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• W2065226504 abstract "Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [3H]leukotriene C4 (LTC4), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC4 labels sites in the NH2- and COOH-proximal halves of MRP1, (ii) labeling of the NH2-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH2-proximal site is preferentially inhibited byS-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14–17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC4 is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC4 binding to the NH2-proximal site, and (vii) the attenuation of LTC4 binding to the NH2-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC4 binds to one of two sites composed, at least in part, of elements in the NH2-proximal half of the protein. Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [3H]leukotriene C4 (LTC4), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC4 labels sites in the NH2- and COOH-proximal halves of MRP1, (ii) labeling of the NH2-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH2-proximal site is preferentially inhibited byS-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14–17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC4 is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC4 binding to the NH2-proximal site, and (vii) the attenuation of LTC4 binding to the NH2-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC4 binds to one of two sites composed, at least in part, of elements in the NH2-proximal half of the protein. P-glycoprotein multidrug resistance protein leukotriene C4 Spodoptera frugiperda reduced glutathione adenosine 5′-O-(3-thiotriphosphate) polyacrylamide gel electrophoresis monoclonal antibody nucleotide binding domain membrane-spanning domain cytoplasmic loop 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid N-(hydrocinchonidin-8′-yl)-4-azido-2-hydroxybenzamide iodoaryl azidorhodamine 123 agosterol-A transmembrane domain Development of multidrug resistance is a frequent impediment to the effective treatment of cancer. Although many different mechanisms are involved, multidrug resistance in cultured tumor cells appears most frequently to be associated with increased expression of the ATP-binding cassette transporter proteins, P-glycoprotein (P-gp)1 and/or multidrug resistance protein (MRP) 1 (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 (2995) Google Scholar, 2Deeley R.G. Cole S.P.C. Semin. Cancer Biol. 1997; 8: 193-204Crossref PubMed Scopus (164) Google Scholar, 3Cole S.P.C. Deeley R.G. Bioessays. 1998; 20: 931-940Crossref PubMed Scopus (332) Google Scholar). As in cells that overexpress P-gp, drug accumulation in cells with elevated levels of MRP1 is reduced, supporting the notion that the multidrug resistance phenotype caused by both of these proteins involves increased drug extrusion (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, 5Zaman 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 (697) Google Scholar). However, in contrast to P-gp, demonstration of MRP1-mediated active transport of unmodified chemotherapeutic drugs such as vincristine and daunorubicin in vitro, using inside-out membrane vesicle systems, requires the presence of GSH in addition to ATP (6Loe 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 (544) Google Scholar, 7Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar, 8Renes J. de Vries E.G. Nienhuis E.F. Jansen P.L. Muller M. Br. J. Pharmacol. 1999; 126: 681-688Crossref PubMed Scopus (244) Google Scholar, 9Ding G.Y. Shen T. Center M.S. Anticancer Res. 1999; 19: 3243-3248PubMed Google Scholar). More recently, we have shown that stimulation of MRP1-mediated transport by GSH or certain of its analogs is not restricted to unmodified hydrophobic drugs but may also include some organic anion conjugates, such as estrogen sulfates and a glucuronidated tobacco-derived nitrosamine (10Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, unlike xenobiotics such as vincristine and verapamil (7Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar, 12Mao Q. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2000; 275: 34166-34172Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), these anionic conjugates appear not to stimulate GSH transport (10Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The mechanisms by which compounds such as vincristine and verapamil stimulate GSH transport remain unclear, although several hypotheses have been proposed (3Cole S.P.C. Deeley R.G. Bioessays. 