Physiol Rev 86: 849 – 899, 2006;
doi:10.1152/physrev.00035.2005.
Transmembrane Transport of Endo- and Xenobiotics
by Mammalian ATP-Binding Cassette Multidrug
Resistance Proteins
ROGER G. DEELEY, CHRISTOPHER WESTLAKE, AND SUSAN P. C. COLE
Division of Cancer Biology and Genetics, Cancer Research Institute, and Departments of Biochemistry
and of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario, Canada
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I. Introduction
II. Discovery of the Multidrug Resistance Protein Family of Transporters
A. MRP1 (ABCC1)
B. MRP2 (ABCC2)
C. MRPs 3– 6 (ABCC3– 6)
D. MRPs 7–9 (ABCC10 –12) and MRP10 (ABCC13)
III. Evolution of the “C” Branch of the ABC Superfamily
A. Long and short ABCC proteins
B. ABCC proteins are characterized by an atypical NH2-proximal NBD
IV. Topology of the MRP1-Like Multidrug Resistance Proteins
A. Experimental evidence for 3 MSDs, 17 transmembrane helices, and extracellular NH2 termini
B. What are the functions of MSD0 and the cytoplasmic MSD0-MSD1 linker?
V. Expression and Membrane Localization of the Multidrug Resistance Proteins
A. MRP1
B. MRP2
C. MRP3
D. MRPs 4 and 5
E. MRP6
F. MRPs 7–9 and ABCC13
VI. Substrate Specificities and Physiological Functions of the Multidrug Resistance Proteins
A. MRP1 and MRP2
B. Transport and the role of GSH
C. Physiological roles of MRP1 and MRP2
D. MRP3
E. MRPs 4 and 5
F. MRP6
G. MRPs 7 and 8
VII. In Vitro Drug Resistance Profiles and Inhibitors of the Multidrug Resistance Proteins
A. Drug resistance profiles
B. Inhibitors and reversing agents
VIII. Clinical Relevance of the Multidrug Resistance Proteins
A. Background
B. MRP1 and solid tumors
C. MRP1 and hematological malignancies
D. Clinical relevance of other MRPs
IX. Mechanism of Transport
A. ATPase activities of purified ABCC proteins
B. ABC NBDs functional cooperatively
C. Stoichiometry of ATP hydrolysis and substrate transport
D. Experimental approaches used to study ATP binding and hydrolysis
E. Functionally important structural differences between ABCC NBDs
F. High- and low-affinity substrate binding states
G. Interactions between MSDs and NBDs
H. The transport cycle of MRPs
X. Substrate Recognition and Binding by Multidrug Resistance Proteins 1, 2, and 3
A. Experimental approaches used to investigate substrate recognition
B. Photoaffinity labeling studies
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C. Mutagenesis studies
D. Is amino acid sequence conservation predictive of substrate specificity?
XI. Higher Order Structure of Multidrug Resistance Protein 1
A. Molecular modeling
B. Electron crystallographic studies
XII. Conclusion and Perspectives
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I. INTRODUCTION
Development of multidrug resistance (MDR) occurs
during treatment of many forms of malignant and infectious disease and is often the ultimate cause of treatment
failure. Although MDR may develop in response to a
specific drug or drug combination, it often encompasses
agents to which individuals have not been previously
exposed, and which may or may not share targets and
mechanisms of action with those that elicited development of resistance. In the case of cancer, these agents
include conventional cytotoxic natural product type
drugs (e.g., the Vinca alkaloids, including vinblastine and
vincristine, and anthracyclines, such as doxorubicin and
daunorubicin), alkylating agents (e.g., melphalan), platinum-containing compounds (e.g., cisplatin and carboplatin), antimetabolites (e.g., methotrexate), and nucleoside/
nucleotide analogs (e.g., gemcitabine and cytosine arabinoside). However, it is becoming increasingly apparent
that resistance can also limit the efficacy of newer, socalled targeted therapeutic agents, such as imatinib mesylate (Gleevec) (133, 264) and various anti-human immunodeficiency virus (HIV) drugs (167, 235, 263, 265). Not
surprisingly, no single mechanism has been identified that
can account for resistance to the entire spectrum of anticancer drugs in common use, and it is widely accepted
that clinical resistance is multifactorial. However, the
discovery more than 30 years ago of P-glycoprotein (Pgp/MDR1) demonstrated that it was possible for a single
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protein to confer resistance to a relatively large number of
structurally diverse drugs with different mechanisms of
action (219). The cross-resistance spectrum conferred by
P-gp/MDR1 typically encompasses a broad range of natural
product type drugs (151, 502), but it does not include other
clinically important chemotherapeutic agents, such as platinum compounds, nucleoside analogs, or alkylating agents.
