041" ref-type="bibr"></xref>
</italic>
) and visualized by silver staining.
Comparison of H69 and H69/AR proteins did not show
significant differences, except for the broad band at ∼190
kDa (Figure <xref rid="bi9922188f00002"></xref>
B). Interestingly, the region where MRP
photoaffinity labeling is observed, between 113 and 200 kDa
(Figure <xref rid="bi9922188f00002"></xref>
A), shows no photoaffinity labeling of some
abundantly expressed proteins. High levels of protein expression are seen for both H69 and H69/AR cells between the
113 and 50 kDa molecular mass markers (Figure <xref rid="bi9922188f00002"></xref>
B). This
may account for some of the photolabeling of certain
abundantly expressed proteins seen below the 113 kDa
molecular mass marker. To further confirm the binding
specificity of IACI toward the 190 kDa protein, H69/AR
membranes were photoaffinity labeled with increasing
concentrations (from 0.25 to 4.0 μM) of IACI. The inset of
Figure <xref rid="bi9922188f00002"></xref>
C shows that the photoaffinity labeling of the 190
kDa protein is saturable at 4.0 μM drug. Furthermore, the
specificity of IACI toward the 190 kDa protein was
confirmed by photolabeling in the presence of a molar excess
(40−400-fold) of the uniodinated IACI. Lanes d and e of
Figure <xref rid="bi9922188f00002"></xref>
D show marked decrease in the extent of photolabeling of the 190 kDa protein in the presence of a molar
excess of IACI. The photolabeling which is evident in some
lower-molecular mass proteins was not significantly affected
with excess uniodinated IACI (Figure <xref rid="bi9922188f00002"></xref>
D).
</p>
<p><italic toggle="yes">Inhibition of Photoaffinity Labeling of the MRP.</italic>
To
determine if IACI binds to physiologically relevant site(s)
in the MRP, membranes from H69/AR cells were
photolabeled with IACI in the presence of a molar excess of
colchicine, chloroquine, doxorubicin, MK571, and LTC<sub>4</sub>
. The
photoaffinity labeling of MRP was inhibited with a 160-fold molar excess of LTC<sub>4</sub>
(Figure <xref rid="bi9922188f00003"></xref>
A). Similarly, a molar
excess (100−150-fold) of MK571 also led to a dramatic
decrease in the extent of photolabeling of the MRP with
IACI. Colchicine, chloroquine, and doxorubicin at 1000-fold
molar excess were similar to MK571 at 150-fold and LTC<sub>4</sub>
at 160-fold, consistent with the higher affinity of MK571
and LTC<sub>4</sub>
for the MRP (Figure <xref rid="bi9922188f00004"></xref>
A). The latter results are in
accord with the previous findings that LTC<sub>4</sub>
is the substrate
with the highest affinity for MRP (<italic toggle="yes">11</italic>
,<italic toggle="yes"> 21</italic>
,<italic toggle="yes"> 22</italic>
). Together,
these results confirm the specificity of IACI toward MRP
and suggest that IACI binding to MRP occurs at the same
or overlapping site(s) as MK571 and LTC<sub>4</sub>
.
<fig id="bi9922188f00003" position="float" orientation="portrait"><label>3</label>
<caption><p>Effects of diverse drugs on the photoaffinity labeling of the MRP by IACI<italic toggle="yes">.</italic>
H69 or H69/AR cells were photoaffinity
labeled with IACI in the absence or presence of a molar excess
(300−1000-fold) of colchicine (COL), chloroquine (CQ), doxorubicin (DOXO), MK571 (100−150-fold), and LTC<sub>4</sub>
(80−160-fold).
Panel B shows a plot of the relative decrease in the extent of
photolabeling of the MRP with IACI in the presence of the drugs
listed above.</p>
</caption>
<graphic xlink:href="bi9922188f00003.gif" position="float" orientation="portrait"></graphic>
</fig>
<fig id="bi9922188f00004" position="float" orientation="portrait"><label>4</label>
<caption><p>Drug accumulation in H69 and H69/AR cells. Cells were preincubated in 10 mM glucose or 2-deoxyglucose and sodium azide prior to the addition of 1 μM IACI. Drug accumulation in cells was assessed 0 and 60 min after the addition of IACI. Cells were lysed, and the amounts of accumulated radiolabel were determined by fluorography. Each value is the mean ± the standard deviation of the two experiments carried out in triplicate.</p>
</caption>
<graphic xlink:href="bi9922188f00004.gif" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p><italic toggle="yes">IACI Accumulation in H69 and H69/AR Cells.</italic>
Given the
results described above, it was of interest to determine if
IACI is a substrate for MRP transport function. Figure <xref rid="bi9922188f00004"></xref>
shows the accumulation of IACI in H69 and H69/AR cells
in the presence of 10 mM glucose. H69/AR cells exhibit
lower steady-state levels of drug accumulation of IACI than
H69 cells (Figure <xref rid="bi9922188f00004"></xref>
). Moreover, preincubation of cells with
10 mM 2-deoxyglucose and 100 nM sodium azide, which
depletes ATP levels, increased the level of accumulation of
IACI in H69/AR cells to the same level as that of the H69
parental cells (Figure <xref rid="bi9922188f00004"></xref>
). Taken together, these results show
that the accumulation of IACI in MRP-expressing cells is
ATP-dependent.
</p>
<p><italic toggle="yes">Three Peptides of the MRP Are Photoaffinity Labeled by
IACI.</italic>
Several reports on MRP secondary structure and
topology have suggested the presence of five N-terminal
transmembrane helices (MSD1), followed by an MDR-like
core of six duplicated transmembrane domains and an ATP
binding site (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00016" ref-type="bibr"></xref>
−<xref rid="bi9922188b00017" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
). Limited proteolysis of MRP has
revealed two trypsin sensitive sites (L1 and L2; see Figure
<xref rid="bi9922188f00005"></xref>
B) that connect MSD1 to MSD2 and MSD3, and MSD1
and MSD2 to MSD3 sequences (<italic toggle="yes"><xref rid="bi9922188b00048" ref-type="bibr"></xref>
</italic>
). The L2 sequence was
shown to be more sensitive to trypsin cleavage than the L1
sequence (<italic toggle="yes"><xref rid="bi9922188b00048" ref-type="bibr"></xref>
</italic>
). Therefore, limited proteolysis of MRP with
trypsin showed two polypeptides with molecular masses of
120 and 75−80 kDa containing MSD1, MSD2, and NBD1,
and MSD3 and NBD2, respectively (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00016" ref-type="bibr"></xref>
−<xref rid="bi9922188b00017" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
). However,
further digestion with trypsin led to the cleavage of the 120
kDa polypeptide into two smaller peptides (40−60 and 57
kDa) that correspond to MSD1, and MSD2 and NBD1,
respectively (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00016" ref-type="bibr"></xref>
−<xref rid="bi9922188b00017" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
). Taking advantage of the trypsin
sensitive sites in the MRP, it was of interest to map IACI
photoaffinity-labeled peptides in the MRP. Figure <xref rid="bi9922188f00005"></xref>
A shows
the results of subjecting the IACI-photolabeled MRP to mild
proteolysis with trypsin and the digested products resolved
by SDS−PAGE. Lanes 3 and 4 of Figure <xref rid="bi9922188f00005"></xref>
A show two major
photoaffinity-labeled polypeptides that migrate with apparent
molecular masses of 111 and 85 kDa. Figure <xref rid="bi9922188f00005"></xref>
C shows
Western blots of the same samples as in Figure <xref rid="bi9922188f00005"></xref>
A probed
with MRPr1, QCRL-1, and MRPm6 Mabs. The Mabs
QCRL-1 and MRPm6 recognized the 85 kDa polypeptide,
while MRPr1 recognized the 111 kDa fragment (Figure <xref rid="bi9922188f00005"></xref>
C).
