Mutation of a Single Conserved Tryptophan in Multidrug Resistance Protein 1 (MRP1/ABCC1) Results in Loss of Drug Resistance and Selective Loss of Organic Anion Transport

Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17β-estradiol 17-(β-d-glucuronide) (E217βG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C4- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C4transport by the W1246C-MRP1 protein was no longer inhibitable by E217βG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp1246 was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp1246 mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E217βG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to theVinca alkaloid vincristine and accumulated levels of [3H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter. Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17β-estradiol 17-(β-d-glucuronide) (E217βG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C4- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C4transport by the W1246C-MRP1 protein was no longer inhibitable by E217βG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp1246 was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp1246 mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E217βG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to theVinca alkaloid vincristine and accumulated levels of [3H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter. multidrug resistance protein ATP-binding cassette membrane-spanning domain leukotriene C4 17β-estradiol 17-(β-d-glucuronide) transmembrane kilobase green fluorescent protein monoclonal antibody phosphate-buffered saline The 190-kDa multidrug resistance protein 1 (MRP1)1 is a member of a branch of the ATP-binding cassette (ABC) superfamily of transport proteins designated ABCC (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar, 2Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar, 3Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Crossref PubMed Scopus (583) Google Scholar). When overexpressed in tumor cells, MRP1 (gene symbol ABCC1) confers resistance to anticancer drugs and other xenobiotics of remarkably diverse structures and charge. Thus, the resistance spectrum associated with MRP1 expression extends from cationic and neutral natural product drugs (e.g. vincristine, doxorubicin, and VP-16) to the antimetabolite methotrexate to arsenical and antimonial oxyanions (4Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar). MRP1 is also a primary active transporter of conjugated organic anions that include GSH-, glucuronide-, and sulfate-conjugated derivatives of both endo- and xenobiotics, suggesting a role for MRP1 in the disposition and elimination of these compounds (2Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar). The ability of MRP1 to confer drug resistance and transport conjugated organic anions is shared by at least two other proteins belonging to the ABCC subfamily, MRP2 (ABCC2) and MRP3 (ABCC3) (5Konig J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Crossref PubMed Scopus (693) Google Scholar, 6Kool M. van der Linden M. de Haas M. Scheffer G.L. de Vree J.M.L. Smith A.J. Jansen G. Peters G.J. Ponne N. Scheper R.J. Oude Elferink R.P.J. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6914-6919Crossref PubMed Scopus (591) Google Scholar). These three proteins share a common five-domain structure that distinguishes them from most other ABC transporters, which more typically have four structural domains. Thus, MRP1, -2, and -3 have a third membrane-spanning domain (MSD) with an extracytosolic N terminus that precedes a core structure consisting of two tandemly arranged units containing an MSD and a nucleotide-binding domain (see Fig.1 A) (7Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 8Cui Y. Konig J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar). The drug resistance profiles and the specificity and relative affinities of the three proteins for organic anions are similar, but not identical (5Konig J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Crossref PubMed Scopus (693) Google Scholar, 8Cui Y. Konig J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar, 9Hirohashi T. Suzuki H. Sugiyama Y. J. Biol. Chem. 1999; 274: 15181-15185Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 10Bakos E. Evers R. Sinkule J.A. Varadi A. Borst P. Sarkadi B. Mol. Pharmacol. PubMed Scopus Google Scholar). resistance is associated with expression of but not MRP1 or MRP3 Y. Konig J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar, K. M. K. T. T. M. K. K. S. M. Cancer Res. Google Scholar). MRP3 is a transporter of organic anions with MRP1 and MRP2 T. Suzuki H. Sugiyama Y. J. Biol. Chem. 1999; 274: 15181-15185Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). in substrate specificity are not that the of MRP2 and MRP3 are only and to that of MRP1. of the of MRP1 are the leukotriene and the 17β-estradiol 17-(β-d-glucuronide) (E217βG) (5Konig J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Crossref PubMed Scopus (693) Google Scholar, Deeley R.G. Cole S.P.C. J. Biol. Chem. Scholar). MRP1 also in to transport xenobiotics, but only in the of D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D.W. Stewart Deeley R.G. Cole S.P.C. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, J. de Jansen M. J. Pharmacol. 