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Biochemistry and Biophysics |
Medical Sciences Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [H. Z., G. D. K.], and the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 [G. L., P. A. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-sulfatolithocholyltaurine (7, 8, 9, 10, 11)
. Another anion transporter related to MRP1, cMOAT (or MRP2), has since been isolated (12, 13, 14) and demonstrated in experiments using the cloned human, rat, and rabbit cDNAs to share the biochemical substrate selectivity of MRP1 with regard to glutathione and glucuronate conjugates (15, 16, 17, 18, 19, 20) . cMOAT, however, differs from MRP1 in its tissue distribution. Whereas MRP1 is expressed in a broad range of tissues (21) , cMOAT is primarily expressed in liver canalicular membranes, where it functions as an apical anion efflux pump (22) . In mutant TR- and Eisai hyperbilirubinemic rats and in humans with Dubin-Johnson syndrome, hereditary defects in cMOAT are characterized by mild elevations in conjugated bilirubin and the inability to excrete certain exogenous organic anions (13 , 14 , 23, 24, 25) . Transfection studies indicate that cMOAT is capable of conferring resistance to anticancer agents. However, its drug resistance profile is distinct from that of MRP1 (19 , 20 , 26) .
As first suspected from the results of biochemical investigations (27) and confirmed by analyses of expressed sequence tags and other partial sequences, it is now clear that there are several human MRP subfamily members in addition to MRP1 and cMOAT (28, 29, 30) . At the time of writing, the complete coding sequences and putative topologies have been determined for four MRP subfamily members from humans: MRP3 (MOAT-D, cMOAT2); MRP4 (MOAT-B); MRP5 (MOAT-C, SMRP); and MRP6 (MOAT-E, ARA; Refs. 31, 32, 33, 34, 35, 36, 37 ), and two from rat: MRP3 and MRP6 (38) . Of these, MRP3 bears the closest resemblance to MRP1 in terms of overall sequence alignment (58% overall amino acid identity; 71 and 74% identity in its first and second nucleotide binding folds, respectively) and its possession of the third (NH2-terminal) membrane-spanning domain also found in cMOAT and MRP6 but not in MRP4 and MRP5 (33) .
The functional characteristics of human MRP3 have yet to be defined in any detail. The ability of MRP3 to confer resistance to certain natural product drugs and other anticancer agents has been assessed to some degree in transfected cell lines (39 , 40) , but nothing is known of its in vitro transport properties, substrate selectivity profile, or physiological functions. In the report that follows, we address these questions by the analysis of MRP3-mediated transport in membrane vesicles prepared from transfected HEK293 cells. In so doing, we demonstrate that MRP3 not only catalyzes the MgATP-energized transport of glutathione and glucuronate conjugates but also the high-capacity transport of the antimetabolite methotrexate and the bile acid glycocholate. In comparison to MRP1, MRP3 is a low-affinity glutathione and glucuronate conjugate transporter. However, unlike MRP1 and any other characterized mammalian ABC transporter, MRP3 has the facility for transporting glycocholate, a major constituent of human bile.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoblot Analysis.
Membrane proteins were separated by SDS-PAGE (6% acrylamide) as
described (42)
. Proteins were transferred to
nitrocellulose filters using a wet transfer system (43)
,
and MRP3 was detected using polyclonal MRP3 (1:1000) antibody and
alkaline phosphatase-conjugated secondary antibody.
Preparation of Membrane Vesicles and Transport Experiments.
Membrane vesicles were prepared as described (8)
and
stored in liquid nitrogen. Transport experiments were performed using
the rapid filtration method, as described (8)
, with
modifications. Briefly, 100 µl of reaction buffer [10 mM
Tris-HCl (pH 7.4), 250 mM sucrose, 4 mM ATP or
AMP, 10 mM MgCl2, 10 mM
creatine phosphate, and 100 µg/ml creatine kinase] containing
radiolabeled compounds, with or without unlabeled carrier, was
preincubated at 37°C for 1 min and then rapidly mixed with 5–10 µl
of membrane vesicle suspension (30 µg protein). At the times
indicated, 20-µl aliquots were removed and added to 1 ml of ice-cold
washing buffer [10 mM Tris-HCl (pH 7.4), 250
mM sucrose] and filtered through 0.22-µm pore size GVWP
polyvinylidene fluoride filters (Millipore, Bedford, MA). The filters
were washed once with 5 ml of ice-cold washing buffer, and
radioactivity was measured by liquid scintillation counting. Membrane
protein was estimated by a modification of the method of Lowry et
al. (see Ref. 44
).
