Download Multidrug resistance mediated by the ATP-binding

Review articles
Multidrug resistance mediated
by the ATP-binding cassette
transporter protein MRP
Susan P.C. Cole* and Roger G. Deeley
Summary
Resistance to multiple natural product drugs associated with reduced drug
accumulation in human tumor cells may be conferred by either the 170 kDa
P-glycoprotein or the 190 kDa multidrug resistance protein, MRP. Both MRP and
P-glycoprotein belong to the large and ancient ATP-binding cassette (ABC)
superfamily of transport proteins but share only 15% amino acid identity. Unlike
P-glycoprotein, MRP actively transports conjugated organic anions such as the
cysteinyl leukotriene C4 and glutathione-conjugated aflatoxin B1. Transport of
unconjugated chemotherapeutic agents appears to require cotransport of glutathione. MRP and several more recently discovered ABC proteins contain an additional NH2-proximal membrane-spanning domain not found in previously characterized ABC transporters. This domain, whose NH2-terminus is extracytosolic, is
essential for MRP-mediated transport activity. This review summarizes current
knowledge of the structural and transport characteristics of MRP which suggest
that the physiologic functions of this protein could range from a protective role in
chemical toxicity and oxidative stress to mediation of inflammatory responses involving
cysteinyl leukotrienes. BioEssays 20: 931–940, 1998. r 1998 John Wiley & Sons, Inc.
Introduction
The development of drug resistance is a frequent impediment
to the effective treatment of infectious and malignant diseases. Many different resistance mechanisms have been
described, but those that involve proteins belonging to the
ABC transporter superfamily have been of particular interest
because of the increasingly prominent role these proteins
play in such devastating and widespread diseases as ma-
Cancer Research Laboratories, Queen’s University, Kingston, Ontario,
Canada.
Funding agencies: Medical Research Council of Canada; National
Cancer Institute of Canada; Cancer Care Ontario.
*Correspondence to: S.P.C. Cole, Cancer Research Laboratories,
Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail:
[email protected]
Abbreviations: MRP, multidrug resistance protein; ABC, ATP-binding
cassette; CFTR, cystic fibrosis conductance regulator; MOAT, multispecific organic anion transporter; MSD, membrane-spanning domain;
NBD, nucleotide-binding domain; SUR, sulfonylurea receptor; LTC4,
leukotriene C4; GSH, glutathione; GSSG, glutathione disulfide; BSO,
buthionine sulfoximine; cAMP, cyclic adenosine monophosphate.
BioEssays 20:931–940, r 1998 John Wiley & Sons, Inc.
laria, leishmaniasis, and cancer. Several mammalian ABC
proteins have attracted further attention because of their
involvement in essential physiological processes. Important
examples of such proteins include the sulfonylurea receptor
SUR1, a subunit of the ATP-sensitive K⫹ channel present in
the plasma membrane of pancreatic ␤ cells involved in
modulating insulin secretion;(1) CFTR, a cAMP-regulated
chloride channel;(2) and the transporters associated with
antigen presentation TAP1 and TAP2, proteins essential for
the transport of peptides in association with major histocompatibility class I molecules.(3) Mutations in several ABC genes
are also now known to be the underlying cause of a variety of
human genetic disorders. For example, persistent hyperinsulinemia hypoglycemia of infancy is associated with mutations
in SUR1;(4) congenital hyperbilirubinemia (Dubin-Johnson
syndrome) is associated with mutations in the gene encoding
MOAT;(5) cystic fibrosis is caused by mutations in CFTR; (6)
and recently, the RIM or ABCR protein in the outer segments
of rod photoreceptor cells has been identified as the protein
affected in Stargardt’s disease, characterized by a progressive loss of central vision in children.(7,8)
Structural features common to all known ABC transporter
BioEssays 20.11
931
Review articles
Figure 1. Chemical structures of selected clinically useful natural product drugs to which MRP and P-glycoprotein confer resistance.
proteins include a hydrophobic polytopic MSD with up to six
transmembrane helices and a hydrophilic cytosolic NBD
containing three highly conserved sequence motifs, viz., the
Walker A and B motifs and the so-called active transport
family signature.(9,10) In bacterial ABC proteins, these structural domains are usually encoded as separate polypeptides,
which then associate with one another to form an active
transporter. Eukaryotic ABC transporters, on the other hand,
frequently contain four domains configured either as two
alternating MSDs and NBDs encoded as a single large
polypeptide (e.g., CFTR) or as two independently encoded
‘‘half-molecules’’ which then associate to function as a heterodimer (e.g., TAP1/TAP2). Presently, more than 200 prokaryotic and eukaryotic ABC proteins have been cloned and
characterized to differing degrees. As sequence databases
expand with increasing rapidity, it seems likely that the roster
of ABC transporters will follow suit.(11)
Drug resistance in human cancer
Drugs belonging to many different chemical classes are used
in the treatment of human tumors, and some of the most
powerful of these are the so-called natural product drugs. As
their name implies, these drugs were originally isolated from
plants, bacteria, or fungi. Semisynthetic or synthetic derivatives of these naturally occurring toxins, which are typically
932
BioEssays 20.11
complex multiheterocyclic structures, have become the mainstay of many commonly used and effective therapeutic
protocols. Key among them are the anthracyclines (e.g.,
doxorubicin and daunorubicin), the Vinca alkaloids (e.g.,
vincristine and vinblastine), the epipodophyllotoxins (e.g.,
VP-16 and VM-26), and the taxanes (e.g., paclitaxel) (Fig. 1).
These drugs exert their lethal effects by interacting with
multiple targets within the tumor cell, and to maximize
cytotoxicity, they are almost always administered to patients
in combination with other drugs (e.g., antimetabolites, alkylating agents, etc.).
