Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m206058200
ISSN1083-351X
AutoresYueming Qian, Caroline E. Grant, Christopher J. Westlake, Dawei Zhang, Peter A. Lander, Robert L. Shepard, Anne H. Dantzig, Susan P.C. Cole, Roger G. Deeley,
Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoMultidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed125I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[35S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH2- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1. Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed125I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[35S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH2- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1. Multidrug resistance protein 1 (MRP1/ABCC1) 1The abbreviations used are: MRP1, multidrug resistance protein 1; ABC, ATP-binding cassette; MSD, membrane spanning domain; CL3, cytoplasmic linker region; NBD, nucleotide binding domain; TM, transmembrane; LTC4, leukotriene C4; E217βG, 17β-estradiol 17-β-(D-glucuronide); HEK, human embryonic kidney; IACI, N-(hydrocinchonidin-8′-yl)-4-azido-2-hydroxybenzamide; IAARh123, iodoaryl azido rhodamine 123; AG-A, agosterol-A. is a member of the C branch of the superfamily of ATP-binding cassette (ABC) transmembrane transporters that also includes the cystic fibrosis transmembrane conductance regulator and the sulfonylurea receptors (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 (3022) Google Scholar, 2Deeley R.G. Cole S.P.C. Semin. Cancer Biol. 1997; 8: 193-204Crossref PubMed Scopus (165) Google Scholar, 3Cole S.P.C. Deeley R.G. Bioessays. 1998; 20: 931-940Crossref PubMed Scopus (333) Google Scholar, 4Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Crossref PubMed Scopus (583) Google Scholar). In addition, the ABCC branch includes eight other MRP related proteins, MRP 2–7 and ABCC11 and ABCC12 (5Keppler D. Cui Y. Konig J. Leier I. Nies A.T. Adv. Enzyme Regul. 1999; 39: 237-246Crossref PubMed Scopus (93) Google Scholar, 6Kool M. de Haas M. Scheffer G.L. Scheper R.J. van Eijk M.J.T. Juijn J.A. Baas F. Borst P. Cancer Res. 1997; 57: 3537-3547PubMed Google Scholar, 7Kool M. van der Linden M. de Haas M. Baas F. Borst P. Cancer Res. 1999; : 175-182PubMed Google Scholar, 8Hopper 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, 9Tammur 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. 2001; 273: 89-96Crossref PubMed Scopus (137) Google Scholar). MRP1 is an active transporter of an extremely diverse array of organic anion conjugates as well as a number of anionic non-conjugated compounds. Some of the most well characterized substrates, such as the cysteinyl leukotriene C4 (LTC4), are conjugated to glutathione (10Leier 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, 11Muller M. Meijer C. Zaman G.J.R. Borst P. Scheper R.J. Mulder N.H. de Vries E.G.E. Jansen P.L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13033-13037Crossref PubMed Scopus (640) Google Scholar, 12Loe 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), but the protein also transports glucuronidated (13Loe 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, 14Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and sulfated compounds (15Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar, 16Qian Y.M. Song W.C. Cui H.-R. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The range of MRP1 substrates includes additional non-conjugated neutral or cationic chemotherapeutic drugs as well as certain anionic conjugates that display a dependence on GSH for transport (14Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 16Qian Y.M. Song W.C. Cui H.-R. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 17Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar, 18Loe D.W. Stewart R.K. Massey T.E. Deeley R.G. Cole S.P.C. Mol. Pharmacol. 1997; 51: 1034-1041Crossref PubMed Scopus (190) Google Scholar, 19Renes J. de Vries E.G.E. Nienhuis E.F. Jansen P.L.M. Muller M. Br. J. Pharmacol. 1999; 126: 681-688Crossref PubMed Scopus (245) Google Scholar). In some cases GSH appears to be co-transported with these substrates (17Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar). In others no increase in GSH transport has been detected (14Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 16Qian Y.M. Song W.C. Cui H.-R. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). GSH alone can be transported by MRP1, but with low affinity and efficiency (14Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 16Qian Y.M. Song W.C. Cui H.-R. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The transport of GSH can also be stimulated by certain compounds such as verapamil in the absence of net transport of verapamil itself (20Loe D.W. Deeley R.G. Cole S.P.C. J. Pharmacol. Exp. Therap. 2000; 293: 530-538PubMed Google Scholar). MRP1 and its related ABCC proteins, MRP 2, 3, 6, and 7, differ topologically from most eukaryotic ABC transporters. In addition to the two polytopic membrane spanning domains (MSD) typical of ABC transporters, these proteins contain a third NH2-terminal MSD (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 (3022) Google Scholar, 3Cole S.P.C. Deeley R.G. Bioessays. 1998; 20: 931-940Crossref PubMed Scopus (333) Google Scholar, 21Hipfner D.R. Deeley R.G. Cole S.P.C. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (380) Google Scholar). In the case of MRP1 and 2, this domain has been shown to be comprised of five TM helices with an extracellular NH2 terminus (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, 23Kast C. Gros P. J. Biol. Chem. 1997; 272: 26479-26487Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 24Bakos E. Hegedus T. Hollo Z. Welker E. Tusnady G.E. Zaman G.J.R. Flens M.J. Varadi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 25Cui Y. Konig J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar). The function of this additional domain is presently not known. However, it has been established that MRP1 retains the ability to transport LTC4 even if this domain is removed (26Bakos 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 (271) Google Scholar, 27Qian 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). Whether this is also true of other substrates, particularly those that depend on GSH for their transport, remains to be established. In most cases, MRP1 substrates compete reciprocally for transport regardless of the identity of the conjugate moiety and the lack of structural similarity of the parental compounds. Some substrates that display GSH dependence for transport also compete with those that do not, although as might be expected their inhibitory potency is increased in the presence of GSH (14Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 16Qian Y.M. Song W.C. Cui H.-R. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 17Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar). These observations suggest that structurally diverse substrates interact with partially shared sets of amino acids that collectively form a binding surface or pocket on the protein. Recent studies indicate that a number of amino acids in predicted TM helix 17 and at least one residue in TM helix 14 are important in determining substrate specificity (28Zhang 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, 29Ito 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, 30Zhang 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). Furthermore, it is possible to make compensatory mutations in these two helices, suggesting that they may be in spatial proximity to one another in the native protein (30Zhang 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). As an alternative approach to identifying regions of the protein directly involved in substrate binding, MRP1 has been photolabeled by its known high affinity physiological substrate LTC4 as well as by compounds that are less well characterized with respect to binding and transport (10Leier 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, 12Loe 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, 27Qian 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, 31Daoud R. Julien M. Gros P. Georges E. J. Biol. Chem. 2001; 276: 12324-12330Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 32Daoud R. Kast C. Gros P. Georges E. Biochemistry. 2000; 39: 15344-15352Crossref PubMed Scopus (75) Google Scholar, 33Daoud R. Desneves J. Deady L.W. Tilley L. Scheper R.J. Gros P. Georges E. Biochemistry. 