These results are consistent with earlier proteolysis of MRP
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00016" ref-type="bibr"></xref>
−<xref rid="bi9922188b00017" specific-use="suppress-in-print" ref-type="bibr"></xref>
<xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
) whereby the 85 kDa polypeptide contains the
QCRL-1 and MRPm6 epitopes while the 111 kDa polypeptide contains the MRPr1 epitope (<italic toggle="yes"><xref rid="bi9922188b00048" ref-type="bibr"></xref>
</italic>
). Therefore, the two
photoaffinity-labeled polypeptides (111 and 85 kDa) correspond to the MRP sequence containing MSD1, MSD2, and
NBD1, and MSD3 and NBD2, respectively. Probing the
same nitrocellulose membrane with goat anti-mouse peroxidase second antibody alone did not show any reactive
proteins (data not shown). Taken together, these results
demonstrate the photoaffinity labeling of two different
domains in the MRP.
<fig id="bi9922188f00005" position="float" orientation="portrait"><label>5</label>
<caption><p>Photoaffinity labeling of two large polypeptides of the MRP by IACI. The MRP photoaffinity labeled by IACI was purified from H69/AR membranes and subjected to mild tryptic digestion. The photolabeled, radiolabeled products were split in two halves; one-half was resolved by SDS−PAGE (A), while the second half was resolved by SDS−PAGE and transferred to nitrocellulose for Western blotting (C). Lanes 2−4 of panel A show the signal from the purified IACI-photolabeled MRP incubated in the absence and in the presence of 8 and 16 ng of trypsin, respectively. Panel C shows the Western blot of the IACI-photolabeled MRP tryptic digest probed separately with MRPr1, QCRL-1, and MRPm6 Mabs. Panel B shows a schematic of the predicted MRP topology with the three monoclonal antibody epitopes listed above in the MRP relative to the protease hypersensitive sites (L1 and L2). The nucleotide binding domains (NBD1 and NBD2) and the extracellular glycosylation sites (**) are indicated on the schematic of MRP secondary structure (B).</p>
</caption>
<graphic xlink:href="bi9922188f00005.gif" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>To determine the number of IACI-photolabeled sites in
the MRP, the photoaffinity-labeled MRP was immunopurified with QCRL-1 and subjected to in-gel digestion with
increasing concentrations of <italic toggle="yes">S. aureus</italic>
V8 protease (1−20
μg/gel slice). Figure <xref rid="bi9922188f00006"></xref>
A shows the resultant proteolytic
fragments migrating with apparent molecular masses of 6
and 4 kDa. Similarly, the IACI photoaffinity-labeled MRP
was subjected to exhaustive trypsin digestion (see Materials
and Methods), and the resulting digest was resolved by
HPLC using reverse phase chromatography. Figure <xref rid="bi9922188f00006"></xref>
B shows
the eluted IACI-photolabeled, radiolabeled tryptic peptides
resolved on a C<sub>18</sub>
reverse phase column with a 0 to 100%
acetonitrile gradient. The results in Figure <xref rid="bi9922188f00006"></xref>
B show one
minor peak eluting at 60% acetonitrile followed by two major
peaks eluting at 65−72% acetonitrile.
<fig id="bi9922188f00006" position="float" orientation="portrait"><label>6</label>
<caption><p>Complete proteolytic digestion of the IACI photoaffinity-labeled MRP yields two labeled peptides. The purified MRP photoaffinity labeled with IACI was subjected to complete in-gel or solution digestion. The in-gel-digested MRP products were resolved on 15% acrylamide SDS−PAGE, while the solution-digested products were resolved by reverse phase chromatography. Lanes 1−3 of panel A show an in-gel digestion of the IACI-photolabeled MRP with increasing concentrations of<italic toggle="yes">S. aureus </italic>
V8
protease (1−20 μg/well). Panel B shows the separation of MRP
photoaffinity-labeled tryptic peptides on a C<sub>18</sub>
reverse phase column
using a 0 to 100% acetonitrile gradient. The amount of radiolabel
in each eluted fraction was plotted vs time of elution in the gradient.
The peak signal at the beginning of the gradient represents the void
volume.</p>
</caption>
<graphic xlink:href="bi9922188f00006.gif" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>To identify the origin of the 4 and 6 kDa IACI-photolabeled peptides relative to the two large photolabeled
domains of the MRP, IACI-photolabeled 111 and 85 kDa
polypeptides were purified and digested with <italic toggle="yes">S. aureus</italic>
V8
protease. Lanes 2 and 3 of Figure <xref rid="bi9922188f00007"></xref>
A show IACI-photolabeled 111 and 85 kDa polypeptides following immunoprecipitation with MRPr1 and MRPm6 Mabs, respectively.
Exhaustive digestion of the 111 kDa polypeptide which
corresponds to MSD1, MSD2, and NBD1 of the MRP
resulted in two photolabeled peptides which migrate with
apparent molecular masses of 4 and 6 kDa (Figure <xref rid="bi9922188f00007"></xref>
B).
However, digestion of the 85 kDa polypeptide, which
corresponds to MSD3 and NBD2 of the MRP, resulted in
only one photolabeled peptide of 6 kDa (Figure <xref rid="bi9922188f00007"></xref>
B). Taken
together, the results in Figure <xref rid="bi9922188f00007"></xref>
suggest the presence of three
IACI-labeled sites in the MRP.
<fig id="bi9922188f00007" position="float" orientation="portrait"><label>7</label>
<caption><p>Proteolytic cleavage of the immunopurified IACI-photolabeled N- and C-halves of MRP. MRP-enriched membranes were photolabeled with IACI and subjected to mild tryptic cleavage. Lanes 1−3 of panel A show immunoprecipitation of native and trypsin-digested MRP with MRPm6 and MRPr1 Mabs, respectively. Panel B shows complete digestion of immunopurified IACI-photolabeled 111 and 85 kDa polypeptides with<italic toggle="yes">S. aureus </italic>
V8
protease. The digested fragments were resolved on the Fairbanks
gel system. The apparent molecular masses of standard proteins
are indicated to the left of the gels.</p>
</caption>
<graphic xlink:href="bi9922188f00007.gif" position="float" orientation="portrait"></graphic>
</fig>
</p>
</sec>
<sec id="d7e737"><title>Discussion</title>
<p>It is now believed that the overexpression of the MRP in
tumor cell lines can confer resistance to certain natural
product toxins (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00023" ref-type="bibr"></xref>
, <xref rid="bi9922188b00049" ref-type="bibr"></xref>
</named-content>
</italic>
). Moreover, MRP-mediated transport
of normal cell metabolites has been demonstrated in intact
cells and in MRP-enriched membrane vesicles (<italic toggle="yes">11</italic>
,<italic toggle="yes"> 21−23</italic>
).
However, the mechanism of MRP drug binding and transport
remains unclear: (i) MRP's broad substrate specificity, (ii)
MRP's ability to bind and transport unmodified drugs, (iii)
the role of free GSH in MRP drug binding and transport,
and (iv) MRP drug binding site(s). In this report, we have
used a photoreactive quinoline-based drug (IACI) to examine
MRP−drug interactions. Our results show the photoaffinity
labeling of a 190 kDa protein by IACI only in drug resistant
cells (H69/AR). The identity of the IACI-photolabeled
protein, as the MRP, was confirmed by its binding to three
MRP-specific monoclonal antibodies (QCRL-1, MRPr1, and
MRPm6; <italic toggle="yes">48</italic>
). The photolabeling of MRP in enriched
membranes from H69/AR cells suggests direct binding
between the MRP and unmodified IACI. Moreover, the
addition of free GSH (up to 5 mM) did not cause a significant
change in MRP photoaffinity labeling (results not shown).
With respect to the role of GSH in MRP drug transport, ATP-dependent transport of unmodified vincristine into membrane
vesicles was shown only in the presence of GSH (<italic toggle="yes"><xref rid="bi9922188b00030" ref-type="bibr"></xref>
</italic>
). Thus,
although the authors of that study (<italic toggle="yes"><xref rid="bi9922188b00030" ref-type="bibr"></xref>
</italic>
) did not examine
vincristine binding to the MRP, it is likely that drug transport
but not drug binding required the presence of free GSH.
Alternatively, GSH may be required for the binding and
transport of certain classes of drugs. Future experiments will
examine the effect of GSH and other MRP substrates on
IACI transport in membrane vesicles from MRP-expressing
cells. Taken together, these findings demonstrate direct
binding between the MRP and the unmodified drug that is
unaffected by GSH.
