Binding of a photoaffinity analogue of glutathione to MRP1 (ABCC1) within two cytoplasmic regions (L0 and L1) as well as transmembrane domains 10-11 and 16-17

MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural products as well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH is required for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating the influence of GSH on MRP1 is poorly understood, and the location of GSH binding site(s) within MRP1 have yet to be determined. To address these issues, we have synthesized a [(125)I] labeled azido-derivative of GSH (IAAGSH) to photoaffinity label MRP1. Our results revealed that IAAGSH labeled MRP1 with high specificity, and binding was inhibited by MRP1 substrates leukotriene C(4) and MK571. Interestingly, verapamil and vincristine enhanced IAAGSH photolabeling of MRP1, in agreement with observations that both drugs enhance GSH transport. We observed GSH to be the best inhibitor of photoaffinity labeling, as compared to oxidized glutathione (GSSG) and four different GSH alkyl derivatives. These observations indicate that IAAGSH interacted with MRP1 in a similar manner as unmodified GSH. Moreover, using eight MRP1-HA variants, each containing hemagglutinin A (HA) epitopes inserted at different sites in MRP1, we mapped the GSH binding sites in MRP1. Our GSH analogue photoaffinity labeled four MRP1 polypeptides that were located within two cytoplasmic domains in linker sequences (L0 and L1) as well as transmembrane domains 10-11 and 16-17. The photoaffinity labeling of polypeptides within L0 and L1 domains is further confirmed using two MRP1-specific monoclonal antibodies (MRPr1 and QCRL1) with epitopes within the linker domains. Taken together, this study provides the most precise information to date on the location of GSH binding sites in MRP1.     

Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves 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 [(3)H]leukotriene C(4) (LTC(4)), 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) LTC(4) labels sites in the NH(2)- and COOH-proximal halves of MRP1, (ii) labeling of the NH(2)-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 NH(2)-proximal site is preferentially inhibited by S-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 LTC(4) is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC(4) binding to the NH(2)-proximal site, and (vii) the attenuation of LTC(4) binding to the NH(2)-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 LTC(4) binds to one of two sites composed, at least in part, of elements in the NH(2)-proximal half of the protein.     

Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1)

The mechanisms of MRP1-drug binding and transport are not clear. In this study, we have characterized the interaction between MRP1 and rhodamine 123 (Rh123) using the photoreactive-iodinated analogue, [(125)I]iodoaryl azido-rhodamine 123 (or IAARh123). Photoaffinity labeling of plasma membranes from HeLa cells transfected with MRP1 cDNA (HeLa-MRP1) with IAARh123 shows the photolabeling of a 190 kDa polypeptide not labeled in HeLa cells transfected with the vector alone. Immunoprecipitation of a 190 kDa photolabeled protein with MRP1-sepcific monoclonal antibodies (QCRL-1, MRPr1, and MRPm6) confirmed the identity of this protein as MRP1. Analysis of MRP1-IAARh123 interactions showed that photolabeling of membranes from HeLa-MRP1 with increasing concentrations of IAARh123 was saturable, and was inhibited with excess of IAARh123. Furthermore, the photoaffinity labeling of MRP1 with IAARh123 was greatly reduced in the presence of excess Leukotreine C(4) or MK571, but to a lesser extent with excess doxorubicin, colchicine or chloroquine. Cell growth assays showed 5-fold and 14-fold increase in the IC(50) of HeLa-MRP1 to Rh123 and the Etoposide VP16 relative to HeLa cells, respectively. Analysis of Rh123 fluorescence in HeLa and HeLa-MRP1 cells with or without ATP suggests that cross-resistance to Rh123 is in part due to reduced drug accumulation in the cytosol of HeLa-MRP1 cells. Mild digestion of purified IAARh123-photolabeled MRP1 with trypsin showed two large polypeptides (approximately 111 and approximately 85 kDa) resulting from cleavage in the linker domain (L1) connecting the multiple-spanning domains MSD0 and MSD1 to MSD2. Exhaustive proteolysis of purified IAARh123-labeled 85 and 111 kDa polypeptides revealed one (6 kDa) and two (approximately 6 plus 4 kDa) photolabeled peptides, respectively. Resolution of total tryptic digest of IAARh123-labeled MRP1 by HPLC showed three radiolabeled peaks consistent with the three Staphylococcus aureus V8 cleaved peptides from the Cleveland maps. Together, the results of this study show direct binding of IAARh123 to three sites that localize to the N- and C-domains of MRP1. Moreover, IAARh123 provides a sensitive and specific probe to study MRP1-drug interactions.     