2000; 39: 6094-6102Crossref PubMed Scopus (42) Google Scholar, 34Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Although these compounds compete with LTC4 for binding to MRP1, their affinities appear to be considerably lower, and they also interact with the distantly related P-glycoprotein. Two of the compounds,125I-labeled N- (hydrocinchonidin-8′-yl)-4-azido-2-hydroxybenzamide (IACI) and iodoaryl azido rhodamine123 (IAARh123), bind to the protein in a GSH-independent manner and label sites in both the NH2- and COOH-proximal halves of the protein (32Daoud R. Kast C. Gros P. Georges E. Biochemistry. 2000; 39: 15344-15352Crossref PubMed Scopus (75) Google Scholar, 33Daoud R. Desneves J. Deady L.W. Tilley L. Scheper R.J. Gros P. Georges E. Biochemistry. 2000; 39: 6094-6102Crossref PubMed Scopus (42) Google Scholar, 35Gustafsson A. Pettersson P.L. Grehn L. Jemth P. Mannervik B. Biochemistry. 2001; 40: 15835-15845Crossref PubMed Scopus (35) Google Scholar) as does [3H]LTC4 (27Qian 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). Both iodinated compounds have been shown to cross-link preferentially to proteolytic peptides that contain either TM helices 10 and 11 or helices 16 and 17 (31). A third non-physiological compound, an azido derivative of the marine sponge polyhydroxylated sterol acetate, agosterol-A (AG-A), has also been used to photolabel MRP1 (34Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In contrast to the other compounds, binding of AG-A is GSH dependent, and labeling is restricted to a site in the COOH-proximal half of the protein (34Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). On the basis of these results it has been suggested that the site labeled by AG-A has a high affinity for drugs and that GSH binds to a site presumed to be in the NH2-terminal half of the protein, possibly involving the cytoplasmic linker (CL3) region between MSD1 and MSD2 (34Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, direct binding studies with photoactivateable derivatives of GSH have not been reported. In this study we have used various combinations of MRP1 fragments co-expressed in insect Sf21 cells together with human and murine hybrid and mutant proteins to investigate the binding of a new and highly specific inhibitor of MRP1. LY475776 is an iodinated azido tricyclic isoxazole that displays essentially complete dependence on GSH for binding to MRP1, and which has a higher affinity for the protein than LTC4 2A. H. Dantzig et al., personal communication. (36Mao Q. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; (in press)Google Scholar, 37Norman B.H. Gruber J.M. Hollinshead S.P. Wilson J.W. Starling J.J. Law K.L. Self T.D. Tabas L.B. Williams D.C. Paul D.C. Wagner M.M. Dantzig A.H. Bioorg. Med. Chem. Lett. 2002; 12: 883-886Crossref PubMed Scopus (64) Google Scholar). Concurrent with these studies we have also examined the binding of azidophenacyl-GSH, which we show can effectively support transport of a GSH-dependent substrate such as estrone sulfate as well as the photolabeling of MRP1 by LY475776. We have recently shown by limited trypsinolysis of full-length MRP1 that the major site photolabeled by LY475776 resides in the COOH-proximal segment of MSD3 (36Mao Q. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; (in press)Google Scholar). Using baculovirus co-expressed fragments of MRP1, we demonstrate that both halves of the protein are required for photolabeling of the site in MSD3, although photolabeling can occur in the absence of MSD1 (amino acids 1–203). However, a region in the CL3 connecting MSD1 and MSD2 (amino acids 204–280) is essential for photolabeling. In contrast to the photolabeling profile of LY475776, azidophenacyl-GSH labels sites in both halves of the protein. Labeling of the COOH-proximal site, but not the NH2-proximal site, markedly stimulated the GSH-dependent substrate estrone 3-sulfate. Although CL3 is required for photolabeling of MRP1 by both LY475776 and azidophenacyl-GSH, neither compound photolabels this region of the protein. Finally, we demonstrate that LY475776 has a higher affinity for MRP1 when compared with its murine ortholog mrp1 and identify non-conserved amino acids in MSD3 that contribute to the binding of this compound by the human protein. GSH, 4-azidophenacylbromide, 4-azidophenacyl-GSH, GSH reductase, NADPH, verapamil, and estrone 3-sulfate were purchased from Sigma. [35S]GSH (835.5 Ci/mmol) and [3H]estrone 3-sulfate (40 Ci/mmol) were obtained from PerkinElmer Life Sciences, and [3H]leukotriene C4 (38 