The first ABC protein demonstrated to confer resistance to
multiple natural product drugs used in the treatment of cancer
was the 170 kDa P-glycoprotein, originally described in
1976.(12,13) In humans, this protein is encoded by the MDR1
gene and in mice by the mdr1a (also called mdr1) and mdr1b
(also called mdr3) genes. For more than 10 years, these
so-called type I isoforms of P-glycoprotein were widely
believed to be the only proteins capable of conferring multidrug resistance in mammalian cells. However, several reports of human tumor cell lines displaying multidrug resistance in the absence of P-glycoprotein overexpression(14–16)
together with studies that failed to detect P-glycoprotein in a
variety of human tumors,(17) pointed to the existence of other
multidrug resistance-conferring proteins. The cloning in 1992
Review articles
Figure 2. Domain organization of MRP
and its related proteins. The figure illustrates two models of membrane topologies
of MRP and its related proteins, both of
which contain three membrane-spanning
domains (MSD1, MSD2, and MSD3) and
two nucleotide binding domains (NBD1
and NBD2). A multiple sequence alignment
of human and mouse MRP/mrp, human
and rabbit MOAT/EBCR, and YCF1 was
used as input for profile-based neural network topology prediction by the PredictProtein server.(82) The model shown in A is the
most compatible with the neural network
output and consists of five transmembrane
helices in MSD1, six in MSD2, and four in
MSD3. This topology correctly places the
NH2-terminus on the extracytoplasmic side
of the membrane. The model of MSD3 in B
shows a more conventional ABC transporter configuration of six transmembrane
segments.
of the cDNA encoding the 190 kDa MRP from a doxorubicinselected lung cancer cell line followed shortly thereafter by
MRP cDNA transfection experiments provided unequivocal
evidence that overexpression of a second ABC protein could
cause multidrug resistance in mammalian cells.(18–20) Multidrug resistance conferred by P-glycoprotein and MRP is
characterized by an ATP-dependent reduction in drug accumulation. However, despite this common ability to mediate
multidrug resistance, there is now a preponderance of evidence demonstrating that these two mammalian ABC proteins differ substantially with respect to a number of important
structural, mechanistic, and pharmacological properties.
MRP and its relationship to the ABC superfamily
On the basis of its deduced amino acid sequence, MRP was
predicted to be an integral membrane ATP-binding, N-glycoyslated phosphoprotein of 1,531 amino acids with a polypeptide
molecular weight of 171 kDa.(18,21) Studies in both drugselected and MRP-transfected cells from several laboratories
have provided supporting experimental evidence demonstrating that MRP does indeed possess many of the biochemical
features predicted by its primary structure.(22,23) In addition, it
has become apparent that MRP has several structural features that distinguish it from previously known ABC proteins.
Prior to the cloning of MRP, ABC proteins were believed to
contain a maximum of two MSDs. The presence of a third
NH2-proximal hydrophobic region was first noted in MRP(18)
and subsequently observed in several other more recently
characterized ABC proteins (Fig. 2). All of the proteins that
contain a third MSD are more closely related to MRP and to
each other than they are to any other member of the ABC
superfamily.(24,25) In addition to the mammalian SUR and
MOAT proteins, these MRP-related transporters have been
identified in a variety of species, such as the heavy metal
resistance protein MRP1 in Caenorhabditis elegans,(26) the
cadmium resistance factor protein YCF1 and oligomycin
resistance protein YOR1/YRS1 in Saccharomyces cerevisiae,(27,28) and the GSH conjugate transporter AtMRP1 from
the plant Arabidopsis thaliana.(29) Thus, there are now at least
a dozen ABC proteins with a third NH2-terminal MSD which
together comprise the MRP branch of the superfamily (Table
1). The amino acid similarity/identity of these proteins to MRP
is relatively high and ranges from 50%/30% (Yor1/Yrs1) to
67%/49% (human MOAT). In contrast, these MRP-related
transporters all share less than 20% amino acid identity with
the P-glycoproteins.
Comparison of the primary structures of MRP-related
proteins and, where available, the intron/exon organization of
their respective genes suggests that they may have evolved
by fusion of a gene encoding a common, four-domain
ancestor with a gene encoding the additional NH2-terminal
MSD.(30,31) Similar comparisons of members of the MRP
branch of the superfamily with ABC proteins containing four
domains indicate that they are most likely to have shared this
ancestor with CFTR. It is difficult to predict whether or not
there was in fact a common five-domain ancestor for the
MRP-related proteins because the primary structures of the
NH2-terminal MSDs are considerably more divergent than the
remainder of the proteins.(29) Consequently, the MRP branch
may have evolved as the result of several independent gene
fusion events. Alternatively, functional constraints on the
BioEssays 20.11
933
Review articles
TABLE 1. Similarity Analyses and Functions of Human MRP and its Most Closely Related ABC Proteins
Protein/gene (species)
Function
% Identity
% Similarity
MRP (human)
mrp (mouse)
MOAT (human)/MRP2
EBCR (rabbit)
C. elegans mrpl (nematode)
C. elegans mrp2 (nematode)
MRP6 (human)
YCF1 (yeast)
AtMRP1 (Arabidopsis)
SUR1 (human)
sur2 (rat, mouse)
YOR1/YRS1 (yeast)
LtpgpA (leishmania)
GS-X pump, anionic conjugate transporter, multidrug resistance
GS-X pump, anionic conjugate transporter, multidrug resistance
GS-X pump, anionic conjugate transporter (hepatocanaliculi)
Probable MOAT ortholog
Heavy metal resistance
Unknown
Unknown
Cadmium resistance, vacuolar GS-X pump
GS-X conjugate pump
Sulfonylurea receptor, K⫹ channel regulator (pancreas)
Sulfonylurea receptor, K⫹ channel regulator (brain, heart)
Oligomycin resistance
Resistance to antimonial and arsenical oxyanions
100
89.9
48.7
48.7
46.3
46.6
42.1
40.2
36.0
33.1
32.5
30.3
30.0
100
96.1
66.9
66.6
64.2
63.6
60.6
59.9
55.0
53.2
53.1
50.0
47.9
Sequences were aligned along their entire length with MRP using CLUSTAL W(1.6) multiple sequence alignment. Sequence data were obtained using the
following accession numbers: MRP, L05628/P33527; mrp, AF022908/1488428; MOAT, U49248/U63970; EBCR, 1430907/Z49144; C. elegans mrp1,
U66260; C. elegans mrp2, U66261; MRP6, U91318; YCF1, L35327/Z48179; AtMRP1, AF008124; SUR1, L78207/U63421; sur2, D83598/D86037;
YOR1/YRS1, Z73066; LtpgpA, X17154. Several additional MRP-related proteins were not included because their complete cDNA sequences have not yet
been published.
primary structure of the additional MSDs may be low compared with other regions of the proteins. As we learn more
about the structure/function relationships of the third MSD, it
may be possible to make a more educated guess.
A second distinctive structural feature of MRP pertains to
its NBDs. The similarity between the two NBDs of the
P-glycoproteins is quite high, and although cooperativity
between these domains is required for activity,(32) there is no
evidence that they are functionally distinct in any significant
way. In contrast, the two NBDs of MRP (which are also both
required for function)(33) and its related proteins, as well as
CFTR, are considerably less similar to each other. Furthermore, there is convincing experimental evidence indicating
that NBD1 and NBD2 of CFTR are functionally distinct, with
NBD1 believed to be important in Cl– channel opening and
NBD2 important in the prolongation of the open channel state
and channel closing.(34) Whether or not the two NBDs of MRP
are also functionally distinct remains to be determined.