1999; PubMed Scopus Google Scholar, D.W. Deeley R.G. Cole S.P.C. Cancer Res. Google Scholar). transport of by MRP1 is by a of structurally diverse molecules that or not with this D.W. Deeley R.G. Cole S.P.C. Cancer Res. Google Scholar, D.W. Deeley R.G. Cole S.P.C. J. Pharmacol. Google Scholar). The by which transport of conjugated and is that MRP1 a that the structural and of P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Crossref PubMed Scopus (583) Google Scholar, D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.H. M. J. 1997; PubMed Scopus Google Scholar). The in the and transport of MRP1 are In the of human MRP1 and to confer resistance to drugs have to a residue at position in putative transmembrane segment in the third MSD of human MRP1 that is for ability to confer resistance to this of drugs D. Cole S.P.C. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and at and for a to in conferring resistance to these drugs R. E. R.G. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). In this study, we have a highly conserved tryptophan residue in the last segment in that is not only for the transport of the conjugated organic E217βG, but also for the ability of MRP1 to confer resistance to both cationic and electroneutral natural product and were from and were from was from and E217βG, and were from and heavy metal oxyanions in were (4Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar). The MRP1 expression vector was by the from with a of the eukaryotic expression vector C.E. G. D.R. Almquist K.C. Cole S.P.C. Deeley R.G. Cancer Res. 1994; 54: Google Scholar). of Trp1246 in MRP1 were the The for was prepared by a from of the MRP1 was to the with the are and by or a was and the in the were The of with was to the of MRP1 to a wild-type the of MRP1 was eliminated to a MRP1 and at the and with and are in and a product with at the and was prepared the from P. and and the two were and and with and the was of to a vector designated of the product were by proteins were by the in the with the comparable containing the or and designated and wild-type and mutant expression were transfected human embryonic kidney cells cells were in and was to the the cells were and membrane vesicles were prepared D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). vector and vector containing the wild-type were in of wild-type and mutant MRP1 proteins were by HeLa cells were in of a and was with the cells were and the was replaced with containing were and in of MRP1 protein in were by and the of cells expressing MRP1 was by by by The levels of wild-type and mutant MRP1 proteins were by of membrane protein from transfected cells D.R. Leslie E.M. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). were a and to a membrane. were with for 1 by with primary and mutant MRP1 proteins were with the human which a consisting of D.R. Almquist K.C. Deeley R.G. Cole S.P.C. Cancer Res. Google Scholar). were with antibody by of substrate levels of protein expression were by a cells were at in a with in of containing and transiently transfected with the cDNA of the were with and with for at with the cells were by in The were in A in and in for at The were and were with 1 of in for in the the were containing 1 of and cells were a with an at were and the of stably transfected cells expressing MRP1, of HeLa cells in for were by with the which a the first nucleotide-binding domain of MRP1 D.R. Leslie E.M. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with and with in for at with and in cells were with for 1 The cells were with and for with in two with cells were in in and a containing the of MRP1 with the of cells were by and to with of the cells expressing wild-type or mutant MRP1. membrane vesicles were prepared from transiently transfected cells or stably transfected HeLa cells, and transport of by the membrane vesicles was a D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transport were at in a containing or and of protein in transport was at by in and the was that had in transport was by were for the of that remained to the which was of the in the of was from transport in the of to transport were in and results are of was in a similar that membrane vesicles of were at in a of containing E217βG and the for was also by with membrane vesicles of at in a with D.W. Deeley R.G. Cole S.P.C. J. Pharmacol. Google Scholar). by transport, were in for at to in the of D.W. Deeley R.G. Cole S.P.C. Cancer Res. Google Scholar). The relative drug resistance of the HeLa stably transfected with wild-type and mutant MRP1 was a S.P.C. Cancer Pharmacol. PubMed Scopus Google Scholar). Cells were in at for the addition of and for a the addition of of at and were to the and the at was an were from the were in and resistance was the of the wild-type or mutant HeLa by the the HeLa transfected with vector in cells was S.P.C. Gerlach J.H. Cancer Res. Google Scholar). HeLa cells were at in the of 1 [3H]vincristine in with and of cells were at and and of [3H]vincristine was by Cells were with and the were in was by the of a of in MRP1, we an MRP1 mutant in which at position was replaced with When stably in HeLa cells, the ability of this mutant to transport organic anions was and no longer resistance to natural product In contrast, a mutant HeLa a phenotype similar to that of cells expressing wild-type MRP1. R. G. and S. P. three of the of the wild-type MRP1 and the first mutant were by The were of the and when was in addition to the the transfected of MRP1 cDNA in the first mutant with an phenotype a C in substitution of cysteine for tryptophan at position This with the highly conserved of Trp1246 ABC belonging to the ABCC to a more of the structural and of this a first to the of this was with cysteine in by and the was transiently transfected cells. with the wild-type and the vector were at the vesicles were and protein expression levels were by the in the levels of W1246C-MRP1 in the were those of wild-type MRP1 A of was and the levels of by the W1246C-MRP1 mutant relative to wild-type