Data Analysis.
Kinetic parameters were computed by nonlinear least squares analysis
(45)
using the Ultrafit computer software package
(BioSoft, Ferguson, MO).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoblot analysis of membranes prepared from HEK/MRP3–5 cells
revealed a broad, intensely immunoreactive
Mr 171,000–192,000 band after
reaction with polyclonal MRP3 antibody, which was absent from cells
transfected with empty vector (Fig. 1)
. The higher Mr species in HEK/MRP3–5
membranes was inferred to be a more heavily glycosylated form of the
lower Mr species because the latter
comigrated with recombinant MRP3 expressed in insect (Sf9) cells (Fig. 1)
, which are known to be only partially competent in protein
glycosylation.
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Although all three of these conjugates were subject to
MgATP-stimulated, time-dependent transport by MRP3,
E217ßG was transported at the highest rate
under the conditions used. Whereas little or no
E217ßG uptake into HEK/MRP3–5 vesicles in
media containing MgAMP instead of MgATP or into HEK/pcDNA3 vesicles in
media containing either MgATP or MgAMP was detected, initial rates of
MgATP-dependent uptake of 
3 pmol/mg/min were obtained with
HEK/MRP3–5 vesicles incubated in 380 nM
[3H]E217ßG (Fig. 2A)
. By comparison, the rates and extents of MRP3-specific,
ATP-dependent [3H]DNP-SG and
[3H]LTC4 uptake were
moderate. Although an uptake increment consequent on MRP3 was evident
for both [3H]DNP-SG and
[3H]LTC4, the background
rates of uptake of these compounds were appreciable (Fig. 2, B and C)
. At an initial concentration of 1.6
µM, DNP-SG was transported at 2.1 and 1.2
pmol/mg/min by HEK/MRP3–5 membranes and at 1.5 and 1.4 pmol/mg/min by
HEKpcDNA3 membranes in media containing MgATP and MgAMP, respectively
(Fig. 2B)
. The corresponding values for the uptake of 20
nM LTC4 were 0.20, 0.08,
0.12 and 0.08 pmol/mg/min (Fig. 2C)
.
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, the rate of MgATP-dependent uptake into HEK/MRP3–5 membrane vesicles
increased as a linear function of the reciprocal of the osmoticum
(sucrose) concentration of the uptake medium, suggesting that the
vesicles were osmotically responsive and that the transported species
was delivered into an osmotically active compartment. By contrast,
osmoticum exerted only a modest effect on substrate retention measured
in media containing MgAMP, suggesting that an appreciable fraction of
the apparent uptake measured under non-energized conditions
represented binding to the membrane vesicles and/or membrane filters,
not genuine uptake into the vesicles (Fig. 3)
. The magnitude of the
ordinate intercepts indicated that substrate binding constituted no
more than 10–15% of the radioactivity retained by HEK/MRP3–5
membranes in media containing MgATP but as much as 50% of the
radioactivity retained by the same membranes in media containing MgAMP
(Fig. 3)
.
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. Nonlinear least squares fitting of the uptake data to the
Michaelis-Menten equation (45)
yielded
Km and
Vmax values of 25.6 ± 5.4 µM and 75.6 ± 5.9
pmol/mg/min for
[3H]E217ßG,
5.7 ± 1.7 µM and 3.8 ± 0.1 pmol/mg/min for [3H]DNP-SG, and
5.3 ± 2.6 µM and 20.2 ± 5.9 pmol/mg/min for
[3H]LTC4 (Table 1)
. The efficiencies
(Vmax:Km
ratios) of MgATP-dependent uptake therefore fell in the rank order
LTC4 (3.8) > E217ßG (3.0) >> DNP-SG (0.7; Table 1
). The
transport measured was largely attributable to MRP3 in that membrane
vesicles prepared from HEK/pcDNA3 cells catalyzed negligible
MgATP-dependent uptake of E217ßG, DNP-SG, or
LTC4 at all of the concentrations examined (Fig. 4)
.
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From these experiments, it was determined that MRP3 is not only
competent in the transport of E217ßG, DNP-SG,
and LTC4 but also competent in the transport of
methotrexate and glycocholate. [3H]Methotrexate
(0.5 µM) and [14C]glycocholate
(50 µM) were transported by HEK/MRP3–5 membrane vesicles
at 0.26 pmol/mg/min and 110 pmol/mg/min, respectively, over the first 5
min of the assays, whereas the corresponding rates for HEK/pcDNA3
membranes were 0.12 and 46 pmol/mg/min (Fig. 5, A and B)
. In strict contrast to glycocholate,
the other bile acid examined, [3H]taurocholate
(50 µM) showed little or no evidence of
MgATP-dependent transport by either HEK/MRP3–5 or HEKpcDNA3 membranes
(Fig. 5C)
.