Ci/mmol) was purchased from Amersham Biosciences. LY475776 (N-(4-azido-3-[I]-phenyl)-2-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo[4,3-c]quinolin-5-yl)-cyclohexyl]-acetamide) and [125I]LY475776 (295.2 μCi/ml) were synthesized and purified by Eli Lilly (Indianapolis, IN). The MRP1-specific monoclonal antibodies MRPm6 and MRPr1 were purchased from Alexis Biochemicals. Full-length MRP1 (MRP11–1531) was cloned into pFASTBAC and MRP1 half molecules (MRP11–932, MRP1932–1531), and fragments (MRP11–280, MRP11–932Δ228–280, and MRP1281–1531) were cloned into pFASTBAC DUAL expression vectors (Invitrogen), as described (38Gao 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, 39Gao 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, 40Gao 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). Recombinant bacmids and baculoviruses were generated as previously described (38Gao 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). Following infection of Sf21 cells, membrane vesicles were prepared by nitrogen cavitation and purified by sucrose gradient centrifugation (12Loe 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, 41Leier I. Jedlitschky G. Buchholz U. Keppler D. Eur. J. Biochem. 1994; 220: 599-606Crossref PubMed Scopus (150) Google Scholar). MRP1/mrp1hybrids were constructed in the mammalian episomal expression vector pCEBV-7 and stably transfected in HEK293 cells (42Wilson G.M. Deeley R.G. Plasmid. 1995; 33: 198-207Crossref PubMed Scopus (28) Google Scholar). The transfected cell lines that have been described previously include: human MRP1 and murine mrp1 (43Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar), chimeric mrp1/MRP1s (44Stride B.D. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1999; 274: 22877-22883Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and mutant MRP1 and mrp1 (28Zhang 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, 30Zhang 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). MRP1 half molecules (amino acids 1–932 and 932–1531) were also cloned into the mammalian dual expression vector pBudCE4.1 (Invitrogen). Membrane vesicles were isolated from transiently transfected HEK293 cells. Membrane vesicles were prepared from HEK293 cells as described for Sf21 cells. SDS-PAGE of membrane vesicle preparations was performed as described previously using 5–15% gradient gels (38Gao 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, 45Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar). Following transfer of the membrane proteins to Immobilon-P membranes (Millipore), MRP1 polypeptides were detected using an enhanced chemiluminescence kit (PerkinElmer Life Sciences) and monoclonal antibodies QCRL-1 and MRPm6 for MRP1 and MRPr1, which recognize a common epitope in MRP1 and mrp1 (46Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar, 47Hipfner D.R. Gao M. Scheffer G. Scheper R. Deeley R.G. Cole S.P.C. Br. J. Cancer. 1998; 78: 1134-1140Crossref PubMed Scopus (71) Google Scholar). The ATP-dependent uptake of LTC4 and estrone 3-sulfate was measured using a rapid filtration assay as described previously (12Loe 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, 16Qian Y.M. Song W.C. Cui H.-R. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Azidophenacyl-[35S]GSH was prepared as previously described (48Kunst M. Sies H. Akerboom T.P. Biochim. Biophys. Acta. 1989; 982: 15-23Crossref PubMed Scopus (14) Google Scholar). In brief, 125 μCi of [35S]GSH was extracted with ethyl acetate to remove dithiothreitol before being added to a reaction mixture containing potassium phosphate buffer (50 mm, pH 7.4), 4-azidophenacylbromide (10 mm), GSH reductase (120 milliunits), and NADPH (1 mm). The reaction was allowed to proceed at 22 °C for 1 h, and the products were separated by Silica G thin layer chromatography using 1-propanol/water/acetic acid (12:5:1, v/v). The area on the thin layer chromatography plate containing the radioactivity with an Rf corresponding to unlabeled azidophenacyl-GSH was scraped off and extracted with 400 μl of water six times. The extract was concentrated under a nitrogen stream. Membrane vesicles (75 μg of protein from Sf21 cells or 50 μg of protein from HEK cells in 35 μl of 50 mm Tris·HCl, pH7.4, 250 mm sucrose) were incubated with [125I]LY475776 (0.5 nm) at 37 °C or azidophenacyl-[35S]GSH (0.5μCi) at