There are 11 amino acids present in the NBDs of the
P-glycoproteins that are absent from the comparable location
in NBD1 of most of the MRP-related proteins, which significantly shortens the distance between the Walker A and B
motifs. This alternate spacing of the Walker A and B motifs is
also present in NBD1 of CFTR and is one of the shared
features suggesting evolution of MRP and CFTR from a
common ancestor.(30,31) The distance between the Walker A
and B motifs in NBD1 of SUR is altered in a different manner
from that for the other MRP-related transporters, in that there
are 14 more amino acids (rather than 11 less) at this location
when compared to the P-glycoproteins. Whether or not this
particular spacing has consequences on ATP binding and
934
BioEssays 20.11
hydrolysis as they relate to SUR function remains to be
elucidated. What appears common among all ABC transporters characterized to date, including MRP,(33) is that the
conserved lysine residue in the Walker A motif (GX4GKS/T) in
both NBDs is essential for full transport function, consistent
with the role of this consensus sequence as the amino acid
acceptor site of the phosphoryl moiety of the nucleotide.
Also of possible significance with respect to the binding
and hydrolysis of nucleotide is the observation that the ABC
‘‘active transport family’’ signature (LSSGGQX3RHydXHydA),
which is relatively invariant in both NBD1 and NBD2 of the
P-glycoproteins, is highly conserved in NBD1, but not in
NBD2, of the MRP-related proteins. Mutational studies indicate that this sequence acts as more than just a linker
between the Walker A and B motifs in CFTR,(35) and by
analogy, it seems probable that this motif will also have a
functional role in MRP.(33) Thus, the distinctive structural
features of NBD1 and NBD2 of MRP and its related proteins
are highly likely to have important consequences with respect
to the mechanism of ATP binding and hydrolysis as well as
the presumed coupling of hydrolysis to transport of substrate.(36)
Transport functions of MRP
As mentioned previously, drug resistance conferred by MRP
and the P-glycoproteins is usually associated with an ATPdependent reduced cellular accumulation of drug. It has been
established by several laboratories that human MRPtransfected mammalian cells, like P-glycoprotein-transfected
cells, are resistant to vincristine, doxorubicin, daunomycin,
and VP-16 and accumulate lower steady-state levels of these
Review articles
drugs than do control transfected cells.(20,33,37) Interestingly,
rather lower levels of resistance to vinblastine, colchicine,
and paclitaxel are observed in MRP transfectants than in
P-glycoprotein-overexpressing cells, but the basis of this
differential sensitivity is unknown. Also intriguing, but presently unexplained, is the lack of anthracycline resistance in
cells transfected with the murine ortholog of MRP.(38) Finally,
in addition to chemotherapeutic agents, MRP also confers
resistance to certain antimonial and arsenical oxyanions, a
property not associated with overexpression of P-glycoprotein.(37,38) Thus, although the drug-resistance profiles of these
two drug-resistance proteins are similar, they are not identical.
It has long been noted in cells that overexpress P-glycoprotein that the degree of reduced drug accumulation seldom
correlates well with the relative resistance of the cells. This is
also true of cells that overexpress MRP, and a generally
satisfactory explanation for these observations remains elusive. Enhanced drug efflux is usually easily demonstrable in
P-glycoprotein-overexpressing cells, whereas in MRP-overexpressing cells, enhanced drug efflux is less pronounced, even
in cells that display relatively high levels of resistance (50- to
100-fold) and express substantial amounts of the protein.
Interpretation of these observations is difficult, and it is widely
agreed that, in most cases, drug transport studies and kinetic
analyses in intact cells are fraught with experimental complications that require a number of assumptions to be made, the
validity of which cannot be readily established. To avoid some
of these problems, the transport properties of P-glycoprotein
and MRP have also been studied more directly in inside-out
membrane vesicle systems. This experimental approach
utilizes a rapid filtration technique to measure the uptake of
radiolabeled substrate into vesicles and allows for more
rigorous analyses of kinetic parameters. In contrast to studies
of membrane vesicles enriched for P-glycoprotein,(39,40) it has
not been possible to demonstrate MRP-mediated active
transport of chemotherapeutic drugs such as vincristine,
daunorubicin, and VP-16,(38,41–43) and contrary reports claiming to have shown direct transport of these compounds have
recently been retracted.(44) Consistent with the lack of direct
transport is the inability to label MRP with photoactive
radiolabeled analogs of vinblastine or doxorubicin.(37,45)
A possible explanation for the inability of MRP to bind and
transport these chemotherapeutic agents, and a clue to one
of the protein’s potential physiological roles, was provided by
the demonstration that MRP in inside-out membrane vesicles
can function as a high-affinity, primary active transporter of
the cysteinyl leukotriene, LTC4.(42,46) MRP can also actively
transport a variety of other GSH-conjugated xenobiotics,
including the GSH conjugates of the activated forms of the
potent carcinogen aflatoxin B1 (Fig. 3).(47) These latter findings suggest that MRP may serve a protective role in
chemical carcinogenesis. Several other proteins of the MRP
branch of the superfamily, such as MOAT, YCF-1, and
Figure 3. Chemical structures of examples of anionic conjugated xenobiotics and endobiotics actively transported by
MRP.
AtMRP1, are also known to transport GSH conjugates in
vitro,(29,48,49) indicating that at least some of these proteins
share a similar substrate specificity distinct from that of the
P-glycoproteins and other ABC transporters.
How then to explain the inability of MRP to transport the
drugs to which it confers resistance? Consideration of the
molecular structure of LTC4 prompted the suggestion that
MRP transported anionic conjugates of the drugs rather than
the unmodified drugs themselves. However, there are several
reasons why this is an unlikely general explanation for the
ability of the protein to confer resistance to such a structurally
diverse spectrum of antineoplastic agents in such a wide
variety of cell types. First, and most compelling, is the
well-established observation that, with few exceptions, phase
II conjugation plays a relatively minor role in the in vivo and in
vitro metabolism of these compounds. In addition, phase II
biotransformation reactions are known to occur primarily in
the liver (and to a much lesser extent in other tissues) and it is
BioEssays 20.11
935
Review articles
Figure 4. Models depicting ATP-dependent, MRP-mediated transport of leukotriene C4 and co-transport of GSH and vincristine across the plasma membrane.
highly improbable that all cell types in which overexpression
of MRP causes resistance are competent to carry out such
conjugations with the efficiency and completeness required to
cause resistance. Finally, phase II conjugation usually (but
not always) results in detoxification of xenobiotics, and
consequently, it is difficult to rationalize why enhanced efflux
of the presumably less toxic conjugated metabolites would
have such a pronounced effect on drug sensitivity.