</p>
<p>The questions of whether IACI is a relevant substrate for
the MRP and whether the photoaffinity labeling of the MRP
by IACI occurs at a physiologically relevant site(s) were
addressed by drug transport and drug competition experiments. Our transport results show IACI is a substrate for
MRP drug efflux as it accumulates less in MRP-expressing
cells (H69/AR) than in the parental cells (H69). Furthermore,
depletion of ATP levels by preincubating cells with sodium
azide and 2-deoxyglucose restored IACI accumulation in
H69/AR cells to the same level as in H69 cells. The
possibility that differences in IACI accumulation between
H69/AR and H69 cells are due to changes or ATP-dependent
mechanisms other than the MRP cannot be ruled out
completely on the basis of our drug transport data alone.
However, along with the photoaffinity labeling results,
described in this study, compelling evidence for MRP-mediated transport of IACI exists. The inhibition of MRP
photolabeling by IACI with a molar excess of LTC<sub>4</sub>
indicates
that IACI binds to the MRP at a physiologically relevant
site. Although little is known about MRP drug binding
domain(s), we speculate that IACI binds to the same domain
(or an overlapping one) as that of MK571 or LTC<sub>4</sub>
.
Alternatively, IACI may bind at another site that is allosterically linked to the LTC<sub>4</sub>
binding domain. In a recent
study by Stride et al. (<italic toggle="yes"><xref rid="bi9922188b00050" ref-type="bibr"></xref>
</italic>
), it was shown that substitution of
the carboxyl third of the human MRP with that of mouse
mrp sequences modulated MRP specificity to the anthracycline doxorubicin while LTC<sub>4</sub>
transport was unaffected, thus
confirming further the notion of more than one drug binding
site in the MRP. In addition, support for multiple drug
binding sites in the MRP comes from studies with another
broad spectrum transporter (P-gp1) which is thought to
encode three drug binding sites (<italic toggle="yes"><xref rid="bi9922188b00051" ref-type="bibr"></xref>
</italic>
).
</p>
<p>Studies of MRP topology are in agreement with the two
“6+6” transmembrane helices (or MSD2 and MSD3) and
two NBDs characteristic of many ABC transporters, in
addition to the hydrophobic N-terminal domain predicted to
encode five transmembrane helices (MSD1) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00017" ref-type="bibr"></xref>
, <xref rid="bi9922188b00018" ref-type="bibr"></xref>
</named-content>
</italic>
). A
previous limited proteolysis study (<italic toggle="yes"><xref rid="bi9922188b00048" ref-type="bibr"></xref>
</italic>
) showed that the MRP
contains two hypersensitive regions (L1 and L2) connecting
MSD1 to MS2 and MSD3, and MSD1 and MSD2 to MSD3,
respectively. In this study, we took advantage of the protease
hypersensitive regions in MRP linker domains together with
the positions of three monoclonal antibodies with known
epitope sequences in the MRP to identify the photoaffinity
labeling domains in the MRP. Mild trypsin digestion of IACI-photolabeled MRP produced two large photolabeled polypeptides with apparent molecular masses of ∼111 and ∼85 kDa
on SDS−PAGE. On the basis of the locations of MRPr1,
QCRL-1, and MRPm6 epitopes (at <sup>238</sup>
GSDLWSLNKE<sup>247</sup>
,
<sup>918</sup>
SSYSGD<sup>924</sup>
, and <sup>1511</sup>
PSDLLQQRGL<sup>1520</sup>
, respectively; <italic toggle="yes">48</italic>
)
relative to one of the trypsin hypersensitive sites in the MRP
linker domain (L2) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00017" ref-type="bibr"></xref>
, <xref rid="bi9922188b00048" ref-type="bibr"></xref>
</named-content>
</italic>
), the 111 kDa polypeptide
corresponds to the N-terminal MSD1 and MSD2 and NBD1.
Similarly, the 85 kDa polypeptide should encode MSD3 and
NBD2 as it reacted only with QCRL-1 and MRPm6 Mabs
(see Figure <xref rid="bi9922188f00005"></xref>
). Exhaustive digestion of the IACI-photolabeled
MRP with V8 protease revealed two photolabeled peptides
with apparent molecular masses of ∼6 and ∼4 kDa.
However, digestion of purified 111 and 85 kDa polypeptides
with V8 protease revealed three IACI-photolabeled peptides,
two (6 and 4 kDa) from the 111 kDa polypeptide and one
(6 kDa) from the 85 kDa polypeptide. Although the possibility that the 6 kDa peptide, from the 111 kDa fragment,
results from incomplete digestion cannot be entirely ruled
out, it is unlikely given the exhaustive nature of our digestion
conditions. Furthermore, exhaustive digestion of the IACI-
labeled MRP with trypsin showed two major and one minor
peak (Figure <xref rid="bi9922188f00006"></xref>
B), consistent with a total of three IACI-photolabeled peptides. The photoaffinity labeling of MRP
at multiple sites is consistent with that observed with another
membrane transporter protein, P-gp1 (<italic toggle="yes"><xref rid="bi9922188b00032" ref-type="bibr"></xref>
</italic>
). The exact amino
acid sequence of P-gp1 drug binding sites is presently not
known; however, several reports support the role of transmembrane helices 5 and 6 and 11 and 12 in drug binding
and transport (<italic toggle="yes"><xref rid="bi9922188b00033" ref-type="bibr"></xref>
</italic>
). The photoaffinity labeling of three
peptides in the MRP with IACI suggests MRP drug binding
may involve three domains. Given the hydrophobic nature
of MRP substrates and our knowledge of P-gp1 drug binding
domains, it is tempting to speculate that the three IACI-photolabeled peptides correspond to the three membrane-spanning domains (MSD1−3) in the MRP.
</p>
<p>Although it is likely that the drug binding domains in the
MRP and the photoaffinity-labeled sites are different, earlier
studies with P-gp1 have shown these sites to be the same or
to overlap (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00032" ref-type="bibr"></xref>
, <xref rid="bi9922188b00033" ref-type="bibr"></xref>
</named-content>
</italic>
). Photoaffinity labeling studies of P-gp1
are in agreement with other molecular approaches. Indeed,
results from the photoaffinity labeling studies were used as
a starting point for mutational analyses of P-gp1 sequences.
Furthermore, results from a recent study aimed at localizing
MRP specificity domains toward anthracyclines suggested
a role for the carboxyl third of the MRP (<italic toggle="yes"><xref rid="bi9922188b00050" ref-type="bibr"></xref>
</italic>
). The latter
findings are consistent with our results in this report which
show the photoaffinity labeling of the carboxyl third of MRP
(MSD3 and NBD2) (<italic toggle="yes"><xref rid="bi9922188b00050" ref-type="bibr"></xref>
</italic>
).
</p>
<p>A molar excess of colchicine and doxorubicin also caused
a significant decrease in the extent of MRP photoaffinity
labeling by IACI. These findings are interesting since earlier
analysis of the cross resistance of MRP-expressing cells
showed only low levels of cross resistance to colchicine (<italic toggle="yes"><xref rid="bi9922188b00052" ref-type="bibr"></xref>
</italic>
).
Inhibition of photoaffinity labeling of IACI with MK571 and
CQ is less surprising as both drugs share the quinoline
moiety. The LTD<sub>4</sub>
receptor antagonist, MK571 (<italic toggle="yes"><xref rid="bi9922188b00036" ref-type="bibr"></xref>
</italic>
), was
recently shown to inhibit LTC<sub>4</sub>
and <italic toggle="yes">S</italic>
-(<italic toggle="yes">p</italic>
-azidophenylacyl)glutathione labeling of the MRP (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00011" ref-type="bibr"></xref>
, <xref rid="bi9922188b00046" ref-type="bibr"></xref>
</named-content>
</italic>
) and to reverse
MRP-mediated MDR (<italic toggle="yes"><xref rid="bi9922188b00035" ref-type="bibr"></xref>
</italic>
). Indeed, several quinoline-based
drugs have been reported to interact directly and specifically
with the MRP (<italic toggle="yes">27</italic>
,<italic toggle="yes"> 28</italic>
,<italic toggle="yes"> 53</italic>
), hence establishing this group as
another class of compounds that interact with MRP. Collectively, these findings are important as many therapeutically
important drugs are quinoline-based drugs (<italic toggle="yes"><xref rid="bi9922188b00054" ref-type="bibr"></xref>
</italic>
). For example,
quinoline-based drugs are extensively used in the treatment
of parasitic infections such as malaria (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00055" ref-type="bibr"></xref>
, <xref rid="bi9922188b00056" ref-type="bibr"></xref>
</named-content>
</italic>
), and resistance
to these drugs has rendered this first-choice drug treatment
ineffective (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi9922188b00055" ref-type="bibr"></xref>
, <xref rid="bi9922188b00056" ref-type="bibr"></xref>
</named-content>
</italic>
). Given these findings, we speculate about
an MRP-like mechanism responsible for resistance in this
parasite.