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. The Km and
Vmax values for
[3H]methotrexate and
[14C]glycocholate uptake derived from analyses
of the concentration dependence data (Fig. 6)
were 776 ± 319 µM and 288 ± 54
pmol/mg/min and 248 ± 113 µM
and 183 ± 34 pmol/mg/min, respectively (Table 1)
.
Because the high capacity of MRP3 for the transport of methotrexate and
glycocholate was more than sufficient to offset the low apparent
affinity for uptake of these two compounds, both were transported at
catalytic efficiencies (0.4 and 0.7, respectively) comparable with that
of DNP-SG (Table 1)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although these results indicate that the substrate range of MRP3 is
similar to that of MRP1 and cMOAT, there are significant differences in
the kinetic properties of these transporters (Table 2)
. The cysteinyl leukotriene LTC4 is a
high-affinity substrate of human MRP1
(Km = 0.097
µM; Ref. 8
) and an intermediate
affinity substrate of human cMOAT
(Km = 0.24–1.0
µM; Refs. 19
, 20
) but a markedly
lower affinity substrate of MRP3
(Km = 5.3
µM). Likewise, the
Km for E217ßG
transport by MRP3, 25.6 µM, is considerably
higher than the values of 1.5 and 2.5 µM
reported for MRP1 (10
, 11)
and 7.2
µM reported for cMOAT (19)
. The
smallest difference determined was for DNP-SG, the
Km of which for MRP3-mediated
transport, 5.7 µM, is only slightly higher than
the value of 3.6 µM reported for MRP1 (Ref.
10
; the kinetic parameters of DNP-SG transport have not
been reported for cMOAT).
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The facility with which MRP1 transports amphipathic anions is probably relevant to resistance to natural product drugs, although these compounds are not known to form covalent conjugates within the cell. The results of cellular transport studies using MRP1 deficient-ES cells and in vitro transport measurements indicate that the natural products etoposide and vincristine are cotransported with free glutathione (50 , 51) . Analogous experiments on cMOAT imply a similar mechanism for the transport of vinblastine by this transporter (52) . It has therefore been proposed that MRP1, and by implication cMOAT, has a bipartite substrate-binding pocket consisting of a binding site for glutathione or other bulky organic anions and another binding site for hydrophobic molecules. There are currently no data concerning the biochemical mechanism whereby MRP3 confers resistance to natural product agents. However, if by analogy with MRP1 and cMOAT, MRP3 cotransports natural products with glutathione, its lower affinity for glutathione conjugates might signify a diminished capacity for binding the free glutathione required for transport of hydrophobic drugs.
The substrate selectivity we describe for human MRP3 is different from that described for rat MRP3 (79.6% identity). In transport studies using membrane vesicles prepared from two different rat MRP3-transfected cell lines, uptake of E217ßG and methotrexate was detected, but transport of the glutathione S-conjugates LTC4 and DNP-SG was not (53) . This may simply reflect the fact that there was appreciable uptake of LTC4 and DNP-SG in the control vesicles used for the studies of rat MRP3 that obscured the contribution made by MRP3. Alternatively, these results may denote a genuine difference in substrate selectivity and physiological functions between the rat and human transporters. Consistent with the latter possibility, the reported localization of rat MRP3 in liver differs from that of the human transporter. Rat MRP3 was reported to be localized to canalicular membranes in the liver (54) , whereas human MRP3 has been reported to be localized in basolateral hepatocyte membranes in one study and in basolateral membranes of cholangiocytes with lesser levels in basolateral membranes of hepatocytes surrounding portal tracts in another (40 , 55) . Although the unexpected difference in the localizations of the human and rat proteins warrants further investigation, particularly in view of the potential for immunological cross-reactivity between closely related members of the MRP subfamily, the data currently available suggest that the functions of human and rodent MRP3 are not exactly equivalent.