The alternative possibilities considered were that either
efflux of certain drugs might require activation of MRP by
GSH (or other endogenous small anionic molecule) or else a
cotransport mechanism might be involved (Fig. 4). Indirect
support of these ideas was provided by the observation that
physiological concentrations of GSH significantly enhanced
the ability of vinblastine and vincristine to inhibit MRPmediated, ATP-dependent transport of LTC4.(42) It was then
more directly demonstrated that MRP could transport unmodified vincristine (and later aflatoxin B1 and VP-16) into membrane vesicles in an ATP-dependent manner but only in the
presence of GSH.(42,47) This effect of GSH was concentrationdependent and inhibitable by MRP-specific monoclonal antibodies. It was shown not to be the consequence of activating
the protein by altering its redox state since the nonreducing
methyl-GSH conjugate could partially substitute for GSH but
other sulfhydryl-reducing agents could not.(42,47) Finally, the
observation that the converse stimulation occurs (i.e., vincristine and VP-16 stimulate ATP-dependent GSH uptake(83))
936
BioEssays 20.11
lends further support to the notion that MRP-mediated efflux
of these drugs occurs in association with an efflux of GSH.
Additional evidence favoring a drug/GSH cotransport
mechanism has been obtained from studies using murine
mrp double-knockout (mrp–/–) cell lines.(50,83) Thus, export of
GSH from murine wild-type (mrp⫹/⫹) embryonic stem cells,
but not from the knockout (mrp–/–) cell lines, has been
reported to be increased in the presence of VP-16.(50)
However, this stimulation of GSH export only occurred at drug
concentrations that exceeded the IC50 of VP-16 in these cells
by 50- to100-fold, and it is not clear why such high drug
concentrations are required to observe this stimulatory effect.
Nevertheless, these observations suggest that, since GSH by
itself is not a substrate for MRP,(51) MRP-mediated export of
GSH in the absence of drug or other xenobiotic may occur in
association with an endogenous metabolite(s).(50) This would
provide a possible explanation for the reduced levels of GSH
previously observed in certain MRP-overexpressing cells.(52,53)
Clearly, identification of such an endogenous substrate,
should it exist, would provide additional important information
about the physiological function(s) of MRP.
A further indication of the important role of GSH in drug
transport is the ability of BSO to improve the efficacy of some
natural product drugs in both cultured cells and mice bearing
tumors that express elevated levels of MRP.(52,54,55) BSO
depletes cellular GSH levels by irreversibly inhibiting ␥-glutamylcysteine synthetase, the rate-limiting enzyme of the GSH
Review articles
biosynthetic pathway. Although these observations are intriguing, a general mechansim that explains MRP-mediated resistance remains elusive. This is particularly true with respect to
the anthracycline antibiotics since GSH displays little or no
ability to enhance either their transport directly or their ability
to inhibit ATP-dependent, MRP-mediated transport of LTC4.(42)
We have also found that treatment of either drug-selected or
transfected cells with BSO restores sensitivity to vincristine
far more effectively than to doxorubicin.(52,53) While remaining
incompletely understood, these data are consistent with the
apparently greater importance of GSH in MRP-mediated
transport of Vinca alkaloids compared to anthracyclines.
Despite considerable effort by numerous investigators, it is
still not clear precisely how P-glycoprotein recognizes such a
broad spectrum of structurally diverse hydrophobic substrates.(56) In the case of MRP, and probably its related
proteins, there is the additional complication of understanding
how substrate selectivity with respect to a cotransport mechanism may occur.
Like many other ABC proteins, it is presumed that ATP
hydrolysis provides the energy for MRP-mediated transport
of substrate (with or without GSH), but precisely how transport is coupled to the binding and hydrolysis of nucleotide is
unknown. There is some evidence that MRP-enriched membranes exhibit basal ATPase activity that can be stimulated,
albeit modestly, by certain drugs.(57,58) The level of this activity
appears substantially lower than the constitutive and drugstimulated ATPase activities typically reported for P-glycoprotein and is more similar to the low levels reported for
CFTR.(36,59) This low activity and modest stimulation by
substrate make it difficult to distinguish ATPase activity
attributable to MRP from other ATPase activities present in
the membrane preparations. More definitive studies will
require the purification of MRP and its reconstitution into a
defined liposomal environment. Purification of mammalian
ABC proteins such as P-glycoprotein and CFTR that have
retained their activity has proven to be a challenging task, as
might be expected for any large integral membrane protein,
but a variety of experimental approaches have recently
proven successful.(56,60) One group has recently reported that
histidine-tagged recombinant MRP purified from transfected
hamster cells by Ni2⫹-chelate chromatography exhibited a
basal ATPase activity in the presence of sheep brain lipid with
an estimated Km of 3 mM and Vmax of 0.46 µmol mg-1 min-1
and that this activity could be stimulated by certain MRP
substrates, including (somewhat surprisingly) unmodified
drugs.(61) However, the estimated affinity of the purified
protein for ATP, although comparable to that for P-glycoprotein, is markedly lower than that obtained from transport
assays in which MRP is present in membrane vesicles (Km for
ATP approximately 100 µM). Furthermore, stimulation of the
ATPase activity of the solubilized protein by LTC4 (previously
reported in several different membrane vesicle systems to
exhibit a Km of approximately 50–100 nM) showed no dosedependent increase over a 1,000-fold concentration range (1
nM–1 µM). However, MRP purification and reconstitution
studies are at an early stage, and it is likely that these
apparently discordant findings will be resolved as we learn
more about the influence of the membrane environment on
MRP topology and function.
Physiological functions of MRP
In addition to LTC4, it has been shown that other endogenously formed organic anion conjugates previously proposed as putative physiological substrates of P-glycoprotein,
such as 17␤-estradiol 17-(␤-D-glucuronide), bilirubin glucuronides, and some sulfated bile salts, are actively transported
by MRP in vitro.(43,62,63) However, although MRP may be an
efficient transporter of certain glucuronides, glucuronate itself, unlike GSH, does not stimulate the active transport of
unmodified xeno- or endobiotics. Another endobiotic shown
to be actively transported by MRP in vitro is oxidized GSH or
GSSG.(51) The identification of this compound as a potential
physiological substrate raises the possibility of a role for MRP
in cellular defenses against oxidative stress and perhaps also
the maintenance of intracellular redox potential.
As mentioned previously, the protein with greatest sequence similarity to MRP is MOAT (sometimes referred to as
MRP2), and these two proteins share a very comparable
substrate specificity.(49) There are some data to suggest that
MOAT, like MRP, is capable of drug transport,(64) and it may
also be capable of conferring resistance, at least in some cell
types, since enhanced sensitivity to certain drugs (i.e., cisplatin, vincristine, and doxorubicin but not VP-16) has been
observed in cells transfected with a MOAT antisense cDNA.(65)
On the other hand, the tissue distribution and subcellular
localization of MRP and MOAT are strikingly different, implying different physiological functions. Thus, levels of MRP are
highest in the lung, testis, and muscle and very low in
liver,(18,24,66) whereas MOAT levels are highest in liver and
relatively lower in other tissues.(67) Furthermore, in rat and
human liver, MOAT is predominantly in canalicular (apical)
membranes, while the small amounts of MRP in this tissue
are predominantly on basolateral membranes.(68) MRP has
also been shown to localize to basolateral membranes when
transfected into polarized kidney cells.(64) What structural
features of these two proteins contribute to their different
subcellular locations are presently unknown.