</p>
<p>In conclusion, there is increasing evidence that the MRP
mediates the transport of structurally diverse drugs through
direct binding. Although several studies have demonstrated
direct binding between the MRP and LTC<sub>4</sub>
using a photoaffinity labeling assay, photoaffinity labeling of the MRP
by [<sup>3</sup>
H]LTC<sub>4</sub>
was inefficient, requiring large amounts of
membrane (200 μg/sample) and extremely long exposure
times (more than 4 weeks in our hands). By contrast,
photoaffinity labeling of the MRP by IACI was done using
20 μg of membranes/sample, and exposure for 2−12 h
showed a strong signal for MRP. The availability of a
photoactive radioiodinated drug that binds specifically to the
MRP should facilitate future analysis of MRP−drug interactions. The photoaffinity labeling of the MRP at multiple sites
which map to two domains in the MRP is consistent with
protein−drug interactions seen with other members of the
ABC family of drug transporters, such as P-gp1. Finally,
we show that IACI binding to the MRP is inhibited by known
substrates of MRP, LTC<sub>4</sub>
, and other natural product drugs.
We speculate that IACI binds to the same domain (or an
overlapping domain) as that of LTC<sub>4</sub>
or MK571. Work is in
progress to determine if IACI photoaffinity labels the same
or different sequences as MK571 and LTC<sub>4</sub>
.
</p>
</sec>
</body>
<back><ack><title>Acknowledgments</title>
<p>We thank Joel Karwatsky for his careful reading of the
manuscript.
</p>
</ack>
<ref-list><title>References</title>
<ref id="bi9922188b00001"><element-citation publication-type="journal"><name name-style="western"><surname>Gottesman</surname>
<given-names>M. M.</given-names>
</name>
<name name-style="western"><surname>Pastan</surname>
<given-names>I.</given-names>
</name>
<source>Annu. Rev. Biochem.</source>
<year>1993</year>
<volume>62</volume>
<fpage>385</fpage>
<lpage>427</lpage>
<pub-id pub-id-type="doi">10.1146/annurev.bi.62.070193.002125</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00002"><element-citation publication-type="journal"><name name-style="western"><surname>Endicott</surname>
<given-names>J. A.</given-names>
</name>
<name name-style="western"><surname>Ling</surname>
<given-names>V.</given-names>
</name>
<source>Annu. Rev. Biochem.</source>
<year>1989</year>
<volume>58</volume>
<fpage>137</fpage>
<lpage>171</lpage>
<pub-id pub-id-type="doi">10.1146/annurev.bi.58.070189.001033</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00003"><element-citation publication-type="journal"><name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<source>Cancer Treat. Res.</source>
<year>1996</year>
<volume>87</volume>
<fpage>39</fpage>
<lpage>62</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00004"><element-citation publication-type="journal"><name name-style="western"><surname>Ueda</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Cardarelli</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Gottesman</surname>
<given-names>M. M.</given-names>
</name>
<name name-style="western"><surname>Pastan</surname>
<given-names>I.</given-names>
</name>
<source>Proc. Natl. Acad. Sci. U.S.A.</source>
<year>1987</year>
<volume>84</volume>
<fpage>3004</fpage>
<lpage>3008</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.84.9.3004</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00005"><element-citation publication-type="journal"><name name-style="western"><surname>Grant</surname>
<given-names>C. E.</given-names>
</name>
<name name-style="western"><surname>Valdimarsson</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Hipfner</surname>
<given-names>D. R.</given-names>
</name>
<name name-style="western"><surname>Almquist</surname>
<given-names>K. C.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<source>Cancer Res.</source>
<year>1994</year>
<volume>54</volume>
<fpage>357</fpage>
<lpage>361</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00006"><mixed-citation><name name-style="western"><surname>Schinkel</surname>
<given-names>A. H.</given-names>
</name>
, <name name-style="western"><surname>Smit</surname>
<given-names>J. J. M.</given-names>
</name>
, <name name-style="western"><surname>van Tellingen</surname>
<given-names>O.</given-names>
</name>
, <name name-style="western"><surname>Beijnen</surname>
<given-names>J. H.</given-names>
</name>
, <name name-style="western"><surname>Wagenaar</surname>
<given-names>E.</given-names>
</name>
, <name name-style="western"><surname>van Deemter</surname>
<given-names>L.</given-names>
</name>
, <name name-style="western"><surname>Mol</surname>
<given-names>C. A. A. M.</given-names>
</name>
, <name name-style="western"><surname>var der Valk</surname>
<given-names>M. A.</given-names>
</name>
, <name name-style="western"><surname>Robanus-Maandag</surname>
<given-names>E. C.</given-names>
</name>
, <name name-style="western"><surname>te Riele</surname>
<given-names>H. P. J.</given-names>
</name>
, <name name-style="western"><surname>Berns</surname>
<given-names>A. J. M.</given-names>
</name>
, <name name-style="western"><surname>and Borst</surname>
<given-names>P.</given-names>
</name>
(1994) <italic toggle="yes">Cell</italic>
<italic toggle="yes">77</italic>
, 491−502.<pub-id pub-id-type="doi">10.1016/0092-8674(94)90212-7</pub-id>
</mixed-citation>
</ref>
<ref id="bi9922188b00007"><element-citation publication-type="journal"><name name-style="western"><surname>Lorico</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Rappa</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Flavell</surname>
<given-names>R. A.</given-names>
</name>
<name name-style="western"><surname>Sartorelli</surname>
<given-names>A. C.</given-names>
</name>
<source>Cancer Res.</source>
<year>1996</year>
<volume>56</volume>
<fpage>5351</fpage>
<lpage>5355</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00008"><mixed-citation><name name-style="western"><surname>Wijnholds</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>Evers</surname>
<given-names>R.</given-names>
</name>
, <name name-style="western"><surname>van</surname>
<given-names>L. M.</given-names>
</name>
, <name name-style="western"><surname>Mol</surname>
<given-names>C.</given-names>
</name>
, <name name-style="western"><surname>Zaman</surname>
<given-names>G.</given-names>
</name>
, <name name-style="western"><surname>Mayer</surname>
<given-names>U.</given-names>
</name>
, <name name-style="western"><surname>Beijnen</surname>
<given-names>J.</given-names>
</name>
, <name name-style="western"><surname>van</surname>
<given-names>d. V. M.</given-names>
</name>
, <name name-style="western"><surname>Krimpenfort</surname>
<given-names>P.</given-names>
</name>
, <name name-style="western"><surname>and Borst</surname>
<given-names>P.</given-names>
</name>
(1997) <italic toggle="yes">Nat. Med.</italic>
<italic toggle="yes">3</italic>
, 1275−1279.<pub-id pub-id-type="doi">10.1038/nm1197-1275</pub-id>
</mixed-citation>
</ref>
<ref id="bi9922188b00009"><element-citation publication-type="journal"><name name-style="western"><surname>Ruetz</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Gros</surname>
<given-names>P.</given-names>
</name>
<source>Cell</source>
<year>1994</year>
<volume>77</volume>
<fpage>1071</fpage>
<lpage>1081</lpage>
<pub-id pub-id-type="doi">10.1016/0092-8674(94)90446-4</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00010"><element-citation publication-type="journal"><name name-style="western"><surname>Raggers</surname>
<given-names>R. J.</given-names>
</name>
<name name-style="western"><surname>van Helvoort</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Evers</surname>
<given-names>R.</given-names>
</name>
<name name-style="western"><surname>van Meer</surname>
<given-names>G.</given-names>
</name>
<source>J. Cell Sci.</source>
<year>1999</year>
<volume>112</volume>
<fpage>415</fpage>
<lpage>422</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00011"><element-citation publication-type="journal"><name name-style="western"><surname>Leier</surname>
<given-names>I.</given-names>
</name>
<name name-style="western"><surname>Jedlitschky</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Buchholz</surname>
<given-names>U.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<name name-style="western"><surname>Keppler</surname>
<given-names>D.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1994</year>
<volume>269</volume>
<fpage>27807</fpage>
<lpage>27810</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00012"><element-citation publication-type="journal"><name name-style="western"><surname>Lorico</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Rappa</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Finch</surname>
<given-names>R. A.</given-names>
</name>
<name name-style="western"><surname>Yang</surname>
