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Functional Importance of Polar and Charged Amino Acid Residues in Transmembrane Helix 14 of Multidrug Resistance Protein 1 (MRP1/ABCC1)

2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês

10.1074/jbc.m308403200

ISSN

1083-351X

Autores

Dawei Zhang, Hongmei Gu, Donna Situ, Anass Haimeur, Susan P.C. Cole, Roger G. Deeley,

Tópico(s)

HIV/AIDS drug development and treatment

Resumo

Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17β-estradiol 17-(β-d-glucuronide) (E217βG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217βG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs. Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17β-estradiol 17-(β-d-glucuronide) (E217βG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217βG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs. Human multidrug resistance protein 1 (MRP1), 1The abbreviations used are: MRP, multidrug resistance protein; P-gp, P-glycoprotein; CFTR, cystic fibrosis transmembrane conductance regulator; MSD, membrane-spanning domain; TM, transmembrane; NBD, nucleotide binding domain; mAb, monoclonal antibody; E217βG, 17β-estradiol 17-(β-d-glucuronide); LTC4, leukotriene C4; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; VP-16, etoposide; HEK, human embryonic kidney; ABC, ATP binding cassette; PBS, phosphate-buffered saline. a 1531-amino acid integral membrane phosphoglycoprotein, was originally cloned from the doxorubicin-selected multidrug-resistant human small cell lung cancer cell line, H69AR (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. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3022) Google Scholar). Subsequent to its cloning, expression of MRP1 protein and/or mRNA has been detected in a wide range of solid and hematological tumors (2Filipits M. Suchomel R.W. Dekan G. Haider K. Valdimarsson G. Depisch D. Pirker R. Clin. Cancer Res. 1996; 2: 1231-1237PubMed Google Scholar, 3Nooter K. Brutel de la Riviere G. Look M.P. van Wingerden K.E. Henzen-Logmans S.C. Scheper R.J. Flens M.J. Klijn J.G. Stoter G. Foekens J.A. Br. J. Cancer. 1997; 76: 486-493Crossref PubMed Scopus (136) Google Scholar, 4Young L.C. Campling B.G. Cole S.P.C. Deeley R.G. Gerlach J.H. Clin. Cancer Res. 2001; 7: 1798-1804PubMed Google Scholar, 5Young L.C. Campling B.G. Voskoglou-Nomikos T. Cole S.P.C. Deeley R.G. Gerlach J.H. Clin. Cancer Res. 1999; 5: 673-680PubMed Google Scholar, 6Efferth T. Thelen P. Schulten H.G. Bode M.E. Granzen B. Beniers A.J. Mertens R. Ringert R.H. Gefeller O. Jakse G. Fuzesi L. Int. J Oncol. 2001; 19: 367-371PubMed Google Scholar, 7Yokoyama Y. Sato S. Fukushi Y. Sakamoto T. Futagami M. Saito Y. J. Obstet. Gynaecol. Res. 1999; 25: 387-394Crossref PubMed Scopus (46) Google Scholar). In some cases, MRP1 expression has been shown to correlate with clinical outcome (3Nooter K. Brutel de la Riviere G. Look M.P. van Wingerden K.E. Henzen-Logmans S.C. Scheper R.J. Flens M.J. Klijn J.G. Stoter G. Foekens J.A. Br. J. Cancer. 1997; 76: 486-493Crossref PubMed Scopus (136) Google Scholar, 5Young L.C. Campling B.G. Voskoglou-Nomikos T. Cole S.P.C. Deeley R.G. Gerlach J.H. Clin. Cancer Res. 1999; 5: 673-680PubMed Google Scholar, 7Yokoyama Y. Sato S. Fukushi Y. Sakamoto T. Futagami M. Saito Y. J. Obstet. Gynaecol. Res. 1999; 25: 387-394Crossref PubMed Scopus (46) Google Scholar, 8van der Kolk D.M. de Vries E.G. van Putten W.J. Verdonck L.F. Ossenkoppele G.J. Verhoef G.E. Vellenga E. Clin. Cancer Res. 2000; 6: 3205-3214PubMed Google Scholar, 9Tada Y. Wada M. Migita T. Nagayama J. Hinoshita E. Mochida Y. Maehara Y. Tsuneyoshi M. Kuwano M. Naito S. Int. J. Cancer. 2002; 98: 630-635Crossref PubMed Scopus (88) Google Scholar). In vitro, MRP1 is able to confer resistance to many commonly used, structurally diverse natural product chemotherapeutic agents, including anthracyclines, epipodophyllotoxins, and Vinca alkaloids (10Cole 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, 11Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P.C. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar, 12Zaman G.J. Flens M.J. van Leusden M.R. de Haas M. Mulder H.S. Lankelma J. Pinedo H.M. Scheper R.J. Baas F. Broxterman H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8822-8826Crossref PubMed Scopus (703) Google Scholar, 13Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar, 14Breuninger L.M. Paul S. Gaughan K. Miki T. Chan A. Aaronson S.A. Kruh G.D. Cancer Res. 1995; 55: 5342-5347PubMed Google Scholar). In addition to conferring drug resistance, transport studies using inside-out membrane vesicles prepared from MRP1-transfected cells have shown that MRP1 is capable of transporting glutathione-, glucuronate- and sulfate-conjugated organic anions such as the cysteinyl leukotriene 4 (LTC4), 17β-estradiol 17-(β-d-glucuronide) (E217βG), and estrone-3-sulfate (15Leier I. Jedlitschky G. Buchholz U. Cole S.P.C. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar, 16Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 17Loe D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 18Muller M. Meijer C. Zaman G.J. Borst P. Scheper R.J. Mulder N.H. de Vries E.G. Jansen P.L. Proc. Natl. Acad. Sci. U. S. 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MRP1-mediated LTC4 and E217βG transport has further been demonstrated in reconstituted proteoliposomes with immunoaffinity-purified native MRP1, clearly proving that MRP1 alone is sufficient for transport of conjugated organic anions (24Mao Q. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2000; 275: 34166-34172Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). LTC4 is a high affinity endogenous glutathione S-conjugate substrate for MRP1 and plays important roles in the inflammatory response (15Leier I. Jedlitschky G. Buchholz U. Cole S.P.C. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar, 16Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 19Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar). In mMRP1-/- mice, failure to express mMRP1 leads to decreased LTC4 secretion from leukotriene-synthesizing cells and impaired response to an inflammatory stimulus (25Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van der Valk M. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (403) Google Scholar). Studies by Robbiani et al. (26Robbiani D.F. Finch R.A. Jager D. Muller W.A. Sartorelli A.C. Randolph G.J. Cell. 2000; 103: 757-768Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar) found that mMRP1-mediated efflux of LTC4 was also involved in regulating the migration of dendritic cells to lymph nodes. Whether E217βG is a physiological substrate for MRP1 is not yet known, but the protein actively transports this cholestatic conjugated estrogen in vitro with a K m of 1-3 μm (13Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. 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Pastan I. Mol. Med. 2001; 7: 509-516Crossref PubMed Google Scholar, 30Tammur J. Prades C. Arnould I. Rzhetsky A. Hutchinson A. Adachi M. Schuetz J.D. Swoboda K.J. Ptacek L.J. Rosier M. Dean M. Allikmets R. Gene (Amst.). 2001; 273: 89-96Crossref PubMed Scopus (137) Google Scholar, 31Kao H. Huang J. Chang M. Gene (Amst.). 2002; 286: 299-306Crossref PubMed Scopus (33) Google Scholar). The predicted topologies of MRP1 to -9 all contain a typical ABC transporter core region, composed of two hydrophobic membrane-spanning domains (MSDs), each with six transmembrane (TM) α-helices, and two hydrophilic nucleotide-binding domains (NBDs). In addition, MRP1, -2, -3, -6, and -7 have a third, NH2-terminal MSD (MSD1), which consists of five TMs with an extracellular NH2 terminus (Fig. 1) (27Hopper E. Belinsky M.G. Zeng H. Tosolini A. Testa J.R. Kruh G.D. Cancer Lett. 2001; 162: 181-191Crossref PubMed Scopus (175) Google Scholar, 31Kao H. Huang J. Chang M. Gene (Amst.). 2002; 286: 299-306Crossref PubMed Scopus (33) Google Scholar, 32Hipfner D.R. Mao Q. Qiu W. Leslie E.M. Gao M. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1999; 274: 15420-15426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 33Bakos E. Hegedus T. Hollo Z. Welker E. Tusnady G.E. Zaman G.J. Flens M.J. Varadi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 34Hipfner D.R. Gao M. Scheffer G. Scheper R.J. Deeley R.G. Cole S.P.C. Br. J. Cancer. 1998; 78: 1134-1140Crossref PubMed Scopus (71) Google Scholar, 35Hipfner 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, 36Kast C. Gros P. J. Biol. Chem. 1997; 272: 26479-26487Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Previously, we have shown that a negatively charged residue, Glu1089, within predicted TM14 is crucial for MRP1-mediated drug resistance, particularly with respect to the anthracyclines (37Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 13231-13239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We have also demonstrated that a number of polar amino acid residues in predicted TM17 of MRP1 are essential for MRP1 to confer drug resistance and/or to transport E217βG efficiently (38Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2002; 277: 20934-20941Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 39Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 34966-34974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 40Ito K. Olsen S.L. Qiu W. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 15616-15624Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In addition, these studies provided evidence for cross-talk between residues in the two TM helices (39Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 34966-34974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). TM17 of MRP1 is topologically equivalent to TM12 of P-gp, which has been shown to be intimately involved in substrate binding and determination of substrate specificity (41Hafkemeyer P. Dey S. Ambudkar S.V. Hrycyna C.A. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 16400-16409Crossref PubMed Scopus (80) Google Scholar). Similarly, TM 14 corresponds to TM 9 of P-gp, but the involvement of residues in this helix in determining substrate specificity or overall activity has only recently been identified (42Loo T.W. Clarke D.M. J. Biol. Chem. 2002; 277: 44332-44338Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 43Song J. Melera P.W. Mol. Pharmacol. 2001; 60: 254-261Crossref PubMed Scopus (14) Google Scholar). In addition to Glu1089, TM14 of MRP1 contains two charged residues, Asp1084 and Lys1092, as well as four amino acid residues with polar side chains (Fig. 1). We have now characterized the role of all of these residues in determining the substrate specificity and/or overall activity of MRP1. These studies demonstrate that Lys1092, Ser1097, and Asn1100, which are all predicted to be on the same face of the helix, are important for defining the substrate specificity of MRP1 and that Asp1084 is critical for its overall activity. We also show that proteins in which Asp1084 has been mutated to non-carboxylic amino acids retain the ability to bind LTC4 with high affinity but are unable to shift into a low affinity binding state in the presence of ATP and vanadate. Finally, we demonstrate that the inability of the Asp1084 mutant protein to enter a transition state is attributable to a defect in the ability of NBD2 to hydrolyze ATP, as evidenced by a loss of vanadate-dependent trapping of ADP at this NBD. Materials—Culture medium and fetal bovine serum were obtained from Invitrogen. [3H]LTC4 (38 Ci/mmol) and [3H]GSH (52 Ci/mmol) were purchased from Amersham Biosciences, [3H]E217βG (44 Ci/mmol) from PerkinElmer Life Sciences, and 8-azido-[α-32P]ATP (11.8 Ci/mmol) from Affinity Labeling Technologies Inc. (Lexington, KY). Doxorubicin HCl, etoposide (VP-16), and vincristine sulfate were obtained from Sigma. Site-directed Mutagenesis—All mutations were generated using the Transformer™ site-directed mutagenesis kit (Clontech, Palo Alto, CA). Templates were prepared as described previously (37Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 13231-13239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 38Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2002; 277: 20934-20941Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 39Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 34966-34974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Mutagenesis was then performed according to the instructions from the manufacturer using a selection primer, 5′-GAG AGT GCA CGA TAT CCG GTG TG-3′, that mutates a unique NdeI site in the vector to an EcoRV restriction site. Oligonucleotides bearing mismatched bases at the residues to be mutated (underlined) were synthesized by ACGT Corp. (Toronto, Ontario, Canada). They are as follows: T1082A (5′-C TCC AAG GAG CTC GAC GCA GTG GAC TCC-3′), D1084N (5′-CTG GAC ACA GTG AAT TCC ATG ATC CCG-3′), D1084A (5′-CTG GAC ACA GTG AAA TCG ATG ATC CCG-3′), D1084E (5′-CTG GAC ACA GTG GAC TCG ATG ATC CCG-3′), D1084V (5′-CTG GAC ACA GTG GTA TCG ATG ATC CCG-3′), S1085A (5′-CTG GAC ACA GTC GAC GCC ATG ATC CCG G-3′), K1092M (5′-C CCG GAG GTC ATC ATG ATG TTC ATG GGC-3′), K1092A (5′-C CCG GAG GTC ATC GCG ATG TTC ATG GGC-3′), K1092E (5′-C CCG GAG GTC ATC GAG ATG TTC ATG GGC-3′), K1092R (5′-C CCG GAG GTC ATC AGG ATG TTC ATG GGC-3′), S1097A (5′-G ATG TTC ATG GGC GCC CTG TTC AAC-3′), N1100A (5′-TTC ATG GGC TCG CTC TTC GCC GTC ATT GGT G-3′), N1100S (5′-TTC ATG GGC TCG CTC TTC AGT GTC ATT GGT G-3′). After confirming all mutations by DNA Thermo Sequenase Cy5.5 and Cy5.0 dye terminator cycle sequencing (Amersham Biosciences) according to the instructions from the manufacturer, DNA fragments containing the desired mutations were transferred into pCEBV7-MRP1. After reconstructing the mutations into the full-length clones, the integrity of the entire mutated inserts and cloning sites was verified by DNA sequencing (ACGT Corp.). Mutation D1084R was generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). Templates were prepared as described previously (44Koike K. Oleschuk C.J. Haimeur A. Olsen S.L. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; 277: 49495-49503Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Mutagenesis was then performed according to the instructions from the manufacturer. Oligonucleotide bearing mismatched bases at the residues to be mutated (underlined) was synthesized by ACGT Corp. It is as follows: D1084R (5′-CTG GAC ACA GTG CGG TCC ATG ATC CCG-3′). After confirming the mutation by DNA sequencing (ACGT Corp.), DNA fragments containing the desired mutations were transferred into pcDNA3.1-MRP1. Cell Lines and Tissue Culture—Stable transfection of HEK293 cells with the pCEBV7 vector containing the wild type and mutant MRP1 cDNAs has been described previously (13Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar, 37Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 13231-13239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 38Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2002; 277: 20934-20941Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 39Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 34966-34974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, HEK293 cells were transfected with pCEBV7 vectors containing mutant MRP1 using FuGENE 6 (Roche Molecular Biochemicals) according to the instructions from the manufacturer. After ∼48 h, the transfected cells were supplemented with fresh medium containing 100 μg/ml hygromycin B. Approximately 3 weeks after transfection, the hygromycin B-resistant cells were cloned by limiting dilution and the resulting cell lines were tested for high level expression of the mutant proteins. Confocal Microscopy—Confocal microscopy was carried out as described previously (37Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 13231-13239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 38Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2002; 277: 20934-20941Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 39Zhang D.W. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 34966-34974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, ∼5 × 105 stably-transfected HEK293 cells were seeded in each well of a 6-well tissue culture dish on coverslips. When the cells had grown to confluence, they were washed once in PBS and then fixed with 2% paraformaldehyde in PBS, followed by permeabilization using digitonin (0.25 mg/ml in PBS). MRP1 proteins were detected with the monoclonal antibody MRPm6. Antibody binding was detected with Alexa Fluor 488 anti-mouse IgG (H+L) (Fab′)2 fragment. Nuclei were stained with propidium iodide. Localization of MRP1 in the transfected cells was determined using a Meridian Insight confocal microscope (filter, 620/40 nm for propidium iodide and 530/30 nm for Fluor 488). Determination of Protein Levels in Transfected Cells—Plasma membrane vesicles were prepared by centrifugation through sucrose, as described previously (13Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar, 16Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). After determination of protein levels by Bradford assay (Bio-Rad), total membrane protein (0.5, 1.0, and 1.25 μg) from transfectants expressing wild type MRP1 and various mutant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% gel. Proteins were subsequently transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA) by electroblotting. The proteins were detected with mAb MRPm6 (Alexis Biochemicals, San Diego, CA). Antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce), followed by enhanced chemiluminescence detection and X-Omat™ Blue XB-1 films (PerkinElmer Life Sciences). Densitometry of the film images was performed using a Chemi-Imager™ 4000 (Alpha Innotech Corp., San Leandro, CA). The relative protein expression levels were calculated by dividing the integrated densitometry values obtained for 0.5, 1.0, and 1.25 μg of total membrane proteins from transfectants expressing the mutant proteins by the integrated densitometry values obtained for the comparable amounts of total membrane proteins from transfectants expressing the wild type protein. Each comparison was performed at least three times in independent experiments. The results were then pooled and the mean values used for normalization purposes. Expression of the NH 2 - and the COOH-proximal Half-molecules of MRP1 in SF21 Cells—The construction of the dual expression vector pFASTBAC Dual (Invitrogen) encoding the NH2- and the COOH-proximal