Two groups of investigators have recently independently
reported the generation and preliminary characterization of
MRP-deficient mrp( –/–) mice using embryonic stem cell
technology.(69,70) In both cases, the mice were viable and
fertile, indicating that MRP is dispensable for development in
these animals, as was previously demonstrated for the
P-glycoproteins (mdr1a, mdr1b, and mdr2).(71) In the one
study, the response of the mrp knockout mice to an inflamma-
BioEssays 20.11
937
Review articles
tory stimulus was impaired. This was attributed to decreased
LTC4 secretion, which was demonstrated in bone marrowderived mast cells obtained from the mice, thus supporting a
physiological role for MRP as a transporter of cysteinyl
leukotrienes.(69) These same mice were also hypersensitive
to VP-16 but not vincristine, although their mast cells displayed increased sensitivity to both drugs when cultured in
vitro. In the second study,(70) the mrp( –/–) mice were also
found to be hypersensitive to VP-16 but vincristine sensitivity
and sensitivity to an inflammatory stimulus were not tested.
Consequently, it is not known whether the phenotypes of
these independently derived knockout mice are identical.
Further characterization of mrp( –/–) mice should continue to
be enlightening with respect to the physiological function(s) of
MRP, its potential protective role in oxidative stress and
chemical toxicity, and the mechanism by which it affects drug
sensitivity.
MRP topology and higher-order structure
To fully understand how the binding of substrates and
hydrolysis of ATP by MRP (or other ABC transporter) are
coupled to transport will require knowledge of the membrane
topology and higher-order structure of the protein. These
types of investigations are greatly aided by recent and
ongoing developments in computer-assisted hydropathy and
modeling methods to predict structures that can then be
subjected to experimental verification.
One approach that has been useful for distinguishing
among several possible topological models of MRP has been
to use various antibodies against known regions of the
protein combined with limited proteolysis studies to map the
location of specific peptide sequences on one side of the
plasma membrane or the other. Another has been to use
site-directed mutagenesis of potential N-glycosylation sites to
determine which ones are actually used. Thus, by mapping
the cytoplasmic epitope of the MRP-specific monoclonal
antibody QCRL-1 to amino acids 918–924, it was shown that
the linker region connecting the two ‘‘halves’’ of the molecule
is indeed cytosolic, as expected, and, moreover, that this
region is hypersensitive to proteolysis.(72,73) Furthermore,
immunostaining of permeabilized cells, but not intact cells,
with antisera and monoclonal antibodies recognizing NBD1
and NBD2 confirmed the cytoplasmic location of these
hydrophilic regions. Similarly, the sequence linking MSD1 to
MSD2 has been localized to the cytoplasm using a monoclonal antibody that maps to amino acids 238–247 and the
COOH-terminus has been confirmed to be cytoplasmic with a
monoclonal antibody mapping to amino acids 1511–1520.(22,74,75)
Finally, by determining that Asn19 and Asn23 are the only sites
of N-linked glycosylation in the NH2-terminal MSD1, it was
revealed that MRP has an extracytosolic NH2-terminus,(76) a
finding that has been independently confirmed and extended
by epitope insertion studies.(77) These data indicated that
938
BioEssays 20.11
MSD1 of MRP contains an odd number of transmembrane
helices, a somewhat unexpected topology for an ABC protein.
The extracytosolic location of the NH2-terminus was also
unexpected, but since the NH2-termini of murine mrp and rat
SUR are also known to be extracytosolic,(78) it may be that this
topological feature is shared by all proteins in the MRP
subgroup of the ABC superfamily.
A complementary approach to understanding the structure/
function relationships of an ABC protein is to express the
individual structural domains (and variants thereof) by themselves and in various combinations in a heterologous system.
The ability of the coexpressed fragments of the protein to
interact with each other can then be measured using a
functional assay or demonstrated more directly by coimmunoprecipitation procedures. Studies using a baculovirus system
to express MRP (and its component domains) in Sf21 insect
cells have been quite informative with respect to our knowledge of the structural requirements for MRP-mediated LTC4
transport. Thus, for example, we have shown that the two
‘‘halves’’ of MRP need not be covalently linked to one another
for active transport of LTC4 to occur,(79) and indeed, amino
acids 859–931 in the so-called connecting region of the
molecule appear to be dispensable for activity.(80) On the
other hand, a polypeptide missing the first 280 amino acids of
MRP (MRP281–1531) is virtually inactive, indicating that MSD1
is critically important for LTC4 transport function.(80) MSD1 is
the most divergent of the five domains of the MRP-related
ABC proteins,(29) and while the LTC4 transport activity of
MRP281–1531 can be restored by coexpression of MSD1
(MRP1–281), a chimeric protein consisting of the corresponding MSD1 of MOAT fused to MRP281–1531 (MOAT1–280MRP281–1531) is inactive. These data point to a key, and
probably essential, role of the first MSD1 in the active
transport function(s) of MRP and possibly its related proteins
as well.
Finally, an approach to investigating the physical interactions of the various domains of MRP that has not yet been
explored is by electron microscopy and image analysis. This
has recently proven feasible for both detergent-solubilized
and lipid-reconstituted P-glycoprotein, for which an initial
structure to 2.5 nm resolution has been derived that is
consistent with most available biochemical data.(81) Thus,
these studies indicate that P-glycoprotein is a monomeric
cylindrical protein with an external diameter of about 10 nm
and a maximum height of about 8 nm. Traversing this cylinder
is a large central pore with a diameter of about 5 nm that is
closed at the inner (cytoplasmic) face of the membrane, thus
forming an aqueous chamber within the membrane. Further,
the surface area occupied by P-glycoprotein in the membrane
is estimated to be about 60 nm2 and the 12 transmembrane
␣-helices of the protein thus appear to be loosely packed.
Similar investigations of MRP should prove extremely interesting, in view of the significant structural differences between
Review articles
the two proteins that are already known from biochemical
analyses.
Conclusions
Investigations of the function and structure of MRP as well as
its role in drug resistance in clinical oncology have been
greatly aided by reference to the established paradigm of
P-glycoprotein. Nevertheless, despite their shared ability to
confer resistance to multiple natural product antineoplastic
drugs, these two proteins are structurally and mechanistically
quite distinct and almost certainly have different physiological
functions. Unlike P-glycoprotein, MRP has the ability to
transport conjugated organic anions and cotransport of GSH
appears to be required for the transport of certain xenobiotics.