P-gp/MDR1 is a member of the ATP binding cassette
(ABC) superfamily of transmembrane transporters, and it
functions as a direct active transporter of the drugs to
which it confers resistance (10). Thus increased drug
efflux is a defining characteristic of the MDR phenotype
conferred by this transporter protein. In humans, the ABC
superfamily includes 49 genes that have been assigned to
a “family tree” with 7 branches, designated A through G
(94). Although the vast majority of the proteins are energydependent transporters, the superfamily also contains examples of one channel gated by ATP binding and hydrolysis, i.e., the cystic fibrosis transmembrane conductance
regulator (CFTR/ABCC7) (417) and ATP-dependent potassium channel regulators, such as the sulfonylurea receptors SUR1/ABCC8 (3, 203) and SUR2/ABCC9 (204).
The range of exogenous and endogenous substrates transported by mammalian ABC proteins is vast, and defects in
a number of these genes are the cause of inherited disorders, with cystic fibrosis being the most common and
extensively studied (258, 390, 417).
The core functional unit of the ABC transporters
consists of two polytropic membrane spanning domains
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Deeley, Roger G., Christopher Westlake, and Susan P. C. Cole. Transmembrane Transport of Endo- and
Xenobiotics by Mammalian ATP-Binding Cassette Multidrug Resistance Proteins. Physiol Rev 86: 849 – 899, 2006;
doi:10.1152/physrev.00035.2005.—Multidrug Resistance Proteins (MRPs), together with the cystic fibrosis conductance regulator (CFTR/ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) comprise the 13
members of the human “C” branch of the ATP binding cassette (ABC) superfamily. All C branch proteins share
conserved structural features in their nucleotide binding domains (NBDs) that distinguish them from other ABC
proteins. The MRPs can be further divided into two subfamilies “long” (MRP1, -2, -3, -6, and -7) and “short” (MRP4,
-5, -8, -9, and -10). The short MRPs have a typical ABC transporter structure with two polytropic membrane spanning
domains (MSDs) and two NBDs, while the long MRPs have an additional NH2-terminal MSD. In vitro, the MRPs can
collectively confer resistance to natural product drugs and their conjugated metabolites, platinum compounds, folate
antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and,
under certain circumstances, alkylating agents. The MRPs are also primary active transporters of other structurally
diverse compounds, including glutathione, glucuronide, and sulfate conjugates of a large number of xeno- and
endobiotics. In vivo, several MRPs are major contributors to the distribution and elimination of a wide range of both
anticancer and non-anticancer drugs and metabolites. In this review, we describe what is known of the structure of
the MRPs and the mechanisms by which they recognize and transport their diverse substrates. We also summarize
knowledge of their possible physiological functions and evidence that they may be involved in the clinical drug
resistance of various forms of cancer.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
eral, it appears not to be essential (19, 253, 291, 397, 448,
490, 496).
Over the last two decades, it has become evident that
P-gp/MDR1 is far from being the only human ABC transporter that, in vitro at least, can confer resistance to
clinically important chemotherapeutic agents (32, 61, 71,
72, 85, 108, 154, 192, 252, 268, 537). The number of additional transporters has now reached 10, and with the
exception of one protein (BCRP/ABCG2), they all belong
to the multidrug resistance protein (MRP/ABCC) family
(94). In vitro, the MRP-related proteins can collectively
confer resistance to natural product drugs and their conjugated metabolites, as well as platinum-containing compounds, folate antimetabolites, nucleoside and nucleotide
analogs, arsenical and antimonial oxyanions, peptidebased agents and, in concert with changes in conjugating
or biosynthetic enzymes, alkylating agents (Fig. 2) (96,
161, 256, 287). Aside from their potential role in drug
FIG. 1. Topology of short and long ABCC
proteins. The figure illustrates a simple, probable topology of the “long” and “short” multidrug
resistance proteins (MRPs). In the case of
MRP1, the model is supported by considerable
experimental data (see sect. IVA). The NH2 terminus of MRP2 has also been shown to be extracellular. However, the topologies predicted
by different computer-assisted algorithms differ,
and in the case of MRP2 MSD2, several favor a
topology involving 4 rather than 6 transmembrane helices.