<given-names>D.</given-names>
</name>
<name name-style="western"><surname>Flavell</surname>
<given-names>R. A.</given-names>
</name>
<name name-style="western"><surname>Sartorelli</surname>
<given-names>A. C.</given-names>
</name>
<source>Cancer Res.</source>
<year>1997</year>
<volume>57</volume>
<fpage>5238</fpage>
<lpage>5242</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00013"><element-citation publication-type="journal"><name name-style="western"><surname>Rappa</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Lorico</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Flavell</surname>
<given-names>R. A.</given-names>
</name>
<name name-style="western"><surname>Sartorelli</surname>
<given-names>A. C.</given-names>
</name>
<source>Cancer Res.</source>
<year>1997</year>
<volume>57</volume>
<fpage>5232</fpage>
<lpage>5237</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00014"><element-citation publication-type="journal"><name name-style="western"><surname>Higgins</surname>
<given-names>C. F.</given-names>
</name>
<source>Cell</source>
<year>1995</year>
<volume>82</volume>
<fpage>693</fpage>
<lpage>696</lpage>
<pub-id pub-id-type="doi">10.1016/0092-8674(95)90465-4</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00015"><element-citation publication-type="journal"><name name-style="western"><surname>Stride</surname>
<given-names>B.</given-names>
</name>
<name name-style="western"><surname>Grant</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Loe</surname>
<given-names>D.</given-names>
</name>
<name name-style="western"><surname>Hipfner</surname>
<given-names>D.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R.</given-names>
</name>
<source>Mol. Pharmacol.</source>
<year>1997</year>
<volume>52</volume>
<fpage>344</fpage>
<lpage>353</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00016"><element-citation publication-type="journal"><name name-style="western"><surname>Bakos</surname>
<given-names>E.</given-names>
</name>
<name name-style="western"><surname>Hegedus</surname>
<given-names>T.</given-names>
</name>
<name name-style="western"><surname>Hollo</surname>
<given-names>Z.</given-names>
</name>
<name name-style="western"><surname>Welker</surname>
<given-names>E.</given-names>
</name>
<name name-style="western"><surname>Tusnady</surname>
<given-names>G. E.</given-names>
</name>
<name name-style="western"><surname>Zaman</surname>
<given-names>G. J.</given-names>
</name>
<name name-style="western"><surname>Flens</surname>
<given-names>M. J.</given-names>
</name>
<name name-style="western"><surname>Varadi</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Sarkadi</surname>
<given-names>B.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1996</year>
<volume>271</volume>
<fpage>12322</fpage>
<lpage>12326</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.271.4.1877</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00017"><element-citation publication-type="journal"><name name-style="western"><surname>Hipfner</surname>
<given-names>D. R.</given-names>
</name>
<name name-style="western"><surname>Almquist</surname>
<given-names>K. C.</given-names>
</name>
<name name-style="western"><surname>Leslie</surname>
<given-names>E. M.</given-names>
</name>
<name name-style="western"><surname>Gerlach</surname>
<given-names>J. H.</given-names>
</name>
<name name-style="western"><surname>Grant</surname>
<given-names>C. E.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P. C.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1997</year>
<volume>272</volume>
<fpage>23623</fpage>
<lpage>23630</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.272.38.23623</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00018"><element-citation publication-type="journal"><name name-style="western"><surname>Kast</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Gros</surname>
<given-names>P.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1997</year>
<volume>272</volume>
<fpage>26479</fpage>
<lpage>26487</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.272.42.26479</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00019"><element-citation publication-type="journal"><name name-style="western"><surname>Gao</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Yamazaki</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Loe</surname>
<given-names>D.</given-names>
</name>
<name name-style="western"><surname>Westlake</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Grant</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1998</year>
<volume>273</volume>
<fpage>10733</fpage>
<lpage>10740</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.273.17.10733</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00020"><mixed-citation><name name-style="western"><surname>Bakos</surname>
<given-names>E.</given-names>
</name>
, <name name-style="western"><surname>Evers</surname>
<given-names>R.</given-names>
</name>
, <name name-style="western"><surname>Szakacs</surname>
<given-names>G.</given-names>
</name>
, <name name-style="western"><surname>Tusnady</surname>
<given-names>G.</given-names>
</name>
, <name name-style="western"><surname>Welker</surname>
<given-names>E.</given-names>
</name>
, <name name-style="western"><surname>Szabo</surname>
<given-names>K.</given-names>
</name>
, <name name-style="western"><surname>de</surname>
<given-names>H. M.</given-names>
</name>
, <name name-style="western"><surname>van Deemter</surname>
<given-names>L.</given-names>
</name>
, <name name-style="western"><surname>Borst</surname>
<given-names>P.</given-names>
</name>
, <name name-style="western"><surname>Varadi</surname>
<given-names>A.</given-names>
</name>
, <name name-style="western"><surname>and Sarkadi</surname>
<given-names>B.</given-names>
</name>
(1998) <italic toggle="yes">J. Biol. Chem</italic>
. <italic toggle="yes"> 273</italic>
, 32167−32175.<pub-id pub-id-type="doi">10.1074/jbc.273.48.32167</pub-id>
</mixed-citation>
</ref>
<ref id="bi9922188b00021"><element-citation publication-type="journal"><name name-style="western"><surname>Jedlitschky</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Leier</surname>
<given-names>I.</given-names>
</name>
<name name-style="western"><surname>Buchholz</surname>
<given-names>U.</given-names>
</name>
<name name-style="western"><surname>Barnouin</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Kurz</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Keppler</surname>
<given-names>D.</given-names>
</name>
<source>Cancer Res.</source>
<year>1996</year>
<volume>56</volume>
<fpage>988</fpage>
<lpage>994</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00022"><element-citation publication-type="journal"><name name-style="western"><surname>Loe</surname>
<given-names>D. W.</given-names>
</name>
<name name-style="western"><surname>Almquist</surname>
<given-names>K. C.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1996</year>
<volume>271</volume>
<fpage>9683</fpage>
<lpage>9689</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.271.44.27782</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00023"><element-citation publication-type="journal"><name name-style="western"><surname>Zaman</surname>
<given-names>G. J. R.</given-names>
</name>
<name name-style="western"><surname>Flens</surname>
<given-names>M. J.</given-names>
</name>
<name name-style="western"><surname>Vanleusden</surname>
<given-names>M. R.</given-names>
</name>
<name name-style="western"><surname>Dehaas</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Mulder</surname>
<given-names>H. S.</given-names>
</name>
<name name-style="western"><surname>Lankelma</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Pinedo</surname>
<given-names>H. M.</given-names>
</name>
<name name-style="western"><surname>Scheper</surname>
<given-names>R. J.</given-names>
</name>
<name name-style="western"><surname>Baas</surname>
<given-names>F.</given-names>
</name>
<name name-style="western"><surname>Broxterman</surname>
<given-names>H. J.</given-names>
</name>
<name name-style="western"><surname>Borst</surname>
<given-names>P.</given-names>
</name>
<source>Proc. Natl. Acad. Sci. U.S.A.</source>
<year>1994</year>
<volume>91</volume>
<fpage>8822</fpage>
<lpage>8826</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.91.19.8822</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00024"><element-citation publication-type="journal"><name name-style="western"><surname>Ishikawa</surname>
<given-names>T.</given-names>
</name>
<source>Trends Biochem. Sci.</source>
<year>1992</year>
<volume>17</volume>
<fpage>463</fpage>
<lpage>468</lpage>
<pub-id pub-id-type="doi">10.1016/0968-0004(92)90489-V</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00025"><mixed-citation><name name-style="western"><surname>Jansen</surname>