The observation that human MRP3 transports glycocholate at high rates, albeit at low affinity, is an intriguing property of this transporter. Sister of Pgp (also known as the bile salt export pump), which localizes to canalicular membranes of hepatocytes, is the only other mammalian ABC transporter that has been shown to transport monovalent bile acids (56 , 57) . Contrary to human MRP3, which transports glycocholate but not taurocholate, experiments using cloned rat sister of Pgp expressed in Sf9 cells indicate that it transports taurocholate and several unconjugated bile acids but not glycocholate (57) . Moreover, although our experiments show that MRP3 transports glycocholate, the basolateral localization of human MRP3 suggests that it cannot function to transport this compound into the bile. Instead, human MRP3 likely assumes other functions in the liver.
In cholestatic conditions, elevated levels of bile acids appear in the blood. However, the mechanism by which bile acids are transported from hepatocytes into sinusoidal blood is unknown. We therefore suggest that MRP3 may function to transport glycocholate and possibly other bile acids (but not taurocholate in the case of human MRP3) from hepatocytes into the blood under these conditions. Although the localization of rat MRP3 protein requires further investigation, the observation that elevations in its levels occur as a consequence of cholestasis induced by bile duct ligation (38 , 54) adds support to this possibility. That elevated levels of MRP3 expression have been detected in patients that have Dubin-Johnson syndrome and are therefore deficient in cMOAT (55) , as well as in rat strains deficient in cMOAT (38 , 54) , is similarly consistent with the notion that MRP3 may function as a basolateral compensatory mechanism to eliminate from hepatocytes potentially toxic compounds that are ordinarily excreted into bile. We speculate that in Dubin-Johnson syndrome and other cMOAT-deficient conditions, MRP3 functions to transport into sinusoidal blood the glucuronate and glutathione conjugates that are normally exported across bile canaliculi by cMOAT (cMOAT does not transport monovalent bile acids). In the context of the enterohepatic circulation of bile acids, it is also pertinent to note that in addition to liver, high levels of MRP3 transcript are found in small intestine (and several other tissues; Refs. 30 , 32 , 36, and 38 ). Bile acids taken up at the apical surface of enterocytes of the small intestine via the ileal bile salt transporter (58) must then be transported across the basolateral membrane into portal blood. However, a transporter capable of performing this function has yet to be identified. The possibility that MRP3 subserves this function is particularly attractive. These and other questions concerning the physiological roles of MRP3 and its significance for clinical drug resistance are important issues that remain to be resolved.
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FOOTNOTES |
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1 This work was supported by NIH Grant CA73728 and
American Leukemia Society Grant 6351 (to G. D. K.) and by an
appropriation from the Commonwealth of Pennsylvania. This work was
partially supported by United States Department of Agriculture National
Research Initiative Competitive Grants Program Grant
99-35304-8094 (to P. A. R.). 
2 These authors contributed equally.
3 To whom requests for reprints should be
addressed, at Department of Medical Oncology, Fox Chase Cancer Center,
7701 Burholme Avenue, Philadelphia, PA 19111. Phone: (215) 728-5317;
Fax (215) 728-3603; Email: GD_Kruh{at}fccc.edu
4 The abbreviations used: Pgp, P-glycoprotein; ABC
transporter, ATP-binding cassette transporter; cMOAT, canalicular
multispecific organic anion transporter; DNP-SG, 2,4-dinitrophenyl
S-glutathione; E217ßG, estradiol
17-ß-D-glucuronide; HEK293, human embryonic kidney 293;
LTC4, leukotriene C4; MRP, multidrug
resistance-associated protein.
Received 1/19/00. Accepted 7/ 5/00.
