Glutathione-dependent Binding of a Photoaffinity Analog of Agosterol A to the C-terminal Half of Human Multidrug Resistance Protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD0), which is connected to a P-glycoprotein-like core region (ΔMRP) by a cytoplasmic linker domain zero (L0). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD0 and L0 are not known. We synthesized [125I]11-azidophenyl agosterol A ([125I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C932–1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C4. Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1. MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD0), which is connected to a P-glycoprotein-like core region (ΔMRP) by a cytoplasmic linker domain zero (L0). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD0 and L0 are not known. We synthesized [125I]11-azidophenyl agosterol A ([125I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C932–1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C4. Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1. multidrug resistance P-glycoprotein human multidrug resistance protein transmembrane domain nucleotide-binding domain glutathione vincristine adriamycin etoposide leukotriene C4 17-β-estradiol-17 (β-d-glucuronate) agosterol A 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio} -methyl]thio)propanoic acid 4-oxo-8-[p-(4-phenylbutyl-oxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide N-{4-[1-hydroxy-2-(dibutylamino)ethyl] quinolin-8′-yl}-4-azidosalicylamide N-(hydrocinchonidin-8-yl)-4-azido-2-hydroxybenzamide polymerase chain reaction polyacrylamide gel electrophoresis glutathione S-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid linker domain Following exposure to a natural product chemotherapeutic agent, tumor cells often acquire resistance to several structurally and functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major obstacle to successful cancer chemotherapy. Two membrane proteins, P-glycoprotein (P-gp) and the human multidrug resistance protein (MRP1) are frequently overexpressed in MDR cells (for reviews see Refs. 1Gottesman M.M Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3567) Google Scholar, 2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (510) Google Scholar, 3Deeley R.G. Cole S.P. Semin. Cancer Biol. 1997; 8: 193-204Crossref PubMed Scopus (165) Google Scholar, 4Hipfner D.R. Deeley R.G. Cole S.P. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (380) Google Scholar). Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the family of ATP-binding cassette transporters (5Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3384) Google Scholar), they share only 15% amino acid sequence identity (6Cole S.P. 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). The amino acid sequence suggests that P-gp consists of two homologous halves and a variable linker region. Each half of the protein has six transmembrane segments and one nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence of an extra N-terminal extension with five transmembrane segments (TMD0), which is connected to the P-gp-like core (ΔMRP) by a cytoplasmic linker domain zero (L0) (7Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 8Bakos 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). The precise roles of TMD0 and L0 are unknown. Although MRP1 and P-gp both confer multidrug resistance by actively effluxing drugs from the cells (9Zaman G.J. Flens M.J. van Leusden M.R. de Haas M. Mulder H.S. Lankelma J. Pinedo H.M. Scheper Borst P. A. PubMed Scopus Google Scholar, A. M.M. Pastan I. Cancer Google Scholar), is that they in drug (6Cole S.P. 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, S.P. K. Grant C.E. Deeley R.G. Cancer Google Scholar). P-gp confers multidrug resistance by binding and transporting drugs M.M. M.M. Pastan I. A. PubMed Scopus Google Scholar, A. M. Google Scholar). MRP1, is an of a of with and leukotriene C4 is the for MRP1 Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). It has been that GSH plays a role in MRP1-mediated MDR G.J. Lankelma J. van J. Borst A. PubMed Scopus Google Scholar). of GSH in cells by an of GSH in a in the of anticancer agents, and an in to adriamycin vincristine and etoposide G.J. Lankelma J. van J. Borst A. PubMed Scopus Google Scholar). to MRP1 has been to actively only in the presence of GSH Deeley R.G. Cole S.P. Cancer 1998; Google Scholar). is that and GSH are by MRP1 Deeley R.G. Cole S.P. Cancer 1998; Google Scholar). membrane vesicles MRP1 be by and of was by Although of the that GSH is for MRP1-mediated the role of GSH in binding of drugs by MRP1 is to the M. L. E. Biochem. Biophys. 