<given-names>P. L. M.</given-names>
</name>
, <name name-style="western"><surname>and Oude Elferink</surname>
<given-names>R. P. J.</given-names>
</name>
(1993) in<italic toggle="yes"> Defective Hepatic Anion Secretion in Mutant TR-Rats</italic>
, Raven, New York.</mixed-citation>
</ref>
<ref id="bi9922188b00026"><element-citation publication-type="journal"><name name-style="western"><surname>Zaman</surname>
<given-names>G. J. R.</given-names>
</name>
<name name-style="western"><surname>Lankelma</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Tellingen</surname>
<given-names>O. V.</given-names>
</name>
<name name-style="western"><surname>Beijnen</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Dekker</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>Paulusma</surname>
<given-names>C.</given-names>
</name>
<name name-style="western"><surname>Elferink</surname>
<given-names>R. P. J. O.</given-names>
</name>
<name name-style="western"><surname>Baas</surname>
<given-names>F.</given-names>
</name>
<name name-style="western"><surname>Borst</surname>
<given-names>P.</given-names>
</name>
<source>Proc. Natl. Acad. Sci. U.S.A.</source>
<year>1995</year>
<volume>92</volume>
<fpage>7690</fpage>
<lpage>7694</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.92.17.7690</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00027"><element-citation publication-type="journal"><name name-style="western"><surname>Vezmar</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Georges</surname>
<given-names>E.</given-names>
</name>
<source>Biochem. Pharmacol.</source>
<year>1988</year>
<volume>56</volume>
<fpage>733</fpage>
<lpage>742</lpage>
<pub-id pub-id-type="doi">10.1016/S0006-2952(98)00217-2</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00028"><element-citation publication-type="journal"><name name-style="western"><surname>Vezmar</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Deady</surname>
<given-names>L. W.</given-names>
</name>
<name name-style="western"><surname>Tilley</surname>
<given-names>L.</given-names>
</name>
<name name-style="western"><surname>Georges</surname>
<given-names>E.</given-names>
</name>
<source>Biochem. Biophys. Res. Commun.</source>
<year>1997</year>
<volume>241</volume>
<fpage>104</fpage>
<lpage>111</lpage>
<pub-id pub-id-type="doi">10.1006/bbrc.1997.7634</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00029"><element-citation publication-type="journal"><name name-style="western"><surname>Nakamura</surname>
<given-names>T.</given-names>
</name>
<name name-style="western"><surname>Oka</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Aizawa</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Soda</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>Fukuda</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Terashi</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Ikeda</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Mizuta</surname>
<given-names>Y.</given-names>
</name>
<name name-style="western"><surname>Noguchi</surname>
<given-names>Y.</given-names>
</name>
<name name-style="western"><surname>Kimura</surname>
<given-names>Y.</given-names>
</name>
<name name-style="western"><surname>Tsuruo</surname>
<given-names>T.</given-names>
</name>
<name name-style="western"><surname>Kohno</surname>
<given-names>S.</given-names>
</name>
<source>Biochem. Biophys. Res. Commun.</source>
<year>1999</year>
<volume>255</volume>
<fpage>618</fpage>
<lpage>624</lpage>
<pub-id pub-id-type="doi">10.1006/bbrc.1999.0245</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00030"><element-citation publication-type="journal"><name name-style="western"><surname>Loe</surname>
<given-names>D. W.</given-names>
</name>
<name name-style="western"><surname>Almquist</surname>
<given-names>K. C.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1996</year>
<volume>271</volume>
<fpage>9675</fpage>
<lpage>9682</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.271.44.27782</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00031"><element-citation publication-type="journal"><name name-style="western"><surname>Chowdry</surname>
<given-names>V.</given-names>
</name>
<name name-style="western"><surname>Westheimer</surname>
<given-names>F. H.</given-names>
</name>
<source>Annu. Rev. Biochem.</source>
<year>1979</year>
<volume>48</volume>
<fpage>293</fpage>
<lpage>325</lpage>
<pub-id pub-id-type="doi">10.1146/annurev.bi.48.070179.001453</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00032"><element-citation publication-type="journal"><name name-style="western"><surname>Greenberger</surname>
<given-names>L. M.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1993</year>
<volume>268</volume>
<fpage>11417</fpage>
<lpage>11425</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00033"><element-citation publication-type="journal"><name name-style="western"><surname>Loo</surname>
<given-names>T.</given-names>
</name>
<name name-style="western"><surname>Clarke</surname>
<given-names>D.</given-names>
</name>
<source>Methods Enzymol.</source>
<year>1998</year>
<volume>292</volume>
<fpage>480</fpage>
<lpage>492</lpage>
<pub-id pub-id-type="doi">10.1016/S0076-6879(98)92037-7</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00034"><element-citation publication-type="journal"><name name-style="western"><surname>Cole</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R.</given-names>
</name>
<source>BioEssays</source>
<year>1998</year>
<volume>20</volume>
<fpage>931</fpage>
<lpage>940</lpage>
<pub-id pub-id-type="doi">10.1002/(SICI)1521-1878(199811)20:11%3C931::AID-BIES8%3E3.0.CO;2-J</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00035"><element-citation publication-type="journal"><name name-style="western"><surname>Gekeler</surname>
<given-names>V.</given-names>
</name>
<name name-style="western"><surname>Ise</surname>
<given-names>W.</given-names>
</name>
<name name-style="western"><surname>Sanders</surname>
<given-names>K. H.</given-names>
</name>
<name name-style="western"><surname>Ulrich</surname>
<given-names>W. R.</given-names>
</name>
<name name-style="western"><surname>Beck</surname>
<given-names>J.</given-names>
</name>
<source>Biochem. Biophys. Res. Commun.</source>
<year>1995</year>
<volume>208</volume>
<fpage>345</fpage>
<lpage>352</lpage>
<pub-id pub-id-type="doi">10.1006/bbrc.1995.1344</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00036"><element-citation publication-type="journal"><name name-style="western"><surname>Jones</surname>
<given-names>T. R.</given-names>
</name>
<name name-style="western"><surname>Zamboni</surname>
<given-names>R.</given-names>
</name>
<name name-style="western"><surname>Belley</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Champion</surname>
<given-names>E.</given-names>
</name>
<name name-style="western"><surname>Charette</surname>
<given-names>L.</given-names>
</name>
<name name-style="western"><surname>Ford-Hutchison</surname>
<given-names>A. W.</given-names>
</name>
<name name-style="western"><surname>Frenette</surname>
<given-names>R.</given-names>
</name>
<name name-style="western"><surname>Gauthier</surname>
<given-names>J.-Y.</given-names>
</name>
<name name-style="western"><surname>Leger</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Masson</surname>
<given-names>P.</given-names>
</name>
<name name-style="western"><surname>McFarlane</surname>
<given-names>C. S.</given-names>
</name>
<name name-style="western"><surname>Piechuta</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>Rokach</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Williams</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>Young</surname>
<given-names>R. N.</given-names>
</name>
<name name-style="western"><surname>DeHaven</surname>
<given-names>R. N.</given-names>
</name>
<name name-style="western"><surname>Pong</surname>
<given-names>S. S.</given-names>
</name>
<source>Can. J. Physiol. Pharmacol.</source>
<year>1989</year>
<volume>67</volume>
<fpage>17</fpage>
<lpage>28</lpage>
<pub-id pub-id-type="doi">10.1139/y89-004</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00037"><element-citation publication-type="journal"><name name-style="western"><surname>Lin</surname>
<given-names>P. H.</given-names>
</name>
<name name-style="western"><surname>Selinfreund</surname>
<given-names>R.</given-names>
</name>
<name name-style="western"><surname>Wakshull</surname>
<given-names>E.</given-names>
</name>
<name name-style="western"><surname>Wharton</surname>
<given-names>W.</given-names>
</name>
<source>Biochemistry</source>
<year>1987</year>
<volume>26</volume>
<fpage>731</fpage>
<lpage>736</lpage>
<pub-id pub-id-type="doi">10.1021/bi00377a012</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00038"><element-citation publication-type="journal"><name name-style="western"><surname>Lowry</surname>