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 M. E. Mendoza, M. J. Monte, M. A. Serrano, M. Pastor-Anglada, B. Stieger, P. J. Meier, M. Medarde, and J. J. G. Marin Physiological characteristics of allo-cholic acid J. Lipid Res., January 1, 2003; 44(1): 84 - 92. [Abstract] [Full Text] [PDF]
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K. Koike, C. J. Oleschuk, A. Haimeur, S. L. Olsen, R. G. Deeley, and S. P. C. Cole Multiple Membrane-associated Tryptophan Residues Contribute to the Transport Activity and Substrate Specificity of the Human Multidrug Resistance Protein, MRP1 J. Biol. Chem., December 13, 2002; 277(51): 49495 - 49503. [Abstract] [Full Text] [PDF]
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M. G. Belinsky, Z.-S. Chen, I. Shchaveleva, H. Zeng, and G. D. Kruh Characterization of the Drug Resistance and Transport Properties of Multidrug Resistance Protein 6 (MRP6, ABCC6) Cancer Res., November 1, 2002; 62(21): 6172 - 6177. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, K. Lee, S. Walther, R. B. Raftogianis, M. Kuwano, H. Zeng, and G. D. Kruh Analysis of Methotrexate and Folate Transport by Multidrug Resistance Protein 4 (ABCC4): MRP4 Is a Component of the Methotrexate Efflux System Cancer Res., June 1, 2002; 62(11): 3144 - 3150. [Abstract] [Full Text] [PDF]
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D.-W. Zhang, S. P. C. Cole, and R. G. Deeley Determinants of the Substrate Specificity of Multidrug Resistance Protein 1. ROLE OF AMINO ACID RESIDUES WITH HYDROGEN BONDING POTENTIAL IN PREDICTED TRANSMEMBRANE HELIX 17 J. Biol. Chem., May 31, 2002; 277(23): 20934 - 20941. [Abstract] [Full Text] [PDF]
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A. Ilias, Z. Urban, T. L. Seidl, O. Le Saux, E. Sinko, C. D. Boyd, B. Sarkadi, and A. Varadi Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6) J. Biol. Chem., May 3, 2002; 277(19): 16860 - 16867. [Abstract] [Full Text] [PDF]
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D. Rost, S. Mahner, Y. Sugiyama, and W. Stremmel Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine Am J Physiol Gastrointest Liver Physiol, April 1, 2002; 282(4): G720 - G726. [Abstract] [Full Text] [PDF]
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N. Zelcer, T. Saeki, G. Reid, J. H. Beijnen, and P. Borst Characterization of Drug Transport by the Human Multidrug Resistance Protein 3 (ABCC3) J. Biol. Chem., November 30, 2001; 276(49): 46400 - 46407. [Abstract] [Full Text] [PDF]
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K. Ito, H. Suzuki, and Y. Sugiyama Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G1034 - G1043. [Abstract] [Full Text] [PDF]
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H. Zeng, Z.-S. Chen, M. G. Belinsky, P. A. Rea, and G. D. Kruh Transport of Methotrexate (MTX) and Folates by Multidrug Resistance Protein (MRP) 3 and MRP1: Effect of Polyglutamylation on MTX Transport Cancer Res., October 1, 2001; 61(19): 7225 - 7232. [Abstract] [Full Text] [PDF]
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G. Liu, R. Sanchez-Fernandez, Z.-S. Li, and P. A. Rea Enhanced Multispecificity of Arabidopsis Vacuolar Multidrug Resistance-associated Protein-type ATP-binding Cassette Transporter, AtMRP2 J. Biol. Chem., March 16, 2001; 276(12): 8648 - 8656. [Abstract] [Full Text] [PDF]
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E. M. Leslie, K.-i. Ito, P. Upadhyaya, S. S. Hecht, R. G. Deeley, and S. P. C. Cole Transport of the beta -O-Glucuronide Conjugate of the Tobacco-specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the Multidrug Resistance Protein 1 (MRP1). REQUIREMENT FOR GLUTATHIONE OR A NON-SULFUR-CONTAINING ANALOG J. Biol. Chem., July 20, 2001; 276(30): 27846 - 27854. [Abstract] [Full Text] [PDF]
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R. Sanchez-Fernandez, T. G. E. Davies, J. O. D. Coleman, and P. A. Rea The Arabidopsis thaliana ABC Protein Superfamily, a Complete Inventory J. Biol. Chem., August 3, 2001; 276(32): 30231 - 30244. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, K. Lee, and G. D. Kruh Transport of Cyclic Nucleotides and Estradiol 17-beta -D-Glucuronide by Multidrug Resistance Protein 4. RESISTANCE TO 6-MERCAPTOPURINE AND 6-THIOGUANINE J. Biol. Chem., August 31, 2001; 276(36): 33747 - 33754. [Abstract] [Full Text] [PDF]
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K.-i. Ito, C. J. Oleschuk, C. Westlake, M. Z. Vasa, R. G. Deeley, and S. P. C. Cole Mutation of Trp1254 in the Multispecific Organic Anion Transporter, Multidrug Resistance Protein 2 (MRP2) (ABCC2), Alters Substrate Specificity and Results in Loss of Methotrexate Transport Activity J. Biol. Chem., October 5, 2001; 276(41): 38108 - 38114. [Abstract] [Full Text] [PDF]
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) or parental plasmid-transfected HEK293 cells (
, 
) were
incubated at 37°C in uptake media containing 380 nM
[3H]E217ßG (A), 1.6
µM [3H]DNP-SG (B), or 20
nM [3H]LTC4 (C)
and 4 mM MgATP (•, 
) or vesicles from HEK/pcDNA3 cells (