Identification of proline residues in the core cytoplasmic and transmembrane regions of multidrug resistance protein 1 (MRP1/ABCC1) important for transport function, substrate specificity, and nucleotide interactions

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro(343), TM8-Pro(448), TM10-Pro(557), and TM11-Pro(595)) and MSD3 (TM14-Pro(1088)) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro(1150) in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17beta-estradiol-17-beta-(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane.     

Functional reconstitution of substrate transport by purified multidrug resistance protein MRP1 (ABCC1) in phospholipid vesicles

The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C(4) (LTC(4)). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69-82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC(4) uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC(4) uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17 beta-estradiol 17-beta-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K(m) for ATP and LTC(4) were 357 +/- 184 microm and 366 +/- 38 nm, respectively, and 2.14 +/- 0.75 microm for 17 beta-estradiol 17-beta-(d-glucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.     

Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein,MRP1 (ABCC1): effects on protein expression,membrane localization,and transport function

The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

A positively charged amino acid proximal to the C-terminus of TM17 of MRP1 is indispensable for GSH-dependent binding of substrates and for transport of LTC4

MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.     

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.     

Identification of the structural and functional boundaries of the multidrug resistance protein 1 cytoplasmic loop 3

Multidrug resistance protein (MRP) 1 is a member of the ABCC branch of the ATP binding cassette (ABC) transporter superfamily that can confer resistance to natural product chemotherapeutic drugs and transport a variety of conjugated organic anions, as well as some unconjugated compounds in a glutathione- (GSH-) dependent manner. In addition to the two tandemly repeated polytopic membrane-spanning domains (MSDs) typical of ABC transporters, MRP1 and its homologues MRP2, -3, -6, and -7 contain a third NH(2)-terminal MSD. The cytoplasmic loop (CL3) connecting this MSD, but apparently not the MSD itself, is required for MRP1 leukotriene C(4) (LTC(4)) transport activity, substrate binding and appropriate trafficking of the protein to the basolateral membrane. We have used a baculovirus dual-expression system to produce various functionally complementing fragments of MRP1 in insect Sf21 cells to precisely define the region in CL3 that is required for activity and substrate binding. Using a parallel approach in polarized MDCK-I cells, we have also defined the region of CL3 that is required for basolateral trafficking. The CL3 NH(2)- and COOH-proximal functional boundaries have been identified as Cys(208) and Asn(260), respectively. Cys(208) also corresponds to the NH(2)-proximal boundary of the region required for basolateral trafficking in MDCK-I cells. However, additional residues downstream of the CL3 COOH-proximal functional boundary extending to Lys(270) were found to be important for basolateral localization. Finally, we show that regions in CL3 necessary for LTC(4) binding and transport are also required for binding of the photoactivatable GSH derivative azidophenacyl-GSH.     

Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity

The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1.     

Photoaffinity labeling of the multidrug resistance protein 2 (ABCC2/cMOAT) with a photoreactive analog of LTC4

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC₄); however direct binding of the LTC₄ to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC₄ (IAALTC₄) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC₄ in plasma membranes from MDCKII^MRP2 cells. The photoaffinity labeling signal of MRP2 with IAALTC₄ was much lower than that of MRP1, consistent with previous studies whereby the measured K_m values of MRP1 and MRP2 for LTC₄ were 1 μM and 0.1 μM LTC₄, respectively. Competition of IAALTC₄ photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC₄ enhanced the photoaffinity labeling of IAALTC₄ to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC₄ binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC₄ binding to MRP2.     

Identification of domains participating in the substrate specificity and subcellular localization of the multidrug resistance proteins MRP1 and MRP2

The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C4 (LTC4) (Km = 0.1 and 1 microm). To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells. We examined the kinetic properties of each chimeric protein by measuring LTC4 and methotrexate transport in inside-out membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods. The following results were determined in these studies: (i) when the NH2-proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, Km(LTC4) values of the chimera decreased approximately 4-fold and Km(methotrexate) values increased approximately 5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased approximately 3-fold; (ii) when the NH2-proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins. These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC4 and anticancer agents.     

Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.     