<given-names>O. H.</given-names>
</name>
<name name-style="western"><surname>Rosebrough</surname>
<given-names>N. J.</given-names>
</name>
<name name-style="western"><surname>Farr</surname>
<given-names>A. L.</given-names>
</name>
<name name-style="western"><surname>Randall</surname>
<given-names>R. J.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1951</year>
<volume>193</volume>
<fpage>265</fpage>
<lpage>275</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00039"><element-citation publication-type="journal"><name name-style="western"><surname>Desneves</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Thorn</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Berman</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Galatis</surname>
<given-names>D.</given-names>
</name>
<name name-style="western"><surname>La Greca</surname>
<given-names>N.</given-names>
</name>
<name name-style="western"><surname>Sinding</surname>
<given-names>J.</given-names>
</name>
<name name-style="western"><surname>Foley</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Deady</surname>
<given-names>L. W.</given-names>
</name>
<name name-style="western"><surname>Cowman</surname>
<given-names>A. F.</given-names>
</name>
<name name-style="western"><surname>Tilley</surname>
<given-names>L.</given-names>
</name>
<source>Mol. Biochem. Parasitol.</source>
<year>1996</year>
<volume>82</volume>
<fpage>181</fpage>
<lpage>194</lpage>
<pub-id pub-id-type="doi">10.1016/0166-6851(96)02732-6</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00040"><element-citation publication-type="journal"><name name-style="western"><surname>Fairbanks</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Steck</surname>
<given-names>T. L.</given-names>
</name>
<name name-style="western"><surname>Wallach</surname>
<given-names>D. F. H.</given-names>
</name>
<source>Biochemistry</source>
<year>1971</year>
<volume>10</volume>
<fpage>2606</fpage>
<lpage>2617</lpage>
<pub-id pub-id-type="doi">10.1021/bi00789a030</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00041"><element-citation publication-type="journal"><name name-style="western"><surname>Laemmli</surname>
<given-names>U. K.</given-names>
</name>
<source>Nature</source>
<year>1970</year>
<volume>227</volume>
<fpage>680</fpage>
<lpage>685</lpage>
<pub-id pub-id-type="doi">10.1038/227680a0</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00042"><element-citation publication-type="journal"><name name-style="western"><surname>Cleveland</surname>
<given-names>D. W.</given-names>
</name>
<name name-style="western"><surname>Fischer</surname>
<given-names>S. G.</given-names>
</name>
<name name-style="western"><surname>Kirschner</surname>
<given-names>M. W.</given-names>
</name>
<name name-style="western"><surname>Laemmli</surname>
<given-names>U. K.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1977</year>
<volume>252</volume>
<fpage>1102</fpage>
<lpage>1106</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00043"><element-citation publication-type="journal"><name name-style="western"><surname>Georges</surname>
<given-names>E.</given-names>
</name>
<name name-style="western"><surname>Zhang</surname>
<given-names>J.-T.</given-names>
</name>
<name name-style="western"><surname>Ling</surname>
<given-names>V.</given-names>
</name>
<source>J. Cell. Physiol.</source>
<year>1991</year>
<volume>148</volume>
<fpage>479</fpage>
<lpage>484</lpage>
<pub-id pub-id-type="doi">10.1002/jcp.1041480321</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00044"><element-citation publication-type="journal"><name name-style="western"><surname>Liu</surname>
<given-names>Z.</given-names>
</name>
<name name-style="western"><surname>Lheureux</surname>
<given-names>F.</given-names>
</name>
<name name-style="western"><surname>Pouliot</surname>
<given-names>J.-F.</given-names>
</name>
<name name-style="western"><surname>Heckel</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Bamberger</surname>
<given-names>U.</given-names>
</name>
<name name-style="western"><surname>Georges</surname>
<given-names>E.</given-names>
</name>
<source>Mol. Pharmacol.</source>
<year>1996</year>
<volume>50</volume>
<fpage>482</fpage>
<lpage>492</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00045"><element-citation publication-type="journal"><name name-style="western"><surname>Paul</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Breuninger</surname>
<given-names>L. M.</given-names>
</name>
<name name-style="western"><surname>Tew</surname>
<given-names>K. D.</given-names>
</name>
<name name-style="western"><surname>Shen</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>Kruh</surname>
<given-names>G. D.</given-names>
</name>
<source>Proc. Natl. Acad. Sci. U.S.A.</source>
<year>1996</year>
<volume>93</volume>
<fpage>6929</fpage>
<lpage>6934</lpage>
<pub-id pub-id-type="doi">10.1073/pnas.93.14.6929</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00046"><element-citation publication-type="journal"><name name-style="western"><surname>Leier</surname>
<given-names>I.</given-names>
</name>
<name name-style="western"><surname>Jedlitschky</surname>
<given-names>G.</given-names>
</name>
<name name-style="western"><surname>Buchholz</surname>
<given-names>U.</given-names>
</name>
<name name-style="western"><surname>Keppler</surname>
<given-names>D.</given-names>
</name>
<source>Eur. J. Biochem.</source>
<year>1994</year>
<volume>220</volume>
<fpage>599</fpage>
<lpage>606</lpage>
<pub-id pub-id-type="doi">10.1111/j.1432-1033.1994.tb18661.x</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00047"><element-citation publication-type="journal"><name name-style="western"><surname>Safa</surname>
<given-names>A.</given-names>
</name>
<source>Methods Enzymol.</source>
<year>1998</year>
<volume>292</volume>
<fpage>289</fpage>
<lpage>307</lpage>
<pub-id pub-id-type="doi">10.1016/S0076-6879(98)92023-7</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00048"><element-citation publication-type="journal"><name name-style="western"><surname>Hipfner</surname>
<given-names>D.</given-names>
</name>
<name name-style="western"><surname>Almquist</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Stride</surname>
<given-names>B.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S.</given-names>
</name>
<source>Cancer Res.</source>
<year>1996</year>
<volume>56</volume>
<fpage>3307</fpage>
<lpage>3314</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00049"><element-citation publication-type="journal"><name name-style="western"><surname>Almquist</surname>
<given-names>K. C.</given-names>
</name>
<name name-style="western"><surname>Loe</surname>
<given-names>D. W.</given-names>
</name>
<name name-style="western"><surname>Hipfner</surname>
<given-names>D. R.</given-names>
</name>
<name name-style="western"><surname>Mackie</surname>
<given-names>J. E.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<source>Cancer Res.</source>
<year>1995</year>
<volume>55</volume>
<fpage>102</fpage>
<lpage>110</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00050"><element-citation publication-type="journal"><name name-style="western"><surname>Stride</surname>
<given-names>B. D.</given-names>
</name>
<name name-style="western"><surname>Cole</surname>
<given-names>S. P. C.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<source>J. Biol. Chem.</source>
<year>1999</year>
<volume>274</volume>
<fpage>22877</fpage>
<lpage>22883</lpage>
<pub-id pub-id-type="doi">10.1074/jbc.274.32.22877</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00051"><element-citation publication-type="journal"><name name-style="western"><surname>Shapiro</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Fox</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Lam</surname>
<given-names>P.</given-names>
</name>
<name name-style="western"><surname>Ling</surname>
<given-names>V.</given-names>
</name>
<source>Eur. J. Biochem.</source>
<year>1999</year>
<volume>259</volume>
<fpage>841</fpage>
<lpage>850</lpage>
<pub-id pub-id-type="doi">10.1046/j.1432-1327.1999.00098.x</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00052"><element-citation publication-type="journal"><name name-style="western"><surname>Cole</surname>
<given-names>S. P.</given-names>
</name>
<name name-style="western"><surname>Sparks</surname>
<given-names>K. E.</given-names>
</name>
<name name-style="western"><surname>Fraser</surname>
<given-names>K.</given-names>
</name>
<name name-style="western"><surname>Loe</surname>
<given-names>D. W.</given-names>
</name>
<name name-style="western"><surname>Grant</surname>
<given-names>C. E.</given-names>
</name>
<name name-style="western"><surname>Wilson</surname>
<given-names>G. M.</given-names>
</name>
<name name-style="western"><surname>Deeley</surname>