1997; PubMed Scopus Google Scholar, M. E. Biochem. 1998; PubMed Scopus Google that a photolabeled MRP1 in both cells and membrane and that GSH is not required for the binding of the photoaffinity which has a to that of has been to photolabel of MRP1 in the absence of GSH R. J. L. Scheper P. E. PubMed Scopus Google Scholar). the of GSH is for MRP1-mediated is which in the drug is by the drug binding of are for drug binding on Annu. Rev. Biochem. PubMed Scopus Google Scholar). of and reversing agents, M.M. Pastan I. J. Biol. Chem. 1992; Full Text PDF PubMed Google M.M. M.M. Pastan I. A. PubMed Scopus Google Scholar, M.M. Pastan I. J. Biol. Chem. Full Text PDF PubMed Google A. M. Google Scholar), and M. Pastan I. M.M. Ambudkar A. 1997; PubMed Scopus Google Scholar), been to photolabel P-gp and drug binding Although the resistance by of MRP1 are to of P-gp a photoaffinity study using photoaffinity for P-gp to photolabel MRP1 the for P-gp S.P. Deeley R.G. 1998; PubMed Scopus Google Scholar). I. G. Cole S.P. Deeley R.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, I. G. J. Biochem. PubMed Scopus Google that photolabeled the site of on MRP1 has not been we a agosterol A (AG-A), from a We found that AG-A MDR in human cells MRP1 K. A. M. 1998; Scopus Google Scholar), and that AG-A inhibited the of by MRP1 M. K. K. M. J. Scholar). This suggests that AG-A is a MDR-reversing the we synthesized a photoaffinity analog of [125I]11-azidophenyl agosterol A ([125I]azidoAG-A), and to photolabel MRP1. We found that GSH is required for binding to the half of MRP1. to the site of GSH on MRP1, a of and mutated and of MRP1 to see they are photolabeled by the photoanalog of AG-A in the presence absence of GSH. We found that the C-proximal of MRP1 (C932–1531) is photolabeled by the photoanalog of AG-A in the presence of GSH and GSH interacts with the of MRP1. was from AG-A was from a in of and of in a K. A. M. 1998; Scopus Google Scholar). acid was to the of AG-A with a linker and with and B. M. J.E. Chem. 1997; 8: PubMed Scopus Google Scholar). The product was by with a on was for the and A. M. Biochem. Biophys. PubMed Scopus Google Scholar). and from and cells from drugs and from human cells with MRP1 and cells with an in K. K. 1999; PubMed Scopus Google Scholar, A. K. 1997; PubMed Scopus Google Scholar). cells in vesicles from and cells with various A. M. Biochem. Biophys. PubMed Scopus Google Scholar). vesicles in and by the of M.M. Biochem. PubMed Scopus Google Scholar). the MRP1 region was A. M. Biochem. Biophys. PubMed Scopus Google Scholar). a expressing an was using two a sequence and site and of and The was to in which MRP1 amino was The of the was by the presence of the site and sequence for of the and of MRP1, and of MRP1 by from an MRP1 The for the and A was the The for the and the a was The two a to and The and of the by sequence and from the and was to with to The with the MRP1 was the and in the site of to The from of the two of the and and from a was with and by the two to The of the was by of a The of MRP1 was from the a and the and in site of the of the a in the in the of amino was with the of the of the of MRP1, a sequence a was by using a two and the The with to and in the presence of M. PubMed Scopus Google Scholar). Following by the was and with the in the to to the of MRP1 with the of the was with the of the and The for was a sequence The to two and MRP1 and The a which an site and a for the The for was a the and using the to A. M. Biochem. Biophys. PubMed Scopus Google Scholar). vesicles of for with photoanalog of AG-A with in the absence presence of various agents and of GSH Following of the with a for the in by L. Biol. PubMed Scopus (165) Google and to of to on the and the was with an in of the gel in was and from the of the photolabeled by vesicles in of A and for The and the was for with of of The was with of in A for with and the with A The by and vesicles with to (7Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google with membrane vesicles in to a of and with for The reaction was by the of and and the to and A. M. Biochem. Biophys. PubMed Scopus Google Scholar). The and of MRP1 with amino M.J. Scheper G.J. Cancer Google