MRP1 were observed to to relative levels of expression of by the W1246C-MRP1 mutant that for this substrate was with wild-type MRP1 but the for the two proteins were similar R. G. and S. P. in These results that substitution of Trp1246 with the of MRP1 for this but that the transport of the mutant and wild-type proteins are In contrast, of E217βG by the W1246C-MRP1 mutant was and was comparable to that of the have that E217βG is a of transport, with D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the ability of E217βG to transport by the W1246C-MRP1 mutant was and the results are in D. E217βG by wild-type MRP1 by In contrast, E217βG had no transport by indicating that the loss of E217βG transport by the mutant protein is associated with a loss of of this the loss of E217βG transport was to a substitution at position were in of These substitution with a with and vesicles were prepared from cells transiently transfected with these and the transport of and E217βG was in the levels of expression of the mutant proteins were comparable or those of wild-type MRP1. The transport levels of the mutant and the and were similar to those of wild-type MRP1 and the W1246C-MRP1 In contrast, the the mutant not transport E217βG E217βG transport by the and was also of wild-type the Trp1246 substitution the transport of other MRP1 was in membrane vesicles prepared from cells transfected with wild-type and MRP1 have that MRP1 transport that is by the and of D.W. Deeley R.G. Cole S.P.C. J. Pharmacol. Google Scholar, D.W. Deeley R.G. Cole S.P.C. Biophys. Res. PubMed Scopus Google Scholar). transport by wild-type MRP1 and the Trp1246 mutant proteins was in the of in four Trp1246 mutant MRP1 proteins the ability to verapamil-stimulated at levels to the that the loss of transport in the Trp1246 MRP1 was not by of the mutant molecules to the plasma of wild-type MRP1 and and were and transiently transfected cells. When the both the wild-type and mutant MRP1 proteins were observed to to the plasma indicating that in the transiently transfected cells, the were correctly routed to the with and HeLa cells transfected with wild-type cDNA showed that of to the C terminus of wild-type MRP1 not the E217βG transport or the drug of the A. R. G. and S. P. In addition to ability to transport organic anions E217βG, and MRP1 confers resistance to natural product anticancer drugs by drug in cells in which is a role in conferring drug stably transfected were by of HeLa cells with and and by were and of protein expression levels was by and The transfected HeLa were for vincristine resistance a HeLa cells expressing wild-type MRP1 resistance to this drug In contrast, the of the W1246C-MRP1 mutant for vincristine was similar to that of the vector control-transfected When of [3H]vincristine was of the drug in the cells were comparable to those in vector control-transfected cells in wild-type cells, vincristine was to of vector control-transfected with results in (4Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar). The drug resistance phenotype of the cells was by the of these cells to the cationic anthracyclines and the electroneutral epipodophyllotoxin in the of the W1246C-MRP1 mutant cells for three drugs were similar to those of the vector control-transfected cells. Thus, in addition to the ability to bind and transport the conjugated E217βG, the substitution in MRP1 results in loss of resistance to of natural product drugs have that in addition to conferring resistance to anticancer both human and MRP1 confer resistance to arsenical and antimonial oxyanions (4Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar, Grant C.E. Loe D.W. D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar). When for to potassium antimony the of the mutant HeLa cells was comparable to that of cells expressing wild-type MRP1 Thus, both showed a similar of resistance to this heavy metal In contrast, mutant cells resistance to sodium arsenite Thus, the of mutant HeLa cells for sodium arsenite was 1 with for cells expressing wild-type MRP1 and for vector control-transfected cells. MRP1 was first and a protein capable of cationic and electroneutral chemotherapeutic agents and to a transporter of GSH-, glucuronide-, and sulfate-conjugated organic anions (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar, 2Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar, Grant C.E. Loe D.W. D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, G. Leier I. Buchholz U. M. Keppler D. Cancer Res. 1994; 54: Google Scholar, D. Leier I. G. Biol. Chem. 1997; Google Scholar, H. Sugiyama Y. 359-376Crossref PubMed Scopus Google Scholar). from and structurally diverse that MRP1 and structures that the and of and electroneutral Thus, ABC transporters, MRP1 and proteins have a remarkably broad capacity to transport of and the for the of this transport capacity is have that of the first of MRP1 the MSD and the to the MSD are important for expression in ability to transport at least of M. M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Evers R. G. E. K. de Haas M. van Borst P. Varadi A. Sarkadi B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of a of this of MRP1, we a mutant in which was replaced with by K. Leslie E.M. K.E. Deeley R.G. Cole S.P.C. Proc. Cancer Res. Scholar). When stably in HeLa cells, the ability of this MRP1 mutant to transport organic anions was diminished, and in E217βG transport was not this mutant protein to confer drug In contrast, a transfected also expressing an MRP1 protein with the a phenotype similar to that of cells expressing wild-type was that the transfected MRP1 in the first mutant had a the substitution of for at position we a mutant of MRP1 