Furthermore, MRP was the first example of a subgroup of the
ABC superfamily whose members do not conform to the
typical four-domain structure of previously characterized ABC
transporters. Thus, in addition to being of major clinical
relevance, the cloning and characterization of MRP may be
considered a key event in the discovery of a new branch of
this large and ancient superfamily of active transporters that
has facilitated new avenues of investigation.
Acknowledgments
We apologize to the many investigators whose work was not
cited because of space limitations. We thank the members of
our laboratories, both past and present, for their contributions
to some of the work described. Special acknowledgment is
due to Dr. James H. Gerlach for helpful discussions as well as
expert and insightful sequence analyses. We thank Elaine M.
Leslie for assistance in compiling the information in Table 1
and Alexander J. Lang for technical assistance with the
figures.
REFERENCES
1 Bryan J, Aguilar-Bryan L (1997) The ABCs of ATP-sensitive potassium
channels: more pieces of the puzzle. Curr Opin Cell Biol 9: 553–559.
2 Riordan JR, Rommens JM, Kerem B-s, Alon N, Rozmahel R,
Grzelczak Z, Zielenski J, Lok S, Plasvsic N, Chou J-L, Drum ML,
Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic
fibrosis gene: cloning and characterization of complementary DNA. Science
245: 1066–1073.
3 Kelly A, Powis SH, Kerr L-A, Mockridge I, Elliott T, Bastin J,
Uchanska-Ziegler B, Ziegler A, Trowsdale J, Townsend A (1992)
Assembly and function of the two ABC transporter proteins encoded in the
human major histocompatibility complex. Nature 355: 641–644.
4 Dunne MJ, Kane C, Shepherd RM, Sanchez JA, James RFL,
Johnson PRV, Aynsley-Green A, Lu S, Clement JP, Lindley KJ,
Seino S, Aguilar-Bryan L (1997) Familial persistent hyperinsulinemic
hypoglycemia of infancy and mutations in the sulfonylurea receptor. N Engl J
Med 336: 703–706.
5 Kartenbeck J, Leuschner U, Mayer R, Keppler D (1996) Absence
of the canalicular isoform of the MRP gene-encoded conjugate export pump
from the hepatocytes in Dubin-Johnson syndrome. Hepatology 23: 1061–
1066.
6 Zielenski J, Tsui L-C (1995) Cystic fibrosis: genotypic and phenotypic
variations. Annu Rev Genet 29: 777–807.
7 Illing M, Molday LL, Molday RS (1997) The 220-kDa rim protein of
retinal rod outer segments is a member of the ABC transporter superfamily. J
Biol Chem 272: 10303–10310.
8 Azarian SM, Travis GH (1997) The photoreceptor rim protein is an ABC
transporter encoded by the gene for recessive Stargardt’s disease (ABCR).
FEBS Lett 409: 247–252.
9 Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly
related sequences in the ␣- and ␤-subunits of ATP synthase, myosin, kinases
and other ATP-requiring enzymes and a common nucleotide binding fold.
EMBO J 1: 945–951.
10 Higgins CF (1992) ABC transporters: from microorganisms to man.
Annu Rev Cell Biol 8: 67–113.
11 Allikmets R, Gerrard B, Hutchinson A, Dean M (1996) Characterization of the human ABC superfamily: isolation and mapping of 21 new genes
using the Expressed Sequence Tags database. Hum Mol Genet 5: 1649–
1655.
12 Juliano RL, Ling V (1976) A surface glycoprotein modulating drug
permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta
455: 152–162.
13 Gottesman MM, Pastan I, Ambudkar SV (1996) P-glycoprotein
and multidrug resistance. Curr Biol 6: 610–617.
14 Mirski SEL, Gerlach JH, Cole SPC (1987) Multidrug resistance in a
human small cell lung cancer cell line selected in adriamycin. Cancer Res 47:
2594–2598.
15 McGrath T, Center MS (1987) Adriamycin resistance in HL60 cells in
the absence of detectable P-glycoprotein. Biochem Biophys Res Commun
145: 1171–1176.
16 Zijlstra JG, de Vries EGE, Mulder NH (1987) Multifactorial drug
resistance in an adriamycin-resistant human small cell lung carcinoma cell
line. Cancer Res 47: 1780–1784.
17 Goldstein LJ, Galski H, Fojo A, Willingham M, Lai S-L, Gazdar
A, Pirker R, Green A, Crist W, Brodeur GM, Lieber M, Cossman J,
Gottesman MM, Pastan I (1989) Expression of a multidrug resistance
gene in human cancers. J Natl Cancer Inst 81: 116–124.
18 Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE,
Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, Deeley RG
(1992) Overexpression of a transporter gene in a multidrug-resistant human
lung cancer cell line. Science 258: 1650–1654.
19 Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole
SPC, Deeley RG (1994) Overexpression of multidrug resistance-associated
protein (MRP) increases resistance to natural product drugs. Cancer Res 54:
357–361.
20 Zaman GJR, Flens MJ, Van Leusden MR, de Haas M, Mulder
HS, Lankelma J, Pinedo HM, Scheper RJ, Baas F, Broxterman HJ,
Borst P (1994) The human multidrug resistance-associated protein MRP is a
plasma membrane drug-efflux pump. Proc Natl Acad Sci USA 91: 8822–8826.
21 Cole SPC, Deeley RG (1993) Multidrug resistance-associated protein:
sequence correction. Science 260: 879.
22 Almquist KC, Loe DW, Hipfner DR, Mackie JE, Cole SPC,
Deeley RG (1995) Characterization of the 190 kDa multidrug resistance
protein (MRP) in drug-selected and transfected human tumor cells. Cancer
Res 55: 102–110.
23 Krishnamachary N, Center MS (1993) The MRP gene associated
with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane
bound glycoprotein. Cancer Res 53: 3658–3661.
24 Stride BD, Valdimarsson G, Gerlach JH, Wilson GM, Cole SPC,
Deeley RG (1996) Structure and expression of the mRNA encoding the
murine multidrug resistance protein (MRP), an ATP-binding cassette transporter. Mol Pharmacol 49: 962–971.
25 Deeley RG, Cole SPC (1997) Function, evolution and structure of MRP.
Semin Cancer Biol 8: 193–204.
26 Broeks A, Gerrard B, Allikmets R, Dean M, Plasterk RHA (1996)
Homologues of the human multidrug resistance genes MRP and MDR
contribute to heavy metal resistance in the soil nematode Caenorhabditis
elegans. EMBO J 15: 6132–6143.