Scholar, D.R. M. G. Scheper Deeley R.G. Cole S.P. J. 1998; PubMed Scopus Google and M.J. Scheper G.J. Cancer Google D.R. M. G. Scheper Deeley R.G. Cole S.P. J. 1998; PubMed Scopus Google Scholar), of membrane vesicles with was the photolabeling We demonstrated that AG-A MDR in human cells MRP1. AG-A interacts with the binding site for on MRP1 and inhibited the of by MRP1 M. K. K. M. J. Scholar). AG-A to the drug binding site of MRP1, we synthesized the photoaffinity analog of photolabeled the 190-kDa only in the presence of GSH. The was not in the presence of GSH in membrane vesicles from cells that not of MRP1 The photolabeling with of GSH to the 190-kDa was MRP1, photolabeled membrane vesicles from cells with and with a D.R. Deeley R.G. Cole S.P. Cancer Google Scholar). The protein was by the not by that the protein is MRP1 We characterized the of GSH on binding to MRP1. Deeley R.G. Cole S.P. Cancer 1998; Google in to by MRP1. We GSH binding to MRP1. in photolabeling of MRP1 to an to that for GSH. of was and was The role of the of GSH in of the binding was by the of the agents, and and a to for GSH in binding It that the of GSH is not for the to to the of MRP1, studies with a of several and reversing agents and The of and on photolabeling of MRP1 in membrane vesicles from cells is in A. The for agents by of the gel to the on the not and AG-A inhibited the photolabeling in a manner and inhibited by to that of the anticancer agents, and inhibited photolabeling with and and and inhibited the by only and the reversing agents, 4-oxo-8-[p-(4-phenylbutyl-oxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate M. K. K. A. M. Cancer 1998; Scopus Google the and inhibited the acid J. Biochem. Biophys. PubMed Scopus Google Scholar), and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide K. M. A. 1997; Google inhibited the by and the inhibited the photolabeling with (β-d-glucuronate) inhibited the photolabeling by and by that to the site on MRP1. to the drug binding site of AG-A on MRP1, the MRP1 molecule was by and the for binding to vesicles from cells with and with for the and half of MRP1, and M.J. Scheper G.J. Cancer Google Scholar, D.R. M. G. Scheper Deeley R.G. Cole S.P. J. 1998; PubMed Scopus Google Scholar). that both the 190-kDa MRP1 to a was with and a was with Following with the of the and the of was to kDa) and which with and the of the of and with and of that both of from MRP1. are in with studies (7Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google and that the of MRP1 are on the cytoplasmic and L0 of MRP1. and the N-terminal half and the half of MRP1, of and halves of MRP1 by of membrane vesicles with was to to the drug binding on MRP1. vesicles photolabeled with and the for We found that the photoaffinity analog of AG-A MRP1 and a with (7Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, E. Welker E. Tusnady G.E. G.J. Flens M.J. Varadi A. Sarkadi B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), that AG-A photolabeled the half of MRP1. the N-terminal half is photolabeled with of the the of the was from to in the of the of MRP1 and the the of was a photolabeled the the N-terminal We is and not the N-terminal half of MRP1 a of AG-A inhibited the of MRP1 not of A was an to study the binding site of AG-A on MRP1. co-expressed, and mutated in It was was MRP1 in cells with to of MRP1 in and that a be both halves of MRP1 M. Grant C.E. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). that of a in cells with a was with with two M. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). molecule of C-proximal molecule of in a cells with a MRP1, with both and from a with a of of and to the of the in membrane vesicles from the cells. in both and the for the the of the both they from a The by membrane vesicles from cells with a both and was the that with vesicles from cells with a MRP1 not membrane vesicles expressing was only photolabeled by the photoanalog of and the was not by GSH. However, was with the binding of AG-A to was in the presence of the of GSH that GSH AG-A binding to and that is required for AG-A binding to in membrane vesicles from cells of MRP1. membrane vesicles from cells with MRP1 of to and with and of by of by membrane vesicles MRP1 of MRP1 of photolabeled with in the absence presence of the of GSH. to and of vesicles from cells a the in in the to in membrane vesicles both in membrane vesicles both GSH is required for the binding of AG-A to the