so that phenotype with that of the When transiently transfected cells, W1246C-MRP1 at levels comparable to those of wild-type MRP1 and also with a similar verapamil-stimulated transport was intact. observed for the transport of E217βG by W1246C-MRP1 membrane vesicles was not from that with vesicles from vector control-transfected cells, and transport in the no longer by these results to that the phenotype of the cells was to the tryptophan substitution at position 1246 in and not the substitution at position in the and in which Trp1246 was with Ala, Phe, and transport similar to those of indicating that of the tryptophan residue the cysteine was for the the drug resistance phenotype of the stably transfected HeLa cells were In addition to the loss of E217βG transport, we found that cells were no longer resistant to natural product chemotherapeutic the electroneutral and the cationic vincristine and with this loss of drug vincristine in cells expressing W1246C-MRP1 was comparable to that in vector control-transfected cells, which was vincristine in HeLa cells expressing wild-type MRP1. the other the cells were resistant to antimony tartrate and partially resistant to sodium these that the structural determinants in MRP1 for and transport of these heavy metal oxyanions differ from those for the natural product chemotherapeutic agents. of MRP1 are from for which are similar in differ in of in the (7Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, E. Varadi A. Sarkadi B. 1997; PubMed Scopus Google Scholar). with the of the which only four in of the Trp1246 in the last segment to the of the membrane 1 This tryptophan residue is conserved and and MRP1. is also found in the transmembrane the the resistance protein and the transporter is not conserved in human or other of the to which this multidrug resistance protein The last in of MRP1 a highly with Trp1246 and other with of the 1 and in the to in the of the plasma membrane 1 The last of other ABCC in which Trp1246 is conserved MRP2 and MRP3 a similar to the that the capacity of putative an important role in the and transport of these the comparable segment in also to determinants of substrate but of this ABC transporter is of MRP1, -2, and -3 P. S. I. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). This to the in the substrate specificity and transport of MRP1 and (2Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar, 3Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Crossref PubMed Scopus (583) Google Scholar). MRP1 to or of a with the natural product drugs and The that the were at comparable levels and correctly routed to the plasma membrane in the transiently transfected cells and that and verapamil-stimulated transport remained that the not the structural of the The of the Trp1246 MRP1 to transport E217βG suggests that tryptophan that are for and transport of MRP1 Thus, the of E217βG transport and drug resistance in the W1246C-MRP1 mutant by the that cysteine in is tryptophan and the of a or residue at position 1246 also in the loss of E217βG transport, indicating that and are not to the of the in MRP1 for this substrate and that the residue are the first example of a tryptophan residue being critically important for substrate specificity in a eukaryotic ABC transporter. of to the tryptophan residue in other of the ABCC is also important for transport Our that Trp1246 is for the and transport of E217βG by MRP1. the human and of MRP1 differ in ability to transport this conjugated this is conserved in the two suggesting that are important Grant C.E. Loe D.W. D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar). Thus, that conserved and determinants the and of MRP1 to a for this organic that substitution of to E217βG and transport the ability to confer drug Our that wild-type MRP1, but not the of the that Trp1246 is important for the transport of other M. K. P. S. S. R. G. and S. P. for is to the that for are from the of the plasma membrane I. Biol. Scholar). or not this is also for MRP1 substrate is not but the of the and of The that a to the of the protein is of for the and transport of E217βG suggests that this organic by MRP1 from the transport is not by the is is that is more important for of MRP1 with E217βG with In of this are that a in the of the of from the to the the for E217βG transport D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The is and more These of and by the in the of a by of the Trp1246 residue from the membrane we have that the ability of MRP1 to confer resistance to cationic anthracyclines is lost when is with a residue D. Cole S.P.C. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This with that and transport of these drugs not only the of a in putative but also the of the Trp1246 in Thus, these the that and are required for MRP1 to confer drug The comparable in and an not of these two drug resistance proteins with to the by which they and transport for other multidrug R. E. R.G. Sci. Full Text Full Text PDF PubMed Scopus Google MRP1 substrate molecules the core of the they a of van der and with the and Our that for a broad range of MRP1 and with Trp1246 other in are also are with in the of cationic drugs D. Cole S.P.C. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). or not have with in MRP1 is not the of MRP1 and proteins a of and that of the protein MRP2 in which in and have replaced by other transport S. T. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Suzuki H. Sugiyama Y. Proc. Cancer Res. Scholar). Thus, and are to to provide important the structural that MRP1 and both and electroneutral and for and and for and for for and with the of the

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