27 Szczypka MS, Wemmie JA, Moye-Rowley WS, Thiele DJ (1994)
A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated
protein. J Biol Chem 269: 22853–22857.
28 Katzmann EJ, Hallstrom TC, Voet M, Wysock W, Golin J,
Volckaert G, Moye-Rowley WS (1995) Expression of an ATP-binding
cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae. Mol Cell Biol 15: 6875–6883.
29 Lu Y-p, Li Z-s, Rea PA (1997) AtMRP1 gene of Arabidopsis encodes a
glutathione S-conjugate pump: isolation and functional definition of a plant
ATP-binding cassette transporter gene. Proc Natl Acad Sci USA 94: 8243–
8248.
30 Grant CE, Kurz EU, Cole SPC, Deeley RG (1997) Analysis of the
intron-exon organization of the multidrug resistance protein (MRP) gene and
alternative splicing of its mRNA. Genomics 45: 368–378.
31 Tusnady GE, Bakos E, Varadi A, Sarkadi B (1997) Membrane
topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters. FEBS Lett 402: 1–3.
32 Azzaria M, Schurr E, Gros P (1989) Discrete mutations introduced in
the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to
confer multidrug resistance. Mol Cell Biol 9: 5289–5297.
33 Zhu Q, Sun H, Center MS (1997) Functional analysis of the nucleotide
binding domains of the multidrug resistance protein (MRP). Oncol Res 9:
229–236.
34 Carson MR, Travis SM, Welsh MJ (1995) The two nucleotide-binding
domains of cystic fibrosis transmembrane conductance regulator (CFTR)
have distinct functions in controlling channel activity. J Biol Chem 270:
1711–1717.
35 Carson MR, Welsh MJ (1995) Structural and functional similarities
between CFTR and GTP-binding proteins. Biophys J 69: 2443–2448.
36 Senior AE, Gadsby DC (1997) ATP hydrolysis cycles and mechanism
in P-glycoprotein and CFTR. Semin Cancer Biol 8: 143–150.
BioEssays 20.11
939
Review articles
37 Cole SPC, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM,
Deeley RG (1994) Pharmacological characterization of multidrug resistant
MRP-transfected human tumor cells. Cancer Res 54: 5902–5910.
38 Stride BD, Grant CE, Loe DW, Hipfner DR, Cole SPC, Deeley
RG (1997) Pharmacological characterization of the murine and human
orthologs of multidrug resistance protein in transfected human embryonic
kidney cells. Mol Pharmacol 52: 344–353.
39 Schlemmer SR, Sirotnak FM (1994) Functional studies of P-glycoprotein in inside-out plasma membrane vesicles derived from murine erythroleukemia cells overexpressing MDR3. Properties and kinetics of the interaction of
vinblastine with P-glycoprotein and evidence for its active mediated transport.
J Biol Chem 269: 31059–31066.
40 Cornwell MM, Pastan I, Gottesman MM (1987) Certain calcium
channel blockers bind specifically to multidrug-resistant human KB carcinoma
membrane vesicles and inhibit drug binding to P-glycoprotein. J Biol Chem
262: 2166–2170.
41 Muller M, Meijer C, Zaman GJR, Borst P, Scheper RJ, Mulder
NH, de Vries EGE, Jansen PLM (1994) Overexpression of the gene
encoding the multidrug resistance-associated protein results in increased
ATP-dependent glutathione S-conjugate transport. Proc Natl Acad Sci USA
91: 13033–13037.
42 Loe DW, Almquist KC, Deeley RG, Cole SPC (1996) Multidrug
resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles: demonstration of glutathione-dependent
vincristine transport. J Biol Chem 271: 9675–9682.
43 Jedlitschky G, Leier I, Buchholz U, Barnouin K, Kurz G,
Keppler D (1996) Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump. Cancer Res 56:
988–994.
44 Kruh GD (1997) Corrections. Biochemistry 36: 13972.
45 McGrath T, Latoud C, Arnold ST, Safa AR, Felsted RL, Center
MS (1989) Mechanisms of multidrug resistance in HL60 cells: analysis of
resistance associated membrane proteins and levels of mdr gene expression.
Biochem Pharmacol 38: 3611–3619.
46 Jedlitschky G, Leier I, Buchholz U, Center M, Keppler D (1994)
ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein. Cancer Res 54: 4833–4836.
47 Loe DW, Stewart RK, Massey TE, Deeley RG, Cole SPC (1997)
ATP-dependent transport of aflatoxin B1 and its glutathione conjugates by the
product of the MRP gene. Mol Pharmacol 51: 1034–1041.
48 Li Z-s, Szczypka MS, Lu Y-p, Thiele DJ, Rea PA (1996) The yeast
cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. J
Biol Chem 271: 6509–6517.
49 Keppler D, Leier I, Jedlitschky G (1997) Transport of glutathione
conjugates and glucuronides by the multidrug resistance proteins MRP1 and
MRP2. Biol Chem 378: 787–791.
50 Rappa G, Lorico A, Flavell RA, Sartorelli AC (1997) Evidence that
the multidrug resistance protein (MRP) functions as a co-transporter of
glutathione and natural product toxins. Cancer Res 57: 5232–5237.
51 Leier I, Jedlitschky G, Buchholz U, Center M, Cole SPC,
Deeley RG, Keppler D (1996) ATP-dependent glutathione disulfide transport mediated by the MRP gene-encoded conjugate export pump. Biochem J
314: 433–437.
52 Lautier D, Canitrot Y, Deeley RG, Cole SPC (1996) Multidrug
resistance mediated by the multidrug resistance protein (MRP) gene. Biochem Pharmacol 52: 967–977.
53 Cole SPC, Downes HF, Mirski SEL, Clements DJ (1990) Alterations in glutathione and glutathione-related enzymes in a multidrug resistant
small cell lung cancer cell line. Mol Pharmacol 37: 192–197.
54 Versantvoort CHM, Broxterman HJ, Bagrij T, Scheper RJ,
Twentyman PR (1995) Regulation by glutathione of drug transport in
multidrug-resistant human lung tumor cell lines overexpressing multidrug
resistance-associated protein. Br J Cancer 72: 82–89.
55 Vanhoefer U, Cao S, Minderman H, Toth K, Skenderis BS II,
Slovak ML, Rustum YM (1996) D,L-Buthionine-(S,R)-sulfoximine potentiates in vivo the therapeutic efficacy of doxorubicin against multidrug resistance protein-expressing tumors. Clin Cancer Res 2: 1961–1968.
56 Sharom FJ (1997) The P-glycoprotein efflux pump: how does it transport
drugs? J Membr Biol 160: 161–175.