site of of GSH on MRP1, region of MRP1 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 was with the P-gp-like core region of MRP1, from a in cells. The of and by with the MRP1 and and was a by not by was a of by not by was using membrane vesicles expressing both and The in the membrane vesicles expressing both and was to that in membrane vesicles expressing MRP1, was in membrane vesicles expressing not are in with 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, M. M. Grant C.E. Cole S.P. Deeley R.G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). vesicles expressing both and photolabeled with in the absence presence of the of GSH. membrane vesicles both and the was photolabeled by AG-A in the absence of and the was the of GSH was to the absence of in membrane vesicles expressing was photolabeled by that in membrane vesicles both and However, was not by GSH to the binding site of AG-A on which the and of MRP1, was with using a A was to the of core for the of the in was a of by the a by the binding of to both halves of the core region of MRP1. studies using and that the binding of AG-A to the core region of MRP1 was not inhibited by Based on the that of the the binding of AG-A to MRP1, that the GSH site on MRP1 is on the region. GSH is the GSH binding site on MRP1 be in a L0 is the site of we a a the transmembrane domain in membrane vesicles with both and and the to that of MRP1. transporting was in MRP1 not which is in with a 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). was in the absence presence of of GSH. GSH the binding of AG-A to in a with the that is required for binding of AG-A to MRP1, that L0 is the site of GSH on MRP1. are a family of that GSH to a of that an of Scholar). are of two of which one binding site for GSH and a binding site for substrates the amino acid sequence to a Curr. Chem. 1999; 6: Google Scholar). The of the human which has been in is by an and important with the of the of GSH P. R. R. M. G. J. Biol. 1992; PubMed Scopus Google Scholar, A.J. A. G. J. J. Biol. 1997; PubMed Scopus Google Scholar). We the of MRP1 for the presence of that with GSH. are in the L0 region of MRP1, only two of are by of the region that only and in a for GSH This was by and with and and the mutated L0 region in binding The mutated L0 region was to the of to in which has a of to that of with both and was using membrane vesicles in the absence presence of of GSH. and that the binding of AG-A to was only in the presence of GSH with a in the binding of AG-A to This suggests that the L0 region of MRP1 of the GSH binding is that P-gp mediates MDR by binding and transporting natural chemotherapeutic agents in an manner M.M. M.M. Pastan I. A. PubMed Scopus Google A. M. Google Scholar). It has been that MRP1 is an which agents, only in the presence of GSH Deeley R.G. Cole S.P. Cancer 1998; Google Scholar, G. I. K. Kurz G. Cancer 1996; Google Scholar). However, the role of GSH in drug is an was that MRP1 confers resistance by drugs with GSH S.P. Deeley R.G. 1998; PubMed Scopus Google Scholar). However, is of drugs, and not found in the of cells to agents G.J. Lankelma J. van J. Borst A. PubMed Scopus Google Scholar). and that the GSH are to in that the in of the cells from the role of GSH are that GSH is required an binding a D.R. Deeley R.G. Cole S.P. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (380) Google Scholar). to we synthesized a photoaffinity analog of AG-A that MRP1-mediated and demonstrated that GSH is required for the binding of AG-A to MRP1. Deeley R.G. Cole S.P. Cancer 1998; Google found in to not and by MRP1, that the of drug the GSH is not on a The of GSH was not for the binding of AG-A to MRP1, AG-A binding to MRP1 with the GSH and a of agents not for GSH in binding It has been inhibited with of that GSH is a of by membrane vesicles Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We found not AG-A binding to MRP1. the inhibited the AG-A binding to MRP1 in a manner not This to the GSH binding site on MRP1 and inhibited the of GSH with the that GSH be required for the binding of to the drug binding site of MRP1. MRP1 and P-gp to the ATP-binding cassette However, MRP1 is characterized by an extra amino acid which is connected to the P-gp-like core region by a linker region The precise of two MRP1 are not known. the the was truncated, AG-A to both halves of the core and GSH was not required for However, L0 was the P-gp-like core binding