1998; 20: 931-940Crossref PubMed Scopus (332) Google Scholar, 10Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,11Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Versantvoort C.H. Broxterman H.J. Bagrij T. Scheper R.J. Twentyman P.R. Br. J. Cancer. 1995; 72: 82-89Crossref PubMed Scopus (234) Google Scholar, 14Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Crossref PubMed Scopus (581) Google Scholar). The most well characterized substrate of MRP1 is the cysteinyl leukotriene, leukotriene C4 (LTC4). Studies with mrp1−/− mice have confirmed that LTC4 is an endogenous substrate for mrp1. These studies have shown that lack of the protein results in an impaired LTC4-mediated inflammatory response and that mrp1-mediated efflux of LTC4 is involved in regulating dendritic cell migration to lymph nodes (15Wijnholds J. Evers R. van Leusden M.R. Mol C.A. Zaman G.J. Mayer U. Beijnen J.H. van, d. V Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (401) Google Scholar, 16Robbiani 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 (415) Google Scholar). To date, LTC4 remains the highest affinity MRP1/mrp1 substrate that has been identified (Km ∼ 100 nm), and many MRP1 structure-function studies have been based on LTC4transport activity. Examples include reconstitution of LTC4transport activity by heterologous co-expression of the NH2- and COOH-proximal halves of MRP1 (17Gao M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and identification of regions essential or dispensable for function (18Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19Bakos E. Evers R. Szakacs G. Tusnady G.E. Welker E. Szabo K. de Haas M. van Deemter L. Borst P. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Bakos E. Evers R. Calenda G. Tusnady G.E. Szakacs G. Varadi A. Sarkadi B. J. Cell Sci. 2000; 113: 4451-4461Crossref PubMed Google Scholar). Topology studies of MRP1 have revealed that MRP1 and P-gp share a similar core structure consisting of two membrane-spanning domains (MSD2 and MSD3) and two nucleotide-binding domains (NBD1 and NBD2) (21Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar). The primary distinguishing characteristic of MRP1 and its related proteins, MRPs 2, 3, 6, and 7, is an additional NH2-terminal region forming a membrane-spanning domain (MSD1) with five transmembrane helices (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 (2995) Google Scholar, 21Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar, 22Hipfner 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, 23Hopper 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). This region is linked to the remainder of the protein by a relatively large cytoplasmic loop (CL), designated CL3, of ∼130 amino acids (21Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar). We have shown previously that deletion of MSD1 plus ∼35 or 95 amino acids of the CL3 eliminates LTC4 transport activity (18Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). However, transport activity could be restored to both NH2-terminally truncated proteins by co-expressing a fragment that contains MSD1 and the first 95 amino acids of CL3, but not by a fragment containing MSD1 and only the first 35 amino acids of CL3 (18Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Similarly, an internal deletion of 53 amino acids within CL3 also inactivates the protein. These studies strongly suggest that the physical integrity of a certain portion of CL3 is essential for LTC4 transport. A similar conclusion was reached by Bakoset al. (19Bakos E. Evers R. Szakacs G. Tusnady G.E. Welker E. Szabo K. de Haas M. van Deemter L. Borst P. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), who demonstrated that a truncated protein lacking MSD1 but retaining essentially all of predicted CL3, MRP1204–1531, retained considerable LTC4transport activity (19Bakos E. Evers R. Szakacs G. Tusnady G.E. Welker E. Szabo K. de Haas M. van Deemter L. Borst P. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The NH2 terminus of such a truncated protein is very close to that predicted for a common four-domain ancestor of the MRPs and the more distantly related cystic fibrosis transmembrane conductance regulator, suggesting that MRP-related proteins with an additional MSD may have evolved by fusion of a gene encoding an already functional transporter with a gene or genes encoding other integral membrane proteins (24Grant C.E. Kurz E.U. Cole S.P.C. Deeley R.G. Genomics. 1997; 45: 368-378Crossref PubMed Scopus (82) Google Scholar). Although previous studies have defined several regions of MRP1 that are required for LTC4 transport (18Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19Bakos E. Evers R. Szakacs G. Tusnady G.E. Welker E. Szabo K. de Haas M. van Deemter L. Borst P. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Bakos E. Evers R. Calenda G. Tusnady G.E. Szakacs G. Varadi A. Sarkadi B. J. Cell Sci. 2000; 113: 4451-4461Crossref PubMed Google Scholar), it is presently not known whether these regions are essential for initial binding of substrate, or are required for some subsequent step in the transport process. To define regions of MRP1 involved in LTC4binding, we took advantage of single and dual baculovirus expression vectors, either to produce truncated forms of MRP1 in Sf21 cells, or to co-express two/three fragments of the protein, which were then photolabeled by [3H]LTC4. Using this approach, we have shown that: (i) cooperativity between the NH2- and COOH-proximal halves of MRP1 is required for high affinity LTC4 binding, (ii) LTC4 photoaffinity labels sites in both the NH2- and COOH-proximal halves of the protein, (iii) all or part of the region of CL3 between amino acids 204 and 281 is essential for LTC4 binding, although it is not a site of photoaffinity labeling, (iv) the affinity of LTC4 binding to the region containing MSD2 and NBD1 is selectively decreased when Vi-ADP is trapped at NBD2, and (v) the decrease in LTC4 binding observed under vanadate trapping conditions occurs in the absence of ATP hydrolysis by NBD1. [14,15-3H]LTC4 (38 Ci mmol−1) was purchased from PerkinElmer Life Sciences and fluorographic reagent Amplify® from Amersham Pharmacia Biotech (Oakville, Ontario, Canada). Nucleotides, GSH, andS-methyl GSH were purchased from Sigma. GammaBind Plus Sepharose was from Amersham Pharmacia Biotech. Recombinant donor plasmids encoding the full-length MRP1 and either of the NH2-proximal half-molecule and the COOH-proximal half-molecule of MRP1 have been described (17Gao M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To introduce cDNA fragments encoding truncated half-molecules of MRP1 into pFASTBAC Dual vectors, the same strategy as described for generation of a construct capable of expressing both halves of the protein (25Gao 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) was used (Fig.1). The previously described pFB-ΔN(MRP932–1531), pFB-MRP900–1143, pFB-MRP1138–1531, and pFB-MRP1061–1531 were individually linearized with SalI, blunted with Klenow fragment, and then digested with KpnI. The recovered SalI*-KpnI fragments were ligated to pFASTBAC Dual, which had been digested with SmaI andKpnI to give pFBDual-MRP932–1531, pFBDual-MRP900–1143, pFBDual-MRP1138–1531, and pFBDual-MRP1061–1531. These constructs were further digested with SalI and XbaI and ligated to the SalI-XbaI fragments that were isolated from pFB-MRP1–932, pFB-MRP1–1097, and pFB-MRP1–1138 to generate pFBDual-MRP1–932/MRP900–1143, pFBDual-MRP1–932/MRP1138–1531, pFBDual-MRP1–1097/MRP1061–1531, and pFBDual-MRP1–1138/MRP1138–1531. The dual expression vector carrying cDNA fragments encoding the amino acids 204–653 and 932–1531 of MRP1 (pFBDual-MRP204–653/MRP932–1531) was constructed by one-step deletion; pFBDual-MRP204–932/MRP932–1531 was digested withEcoNI and XbaI, made blunt-ended using Klenow fragment, and then ligated after removal of the deleted fragment. Construct pBSMRP-fc-ATG, which was described previously (17Gao M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), was linearized with BamHI, made blunt-ended using Klenow fragment, and then digested with SacI, generating one fragment encoding amino acids 1–281 of MRP1. TheSacI-BamHI* fragment was ligated to pFBDual vector that had a blunted end of HindIII* site and a cohesive end of SacI site to produce pFBDual MRP1–281. Translation of the inserted fragment terminated at a stop codon in the vector, resulting in the addition of six amino acids, QLVEKY. pFBDual MRP1–281 was linearized with SmaI and KpnI and ligated to the SalI*-KpnI fragment isolated from pFB-MRP281–1531 to produce pFBDual MRP1–281/MRP281–1531. Constructs of pFB-MRPΔ(228–280) and pFB-MRP281–1531, which were described previously (18Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), were digested with SalI andSphI to release SalI-SphI fragments. These fragments were ligated to pFBDual-MRP1–932/MRP932–1531 that had been linearized with the same restriction enzymes to produce pFBDual-MRP1–932(Δ228–280)/MRP932–1531 and pFBDual-MRP281–932/MRP932–1531. Generation of recombinant bacmids and baculoviruses and conditions used for viral infection were described previously (17Gao M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Membrane