half-molecules of MRP1 has been described previously (45Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). To generate MRP1D1084N-pFASTBAC Dual vector, pCEBV7-MRP1 containing mutation D1084N was digested with BstEII, and an ∼600-bp fragment comprising nucleotides 3369-3913 of MRP1D1084N was isolated. This fragment was then ligated to an ∼9.4-kb fragment isolated from BstEII-digested MRP1-pFASTBAC Dual vector. Recombinant bacmids and baculoviruses were generated as described previously (45Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The conditions used for viral infection were also similar to those described previously (45Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Plasma membrane vesicles were prepared, and the expression levels of wild type and mutant protein in infected cells were determined, as described previously (13Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar, 16Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). The NH2-proximal proteins were detected with mAb MRPr1 (Alexis Biochemicals), and the COOH-proximal proteins were detected with mAb MRPm6 (Alexis Biochemicals). LTC 4 , E 2 17βG, and GSH Transport by Membrane Vesicles—Plasma membrane vesicles were prepared as described previously, and ATP-dependent transport of [3H]LTC4 into the inside-out membrane vesicles was measured by a rapid filtration technique (16Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 17Loe D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Briefly, vesicles (10 μg of total proteins) were incubated at 23 °C in 100 μl of transport buffer (50 mm Tris-HCl, 250 mm sucrose, 0.02% sodium azide, pH 7.4) containing ATP or AMP (4 mm), 10 mm MgCl2 and [3H]LTC4 (50 nm, 200 nCi). At the indicated times, 20-μl aliquots were removed and added to 1 ml of ice-cold transport buffer, followed by filtration under vacuum through glass fiber filters (type A/E, Gelman Sciences, Dorval, Quebec, Canada). Filters were immediately washed twice with 4 ml of cold transport buffer. The bound radioactivity was determined by scintillation counting. All data were corrected for the amount of [3H]LTC4 that remained bound to the filter in the absence of vesicle protein (usually <5% of the total radioactivity). [3H]LTC4 uptake was expressed relative to the total protein concentration in each reaction. ATP-dependent uptake of [3H]E217βG (400 nm, 120 nCi) was measured as described for [3H]LTC4 except that the reaction was carried out at 37 °C. K m and V max values of ATP-dependent [3H]LTC4 uptake by membrane vesicles (2.5 μg of total proteins) were measured at various LTC4 concentrations (0.01-1 μm) for 1 min at 23 °C. in 25 μl of transport buffer containing 4 mm ATP and 10 mm MgCl2, followed by non-linear regression analyses. Kinetic parameters of ATP-dependent [3H]E217βG (0.1-16 μm) uptake were determined as described for [3H]LTC4 except that the reaction was carried out at 37 °C. GSH uptake was also measured by rapid filtration with membrane vesicles (20 μg of total proteins) incubated at 37 °C for 20 min in a 60-μl reaction volume with [3H]GSH (100 μm, 300 nCi). To minimize GSH catabolism by γ-glutamyltranspeptidase during transport, membranes were preincubated in 0.5 mm acivicin for 10 min at 37 °C prior to measuring [3H]GSH uptake in the presence of verapamil (100 μm). Photoaffinity Labeling of the Wild Type and Mutant Proteins with [3H]LTC 4 —Wild-type and mutant MRP1 membrane proteins were photolabeled with [3H]LTC4 essentially as described previously (16Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 46Qian Y.M. Qiu W. Gao M. Westlake C.J. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 38636-38644Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 47Qian Y.M. Grant C.E. Westlake C.J. Zhang D.W. Lander P.A. Shepard R.L. Dantzig A.H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2002; 277: 35225-35231Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, membrane vesicles (50 μg of total proteins in 35 μl of transport buffer) were incubated with [3H]LTC4 (0.3 μCi, 200 nm) at room temperature for 10 min, frozen in liquid nitrogen, and UV-irradiated. Proteins were analyzed on a 5-15% gradient gel by SDS-PAGE. The gel was then fixed, treated with Amplify (Amersham Biosciences), dried, and exposed to film for 1 week at -70 °C. Photolabeling of MRP1 by 8-Azido-[α-32P]ATP and Orthovanadate-induced Trapping of 8-Azido-[α-32P]ATP by MRP1—Wild-type and mutant MRP1 membrane proteins were photolabeled with 8-azido-[α-32P]ATP essentially as described previously (45Gao M. Cui H.R. Loe D.W.

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