was of two amino in L0 the binding of AG-A to that L0 is the site of GSH on MRP1. the of a study suggested that TMD0 is not required for by MRP1 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). TMD0 was the core region of MRP1, AG-A not to the in the presence of GSH not The photolabeling study suggested that TMD0 is not required for and drug binding to MRP1. The drug binding on MRP1 is unknown. studies using photoaffinity that P-gp two within the transmembrane segments and in N-terminal and in one drug two drug binding M. Pastan I. M.M. Ambudkar A. 1997; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the site of on MRP1 is within is required for the was not photolabeled with the photoanalog of It is not AG-A photolabeled MRP1 in the absence of and was not inhibited by and a from that of MRP1. a drug binding be on It has been demonstrated that confers MDR in human cells Grant C.E. D.R. Cole S.P. Deeley R.G. 1997; PubMed Scopus Google Scholar). However, the protein not confer resistance to studies with demonstrated that amino which are in the half of MRP1, are for resistance Cole Deeley R.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). that the AG-A site on MRP1 is in is with that the site of AG-A binding on MRP1 is to the binding of and It has been that MRP1 is an with one of the MRP1 substrates to Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). with the the agents we in cells and to and not to M. K. K. M. J. Scholar, S.P. K. Grant C.E. Deeley R.G. Cancer Google Scholar). The study that and inhibited the photolabeling of MRP1 by not We demonstrated that AG-A the resistance to and of cells and found that a with a from MDR reversing agents, MRP1-mediated MDR by two was by with MRP1 and the by the of GSH M. K. K. M. J. Scholar). the photolabeling the of was that of and resistance to in was M. K. K. M. J. Scholar). AG-A resistance to by the of GSH. of not be a for MRP1, one drug binding site on MRP1, and the binding site of on MRP1 be from that of studies that MRP1 is photolabeled by I. G. Cole S.P. Deeley R.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, I. G. J. Biochem. PubMed Scopus Google Scholar). However, is to the role of GSH in drug binding using is a GSH drugs been to to MRP1 in the absence of GSH M. L. E. Biochem. Biophys. 1997; PubMed Scopus Google Scholar, M. E. Biochem. 1998; PubMed Scopus Google Scholar, R. J. L. Scheper P. E. PubMed Scopus Google Scholar). demonstrated that cells are to drugs the and that drugs are substrates of MRP1 on the of the of and drug R. J. L. Scheper P. E. PubMed Scopus Google that photolabeled in the and of MRP1 in the absence of GSH. We found that AG-A to the half of MRP1 only in the presence of and that AG-A to both halves of in the absence of GSH. The binding of AG-A to the half of MRP1, not the binding of AG-A to was inhibited by and we that the binding of AG-A to the P-gp-like core region of MRP1, differs from the drug binding site on MRP1. The the drugs photolabel MRP1 in the absence of GSH is unknown. The photoanalog of AG-A is from the drugs in for GSH for binding to the drug binding of MRP1. drugs to of MRP1 the drug binding site in the and that to be by MRP1. AG-A is a for MRP1, we the of the photoanalog of AG-A in and cells in the The of the photoanalog in cells was to that in that to MRP1 was not by The of to the drug binding site of MRP1 be for to be Borst P. Evers R. M. J. Biochim. Biophys. Acta. 1999; 1461: PubMed Scopus Google a for with two drug binding that one site a for GSH and a for drug and the site a for drug and a for GSH that the and the of are in the L0 and half of MRP1, GSH to L0 and a Scheper J. PubMed Scopus Google Scholar), which the binding of AG-A to MRP1. and with not GSH to binding and by MRP1. Borst P. Evers R. M. J. Biochim. Biophys. Acta. 1999; 1461: PubMed Scopus Google suggested that a for both the and and are in the absence of GSH. and the that mutated MRP1 the TMD0 L0 a MRP1 both TMD0 and L0 The role of L0 in has been We that the of transporting in membrane vesicles the P-gp-like core region of MRP1 is to the of the GSH binding of the and of and to the for the of drugs by MRP1. The agents that with GSH for binding to L0 MRP1-mediated drug that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1. We Borst Cancer for the MRP1 P. Cole for the for for cells and of and of for and useful and for the We for and and for

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