vesicles were prepared by nitrogen cavitation and sucrose gradient centrifugation, as described (6Loe 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 (544) Google Scholar, 10Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Membrane vesicle proteins were electrophoresed on 5–15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), transferred to Immobilon-P membranes (Millipore, Bedford, MA), and probed with mAbs QCRL-1, MRPr1, or MRPm6, which we have demonstrated recognize the NH2-proximal part of the protease-sensitive region connecting NBD1 to MSD3 (amino acids 918–924), CL3 (amino acids 228–237), and the COOH terminus of MRP1 (amino acids 1511–1520), respectively (26Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar, 27Hipfner 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). Immunodetection was performed with the enhanced chemiluminescence Western blotting system from Amersham Pharmacia Biotech. LTC4 transport was assayed in membrane vesicles at 23 °C in the presence of 4 mm ATP or AMP using a rapid filtration technique, as described previously (6Loe 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 (544) Google Scholar). Insect cell membrane vesicles (75 µg of protein in 35 µl) were incubated with [3H]LTC4 (0.25 µCi, 200 nm) at room temperature for 10 min, frozen in liquid nitrogen, and UV-irradiated, as described (10Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Radiolabeled vesicles were analyzed on a 5–15% (or as indicated) gel by SDS-PAGE prior to fluorography. [3H]LTC4 photolabeling of tryptic fragments of native MRP1 in cell membranes from the multidrug-resistant small cell lung cancer cell line, H69AR, was also performed as above after membrane vesicles (120 µg of protein) were treated with trypsin at various trypsin/protein ratios (1:800–1:25) (22Hipfner 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). For immunoprecipitation experiments, LTC4 labeling was carried out using 150 µg of membrane proteins and 0.5 µCi of [3H]LTC4. After UV irradiation, membrane proteins were solubilized in phosphate-buffered saline containing 1% CHAPS at 4 °C for 3 h and insoluble fraction was removed by centrifugation. mAbs QCRL-1 and MRPm6 (1 µg each) were then added to the supernatant of solubilized membrane proteins and incubated at 4 °C overnight. Antibody-associated proteins were absorbed with GammaBind Plus Sepharose for 1 h, and the beads were washed four times with cold phosphate-buffered saline. Immunocomplexes were then solubilized and analyzed as above. Initially, membrane vesicles from Sf21 insect cells expressing intact MRP1 were examined to establish that the protein could be specifically and efficiently photolabeled with [3H]LTC4, as we and others have shown previously using MRP1-enriched membrane vesicles from mammalian cells (6Loe 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 (544) Google Scholar, 28Leier 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). A prominently labeled 170-kDa protein, consistent with the size predicted for core glycosylated MRP1 (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 (2995) Google Scholar, 17Gao M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) was detectable by fluorography following SDS-PAGE of total membrane protein from cells infected with baculovirus encoding full-length MRP1, with very little labeling of any other proteins between 70 and 200 kDa. No comparable labeling was observed in membranes prepared from control cells infected with baculovirus encoding β-glucuronidase. In addition, unlabeled LTC4 (6 µm) abolished [3H]LTC4 labeling of the 170-kDa protein confirming the specificity of the binding (Fig.2B). Previously, we have demonstrated that LTC4 selectively stimulates ATP binding by NBD1 of MRP1, but the influence of nucleotide on LTC4binding has not been determined (25Gao 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). Consequently, photoaffinity cross-linking with [3H]LTC4 was performed using previously established ATP binding conditions in the absence and presence of 4 mm ATPγS, a poorly-hydrolyzable ATP analog. No effect of the nucleotide analog on LTC4 binding was observed (Fig. 2C). MRP1 fragments comprising 1–932 and 932–1531 have been shown previously to be capable of associating to form a functional transporter (see Fig. 1 for predicted secondary structure of MRP1 and illustration of various MRP1 constructs) (17Gao M. Loe D.W. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To examine LTC4 binding by these fragments, cells were infected with either a dual expression vector encoding both MRP11–932and MRP1932–1531 or with vectors encoding one or the other fragment. Membrane vesicles were then prepared from the infected cells and immunoblotted with mAbs QCRL-1 and MRPm6 to detect the NH2- and COOH-halves of the protein, respectively (Fig.3A). When membrane vesicles prepared from cells infected with the dual expression vector were photolabeled with [3H]LTC4, strong labeling of a protein corresponding in size to the NH2-proximal fragment and weaker but readily detectable labeling of a protein with the predicted size of the COOH-proximal fragment was observed (Fig. 3B). Confirmation of the identities of the proteins labeled with LTC4 in cells co-expressing both halves of the protein was obtained by immunoprecipitation with a combination of two mAbs, QCRL-1 and MRPm6, recognizing the NH2- and COOH-terminal halves of MRP1, respectively (Fig. 3C). To investigate whether labeling of both half molecules was the consequence of autonomous binding by independent sites in each half of the protein, we examined membranes from cells expressing comparable levels of one or the other fragment. Although much reduced in intensity, weak photolabeling of the NH2-terminal half of the protein could be detected in the absence of the COOH-terminal fragment (Fig. 3B). Labeling of the COOH-terminal half was also markedly decreased. However, because of the presence of endogenous proteins with similar electrophoretic mobility to this fragment that were very weakly labeled with [3H]LTC4, we were unable to definitively determine whether or not a very low level of binding to the COOH-terminal half of the protein may also occur. Overall, these data demonstrate that high affinity binding of LTC4 requires association of both halves of the protein and that the NH2-proximal half alone may be capable of binding LTC4 but with much lower affinity than the complete protein. Co-expression of other combinations of fragments that fail to form a functional transporter, in some cases, displayed labeling on one or two fragments. However, when compared with the results obtained with the full-length protein or by co-expressing fragments MRP11–932 with MRP1932–1531, the intensity of labeling was in general much reduced (summarized in TableI or shown in Fig. 3D).Table ISummary of [3H]LTC4 labeling of membrane vesicles co-expressing two or three fragments of MRP1Protein[3H]LTC4labelingLTC4 transport activityNH2-fragmentCOOH-fragment%MRP11–932/932–1531+++++++++100MRP1281–932/932–1531+++0–10MRP1281–932/932–15311-aThese two fragments were co-expressed with MRP11–281.++++++++60–70MRP1204–563/932–1531+/−+++0–10MRP1932–1531−+0MRP11–932/900–1143++/−0–10MRP11–932/1138–1531+++/−0–10MRP11–932+−0MRP11–281/281–1531−+++++70–80MRP11–1097/1061–1531++++0–10MRP11–1138/1138–1531+−0–101-a These two fragments were co-expressed with MRP11–281. Open table in a new tab To confirm that sites in the NH2- and COOH-proximal halves of the intact MRP1 expressed in mammalian cells were also labeled with LTC4, photolabeling experiments were carried out with membranes prepared from drug resistant H69AR lung cancer cells from which MRP1 was originally cloned (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 (2995) Google Scholar). We have shown that MRP1 constitutes up to 5% of membrane proteins in these cells (29Mao Q. Leslie E.M. Deeley R.G. Cole S.P. Biochim. Biophys. Acta. 1999; 1461: 69-82Crossref PubMed Scopus (86) Google Scholar). When H69AR membranes were subjected to mild trypsinolysis and [3H]LTC4 cross-linking followed by SDS-PAGE and fluorography, two fragments of 75–80 and 55–60 kDa were labeled (Fig. 4). Immunoblotting using MRP1-specific antibodies with known epitopes indicated that these two fragments corresponded to regions between approximately amino acids 900–1531 (MSD3 and NBD2) and 250–900 (MSD2 and NBD1), the fragment with the larger apparent Mr resulting from glycosylation at position 1006 in MSD3 (Ref. 22Hipfner 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 and data not shown). These results are consistent with the experiments using recombinant MRP1 expressed in insect cells as shown above. They confirm that [3H]LTC4 labels sites in both halves of MRP1 and, that the NH2-proximal fragment corresponding to MSD2 and NBD1 (amino acids 320–930) and part of the cytoplasmic loop connecting it to MSD1 (CL3) is more strongly labeled than the COOH-proximal 75–80-kDa tryptic fragment corresponding to MSD3 and NBD2. We have shown previously that removal of amino acids 1–229 or 1–281 of MRP1, or the region" @default.
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