room temperature for 10 min and UV-irradiated for 5 min at 254 nm or 312 nm on ice, for LY475776 and or azidophenacyl-[35S]GSH, respectively. Proteins were solubilized in Laemmli's buffer and analyzed on a 5–15% gradient gel by SDS-PAGE. The gel was then dried onto blotting paper and exposed to x-ray film at room temperature for detection of [125I]LY475776. For the azidophenacyl-[35S]GSH-labeled proteins, gels were treated with Amplify (Amersham Biosciences), dried, and exposed to film at −70 °C. Exposure times were typically 1–3 days. The ability of [125I]LY475776 to label MRP1 was confirmed using plasma membrane vesicles from Sf21 insect cells and stable transfectants of human embryonic kidney (HEK) cells expressing the full-length protein. As observed previously using membranes from multidrug resistant H69AR cells, photolabeling was specific for a protein of the anticipated size of MRP1 and was detectable only in the presence of GSH (Fig. 1, A andC, respectively) (36Mao Q. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; (in press)Google Scholar). No labeling of a comparably sized protein was detected when membranes from cells infected with a control vector encoding β-glucuronidase (β-gus) were used, confirming that the protein was indeed MRP1 (Fig. 1 C). Our recent studies have shown that photolabeling of MRP1 with the high affinity substrate LTC4 results in labeling of a site in the NH2-proximal half of MRP1 and weaker labeling of a site in the COOH-proximal half (27Qian 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). We have also demonstrated that association of the two halves of the protein is a prerequisite for labeling of both sites. To determine whether this was also the case for [125I]LY475776, we examined photolabeling of individually expressed and co-expressed half molecules of MRP1. As observed previously with [3H]LTC4, no binding of [125I]LY475776 was detected when either half-molecule was expressed alone (Fig. 1 B). In contrast to the results obtained with LTC4, when the two halves of the protein were co-expressed, labeling occurred predominantly in the COOH-proximal half of the protein in the region extending from amino acid 932 to 1531, consistent with previous trypsinolysis studies using H69AR membranes (36Mao Q. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; (in press)Google Scholar). With the co-expressed half-molecules, we also detected very weak labeling of the NH2-proximal portion (Fig. 1 B). Although weak, this labeling was GSH dependent and was enhanced in the presence of S-methyl-GSH, which we have shown previously can substitute for GSH in supporting the transport of some MRP1 substrates (Fig.1 B, right panel) (17Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar). Thus, the weak photolabeling of the NH2-half of the protein appears to be the result of a specific GSH-dependent interaction. Previous labeling and transport studies have shown that MSD1 extending from amino acid 1 to 204 is not required for the binding and transport of [3H]LTC4 (26Bakos 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 (271) Google Scholar, 27Qian 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). Deletion of part of CL3 between amino acids 204 and 281 essentially abolishes photolabeling of the region containing MSD2 and NBD1, but its removal has less of an effect on labeling of the COOH-proximal site (27Qian 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, 39Gao 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). Consequently, we examined the extent to which binding and photolabeling with [125I]LY475776 was dependent on the presence of both MSD1 and CL3. MRP1 lacking amino acids 1–204 could be efficiently photolabeled (Fig. 1 C, left panel). However, truncation to amino acid 280 essentially eliminated labeling, and the low level of labeling detected was unaffected by GSH (Fig.1 C). As with LTC4, co-expression of MRP1281–1531 with a fragment comprised of the missing NH2-proximal amino acids restored strong GSH-dependent labeling of the larger fragment despite the fact that LY475776 primarily labels the COOH-proximal region of the protein (Fig. 1 C, right panel). Previous studies have shown that some MRP1 substrates that display a dependence on GSH for transport, such as vincristine, recip
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