<given-names>R. G.</given-names>
</name>
<source>Cancer Res.</source>
<year>1994</year>
<volume>54</volume>
<fpage>5902</fpage>
<lpage>5910</lpage>
</element-citation>
</ref>
<ref id="bi9922188b00053"><element-citation publication-type="journal"><name name-style="western"><surname>Priebe</surname>
<given-names>W.</given-names>
</name>
<name name-style="western"><surname>Krawczyk</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Kuo</surname>
<given-names>M. T.</given-names>
</name>
<name name-style="western"><surname>Yamane</surname>
<given-names>Y.</given-names>
</name>
<name name-style="western"><surname>Savaraj</surname>
<given-names>N.</given-names>
</name>
<name name-style="western"><surname>Ishikawa</surname>
<given-names>T.</given-names>
</name>
<source>Proc. Am. Assoc. Cancer Res.</source>
<year>1997</year>
<volume>38</volume>
<fpage>440</fpage>
</element-citation>
</ref>
<ref id="bi9922188b00054"><element-citation publication-type="journal"><name name-style="western"><surname>Baba</surname>
<given-names>A.</given-names>
</name>
<name name-style="western"><surname>Kawamura</surname>
<given-names>N.</given-names>
</name>
<name name-style="western"><surname>Makino</surname>
<given-names>H.</given-names>
</name>
<name name-style="western"><surname>Ohta</surname>
<given-names>Y.</given-names>
</name>
<name name-style="western"><surname>Taketomi</surname>
<given-names>S.</given-names>
</name>
<name name-style="western"><surname>Sohda</surname>
<given-names>T.</given-names>
</name>
<source>J. Med. Chem.</source>
<year>1996</year>
<volume>39</volume>
<fpage>5176</fpage>
<lpage>5182</lpage>
<pub-id pub-id-type="doi">10.1021/jm9509408</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00055"><element-citation publication-type="journal"><name name-style="western"><surname>Foley</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Tilley</surname>
<given-names>L.</given-names>
</name>
<source>Int. J. Parasitol.</source>
<year>1997</year>
<volume>27</volume>
<fpage>231</fpage>
<lpage>240</lpage>
<pub-id pub-id-type="doi">10.1016/S0020-7519(96)00152-X</pub-id>
</element-citation>
</ref>
<ref id="bi9922188b00056"><element-citation publication-type="journal"><name name-style="western"><surname>Foley</surname>
<given-names>M.</given-names>
</name>
<name name-style="western"><surname>Tilley</surname>
<given-names>L.</given-names>
</name>
<source>Pharmacol. Ther.</source>
<year>1998</year>
<volume>79</volume>
<fpage>55</fpage>
<lpage>87</lpage>
<pub-id pub-id-type="doi">10.1016/S0163-7258(98)00012-6</pub-id>
</element-citation>
</ref>
<ref id="bi9922188n00001"><mixed-citation><comment>Abbreviations: MDR, multidrug resistance; P-gp, P-glycoprotein; MRP, multidrug resistance protein; SCLC, small cell lung cancer; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; ABC, ATP binding cassette.</comment>
</mixed-citation>
</ref>
</ref-list>
</back>
</article>
</istex:document>
</istex:metadataXml>
<mods version="3.6"><titleInfo><title>The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites†</title>
</titleInfo>
<name type="personal"><namePart type="family">DAOUD</namePart>
<namePart type="given">Roni</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<affiliation> Institute of Parasitology, McGill University.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="family">DESNEVES</namePart>
<namePart type="given">Jose</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="family">DEADY</namePart>
<namePart type="given">Leslie W.</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<affiliation> La Trobe University.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="family">TILLEY</namePart>
<namePart type="given">Leann</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<affiliation> La Trobe University.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="family">SCHEPER</namePart>
<namePart type="given">Rik J.</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<affiliation> Free University Hospital.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="family">GROS</namePart>
<namePart type="given">Philippe</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<affiliation> Department of Biochemistry, McGill University.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal" displayLabel="corresp"><namePart type="family">GEORGES</namePart>
<namePart type="given">Elias</namePart>
<affiliation>Institute of Parasitology, Department of Biochemistry, McGill University, Macdonald Campus, Ste-Anne de Bellevue,Quebec, Canada, The School of Chemistry and The School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia,and Department of Pathology, Free University Hospital, Amsterdam, The Netherlands</affiliation>
<affiliation> Institute of Parasitology, McGill University.</affiliation>
<affiliation> To whom correspondence should be addressed: Institute ofParasitology, McGill University, 21, 111 Lakeshore Road, Ste-Annede Bellevue, PQ H9X 3V9. Telephone: (514) 398-8137. Fax: (514)398-7857. E-mail: Elias_Georges@maclan.McGill.CA.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<typeOfResource>text</typeOfResource>
<genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre>
<originInfo><publisher>American Chemical Society</publisher>
<dateCreated encoding="w3cdtf">2000-04-26</dateCreated>
<dateIssued encoding="w3cdtf">2000-05-23</dateIssued>
<copyrightDate encoding="w3cdtf">2000</copyrightDate>
</originInfo>
<note type="footnote" ID="bi9922188AF2"> This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada to E.G. Research at the Institute of Parasitology is partially supported by a grant from the FCAR pour l'aide à la recherche.</note>
<abstract>Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has 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 binding and 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 of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as 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, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation 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 (∼111 and ∼85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids ∼900−1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1−900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and ∼6 plus 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 the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP−drug interactions.</abstract>
<note type="footnote" ID="bi9922188AF2"> This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada to E.G. Research at the Institute of Parasitology is partially supported by a grant from the FCAR pour l'aide à la recherche.</note>
<relatedItem type="host"><titleInfo><title>Biochemistry</title>
</titleInfo>
<titleInfo type="abbreviated"><title>Biochemistry</title>
</titleInfo>
<genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre>
<identifier type="ISSN">0006-2960</identifier>
<identifier type="eISSN">1520-4995</identifier>
<identifier type="acspubs">bi</identifier>
<identifier type="coden">BICHAW</identifier>
<identifier type="uri">pubs.acs.org/biochemistry</identifier>
<part><date>2000</date>
<detail type="volume"><caption>vol.</caption>
<number>39</number>
</detail>
<detail type="issue"><caption>no.</caption>
<number>20</number>
</detail>
<extent unit="pages"><start>6094</start>
<end>6102</end>
</extent>
</part>
</relatedItem>
<identifier type="istex">36ABA24296CC0F5175E3849F9CFC34622FCCF8CA</identifier>
<identifier type="ark">ark:/67375/TPS-J0RG5SH4-V</identifier>
<identifier type="DOI">10.1021/bi9922188</identifier>
<accessCondition type="use and reproduction" contentType="restricted">Copyright © 2000 American Chemical Society</accessCondition>
<recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">ACS</recordContentSource>
<recordOrigin>Copyright © 2000 American Chemical Society</recordOrigin>
</recordInfo>
</mods>
<json:item><extension>json</extension>
<original>false</original>
<mimetype>application/json</mimetype>
<uri>https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/record.json</uri>
</json:item>
</metadata>
<annexes><json:item><extension>gif</extension>
<original>true</original>
<mimetype>image/gif</mimetype>
<uri>https://api.istex.fr/ark:/67375/TPS-J0RG5SH4-V/annexes.gif</uri>
</json:item>
</annexes>
<serie></serie>
</istex>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/ChloroquineV1/Data/Istex/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001A05 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 001A05 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= ChloroquineV1 |flux= Istex |étape= Corpus |type= RBID |clé= ISTEX:36ABA24296CC0F5175E3849F9CFC34622FCCF8CA |texte= The Multidrug Resistance Protein Is Photoaffinity Labeled by a Quinoline-Based Drug at Multiple Sites† }}
This area was generated with Dilib version V0.6.33. |