57 Hooijberg JH, Broxterman HJ, Heijn M, Fles DLA, Lankelma
J, Pinedo HM (1997) Modulation by (iso)flavonoids of the ATPase activity of
the multidrug resistance protein. FEBS Lett 413: 344–348.
58 Taguchi Y, Yoshida A, Takada Y, Komano T, Ueda K (1997)
Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of
nucleotide in multidrug resistance-associated protein (MRP). FEBS Lett 401:
11–14.
59 Li C, Ramjeesingh M, Wang W, Garami E, Hewryk M, Lee D,
Rommens JM, Galley K, Bear CE (1996) ATPase activity of the cystic
fibrosis transmembrane conductance regulator. J Biol Chem 271: 28463–
28468.
60 Shapiro A, Ling V (1994) ATPase activity of purified and reconstituted
P-glycoprotein from Chinese hamster ovary cells. J Biol Chem 269: 3745–
3754.
940
BioEssays 20.11
61 Chang X, Hou Y-X, Riordan JR (1997) ATPase activity of purified
multidrug resistance-associated protein. J Biol Chem 272: 30962–30968.
62 Loe DW, Almquist KC, Cole SPC, Deeley RG (1996) ATPdependent 17␤-estradiol 17-(␤-D-glucuronide) transport by multidrug resistance protein: inhibition by cholestatic steroids. J Biol Chem 271: 9683–9689.
63 Jedlitschky G, Leier I, Buchholz U, Hummel-Eisenbeiss J,
Burchell B, Keppler D (1997) ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocytes canalicular isoform MRP2. Biochem J 327: 305–310.
64 Evers R, Zaman GJR, van Deemster L, Jansen H, Calafat J,
Oomen LCJM, Oude Elferink RPJ, Borst P, Schinkel AH (1996)
Basolateral localization and export activity of the human multidrug resistanceassociated protein in polarized pig kidney cells. J Clin Invest 97: 1211–1218.
65 Koike K, Kawabe T, Tanaka T, Toh S, Uchiumi T, Wada M,
Akiyama S, Ono M, Kuwano M (1997) A canalicular multispecific organic
anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in
human hepatic cancer cells. Cancer Res 57: 5475–5479.
66 Flens MJ, Zaman GJR, van der Valk P, Izquierdo MA, Schroeijers AB, Scheffer GL, van der Groep P, de Haas M, Meijer CJLM,
Scheper RJ (1996) Tissue distribution of the multidrug resistanceassociated protein. Am J Pathol 148: 1237–1247.
67 Paulusma CC, Bosma PJ, Zaman GJR, Bakker CTM, Otter M,
Scheffer GL, Scheper RJ, Borst P, Oude Elferink RPJ (1996)
Congenital jaundice in rats with a mutation in a multidrug resistanceassociated protein gene. Science 271: 1126–1128.
68 Roelofsen H, Vos TA, Schippers IJ, Kuipers F, Koning H,
Moshage H, Jansen PLM, Muller M (1997) Increased levels of the
multidrug resistance protein in lateral membranes of proliferating hepatocytederived cells. Gastroenterology 112: 511–521.
69 Wijnholds J, Evers R, Van Leusden MR, Mol CAAM, Zaman
GJR, Mayer U, Beijnen JH, van der Valk M, Krimpenfort P, Borst P
(1997) Increased sensitivity to anticancer drugs and decreased inflammatory
response in mice lacking the multidrug resistance-associated protein. Nat
Med 3: 1275–1279.
70 Lorico A, Rappa G, Finch RA, Yang D, Flavell RA, Sartorelli AC
(1997) Disruption of the murine MRP (multidrug resistance protein) gene leads
to increased sensitivity to etoposide (VP-16) and increased levels of glutathione. Cancer Res 57: 5238–5242.
71 Borst P, Schinkel AH (1996) What have we learnt thus far from mice
with disrupted P-glycoprotein genes? Eur J Cancer 32A:985–990.
72 Hipfner DR, Gauldie S, Deeley RG, Cole SPC (1994) Detection of
the Mr190,000 multidrug resistance protein, MRP, with monoclonal antibodies.
Cancer Res 54: 5788–5792.
73 Hipfner DR, Almquist KC, Stride BD, Deeley RG, Cole SPC
(1996) Location of a protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRP-specific monoclonal
antibody QCRL-1. Cancer Res 56: 3307–3314.
74 Flens MJ, Izquierdo MA, Scheffer GL, Fritz JM, Meijer CJLM,
Scheper RJ, Zaman GJR (1994) Immunochemical detection of the
multidrug resistance-associated protein MRP in human multidrug-resistant
tumor cells by monoclonal antibodies. Cancer Res 54: 4557–4563.
75 Hipfner DR, Gao M, Scheffer G, Scheper R, Deeley RG, Cole
SPC (1998) Epitope mapping of monoclonal antibodies specific for the 190
kDa multidrug resistance protein, MRP. Br J Cancer (in press).
76 Hipfner DR, Almquist KC, Leslie EM, Gerlach JH, Grant CE,
Deeley RG, Cole SPC (1997) Membrane topology of the multidrug
resistance protein, MRP: a study of glycosylation-site mutants reveals an
extracytosolic NH2-terminus. J Biol Chem 272: 23623–23630.
77 Kast C, Gros P (1997) Topology mapping of the amino-terminal half of
multidrug resistance-associated protein by epitope insertion and immunofluorescence. J Biol Chem 272: 26479–26487.
78 Nelson DA, Bryan J, Wechsler S, Clement JP, Aguilar-Bryan L
(1996) The high-affinity sulfonylurea receptor: distribution, glycosylation,
purification, and immunoprecipitation of two forms from endocrine and
neuroendocrine cell lines. Biochemistry 35: 14793–14799.
79 Gao M, Loe DW, Grant CE, Cole SPC, Deeley RG (1996) Reconstitution of ATP-dependent LTC4 transport by co-expression of both halfmolecules of human MRP in insect cells. J Biol Chem 271: 27782–27787.
80 Gao M, Yamazaki M, Loe DW, Westlake CJ, Grant CE, Cole
SPC, Deeley RG (1998) Multidrug resistance protein: identification of
regions required for active transport of leukotriene C4. J Biol Chem 273:
10733–10740.
81 Rosenberg MF, Callaghan R, Ford RC, Higgins CF (1997) Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined
by electron microscopy and image analysis. J Biol Chem 272: 10685–10694.
82 Rost B, Fariselli P, Casadio R (1996) Topology prediction for helical
transmembrane proteins at 86% accuracy. Protein Sci 5: 1704–1718.
83 Loe DW, Deeley RG, Cole SPC (1998) Characterization of vincristine
transport by the 190 kDa multidrug resistance protein, MRP: evidence for
co-transport with reduced glutathione. Cancer Res 58 (In press Nov 15th
issue).