Major photoaffinity drug binding sites in multidrug resistance protein 1 (MRP1) are within transmembrane domains 10-11 and 16-17

MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown to confer resistance to structurally dissimilar drugs. Studies of MRP1 topology suggested the presence of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (L0). MRP1-mediated multidrug resistance is thought to be due to enhanced drug efflux. However, little is known about MRP1-drug interaction and its drug binding site(s). We previously developed several photoreactive probes to study MRP1-drug interactions. In this report, we have used eight MRP1-HA variants that were modified to have hemagglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exhaustive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA variants revealed the same profile of photolabeled peptides as seen for wild type MRP1. Photolabeling of the different MRP1-HA variants followed by digestion with increasing concentrations of trypsin or Staphylococcus aureus V8 protease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identified two small photolabeled peptides ( approximately 6-7 kDa) from MRP1-HA(574) and MRP1-HA(1222). Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 (Ser(542)-Arg(593)) and transmembranes 16 and 17 of MSD2 (Cys(1205)-Glu(1253)). Interestingly, the same sequences in MRP1 were also photolabeled with a structurally different photoreactive drug, IACI, confirming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug binding. Taken together, the results in this study provide the first delineation of the drug binding site(s) of MRP1. Furthermore, our findings suggest the presence of common drug binding site(s) for structurally dissimilar drugs.     

Structural and functional consequences of mutating cysteine residues in the amino terminus of human multidrug resistance-associated protein 1

Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette membrane transport superfamily and is responsible for multidrug resistance in cancer cells. Currently, there are nine known human MRPs. Distinct from many other members of the ATP-binding cassette superfamily, human MRP1 and four other MRPs have an additional membrane-spanning domain (MSD) with a putative extracellular amino terminus. The functional significance of this additional MSD (MSD1) is currently unknown. To understand the role of MSD1 in human MRP1 structure and function, we studied the amino-terminal 33 amino acids. We found that the amino terminus of human MRP1 has two cysteine residues (Cys(7) and Cys(32)) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys(7) residue is critical for the MRP1-mediated drug resistance and leukotriene C(4) transport activity. On the other hand, mutation of Cys(32) reduced only moderately the MRP1 function. The effect of Cys(7) mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate V(max). We also found that mutation of Cys(7) changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in V(max) of LTC(4) transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys(7) residue plays a critical role in maintaining the proper structure and function of human MRP1.     

Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1

Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.     

GSH-dependent photolabeling of multidrug resistance protein MRP1 (ABCC1) by [125I]LY475776. Evidence of a major binding site in the COOH-proximal membrane spanning domain

Substrates transported by the 190-kDa multidrug resistance protein 1 (MRP1) (ABCC1) include endogenous organic anions such as the cysteinyl leukotriene C(4). In addition, MRP1 confers resistance against various anticancer drugs by reducing intracellular accumulation by co-export of drug with reduced GSH. We have examined the properties of LY475776, an intrinsically photoactivable MRP1-specific tricyclic isoxazole modulator that inhibits leukotriene C(4) transport by this protein in a GSH-dependent manner. We show that [125I]LY475776 photolabeling of MRP1 requires GSH but is also supported by several non-reducing GSH derivatives and peptide analogs. Limited proteolysis revealed that [(125)I]LY475776 labeling was confined to the 75-kDa COOH-proximal half of MRP1. More extensive proteolysis generated two major 125I-labeled fragments of approximately 56 and approximately 41 kDa, and immunoblotting with regionally directed antibodies showed that these fragments correspond to amino acids approximately 1045-1531 and approximately 1150-1531, respectively. However, an approximately 33-kDa COOH-terminal immunoreactive fragment was not labeled, inferring that the major [125I]LY475776-labeling site resides approximately between amino acids 1150-1250. This region encompasses transmembrane (TM) segments 16 and 17 at the COOH-proximal end of the third membrane spanning domain of the protein. [125I]LY475776 labeling of mutant MRP1 molecules with substitutions of Trp(1246) in TM17 were reduced >80% compared with wild-type MRP1, confirming that TM17 is important for LY475776 binding. Finally, vanadate-induced trapping of ADP inhibited [125I]LY475776 labeling, suggesting that ATP hydrolysis causes a conformational change in MRP1 that reduces the affinity of the protein for this inhibitor.     

Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein,MRP1

The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NBDs) are configured MSD1-MSD2-NBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C(4) and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17beta-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C(4) transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TM8), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the Trp(445), Trp(553), and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp(553)). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1.     

Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.