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(MSDs) [each of which typically contains 6 transmembrane (TM) helices but may range from 5 to 10] and two
nucleotide binding domains (NBDs) (171). All four of
these domains can be encoded in a single polypeptide in
the order NH2-MSD-NBD-MSD-NBD-COOH (Fig. 1). Alternatively, the functional transporter may be a homo- or
heterodimer of polypeptides, each of which contributes
an MSD and an NBD. In some cases, the more typical
NH2-MSD-NBD-COOH order may be reversed, as exemplified by breast cancer resistance protein (BCRP/ABCG2)
(108). The NBDs of ABC proteins all contain Walker A and
B motifs first identified in F1 ATPases that are essential
for ATP binding and hydrolysis (522). In addition, they
also contain a motif known as the “C” signature that is
characteristic of the ABC ATPases and that has the core
sequence LSGGQ (170). Although the C signature is
clearly intimately involved in ATP hydrolysis, its role in
nucleotide binding has not been established, and in gen-
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resistance, several MRPs together with P-gp/MDR1 (10,
114) and BCRP/ABCG2 (109) have been recognized as
major components of the distribution and elimination
pathways for a wide range of both anti-cancer and nonanti-cancer drugs and metabolites (161, 256). In addition,
because of their presence in a number of tissue/blood
barriers protecting sanctuary sites in the body, they also
markedly influence the disposition of drugs and other
chemicals (80, 113, 270, 287, 360, 404, 536, 571). In this
review, we focus primarily on the structure of the MRPs
and their mechanisms of substrate recognition and transport. We also summarize the state of knowledge of their
normal physiological functions and the evidence that they
may or may not play a role in the clinical drug resistance
of various forms of cancer.
II. DISCOVERY OF THE MULTIDRUG
RESISTANCE PROTEIN FAMILY
OF TRANSPORTERS
A. MRP1 (ABCC1)
The first member of the MRP transporter family was
cloned in 1992 from the drug-selected human lung cancer
cell line H69AR (72, 342). This cell line is a multidrug
resistant derivative of the small cell lung cancer cell line
H69 that was selected by repeated exposure to the anthracycline doxorubicin. Although selected in a single
drug, H69AR cells display cross-resistance to a wide range
of structurally unrelated natural product cytotoxic drugs
that have a variety of different molecular targets. The
multidrug resistance phenotype of the H69AR cells was
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somewhat similar to that previously, exclusively associated with the overexpression of P-gp/MDR1 (151, 219,
502). However, despite extensive investigation, no evidence of increased expression of P-gp/MDR1 was detectable in the H69AR cells (70, 342), nor was its resistance
phenotype readily reversible by agents that reverse P-gp/
MDR1-mediated resistance (68, 71).
The primary cause of the multidrug resistance phenotype of H69AR cells was ultimately identified by screening for mRNAs that were stably overexpressed following
drug selection (72). Several overlapping cDNA clones
were obtained that corresponded to an mRNA with an
open reading frame encoding a protein of 1,531 amino
acids (72). Cytogenetic analyses indicated that the gene
encoding the overexpressed mRNA was located at chromosome 16p13.1 and was amplified ⬃100-fold in H69AR
and HT1080/DR4 cells (72, 470). Analysis of the predicted
sequence of the protein, initially named multidrug resistance-associated protein (MRP) and subsequently multidrug resistance protein 1 (MRP1), suggested the presence
of numerous TM helices and two cytoplasmic domains
containing motifs associated with the ABC superfamily of
transmembrane transporters. Thus MRP1 (ABCC1) appeared to belong to the same protein superfamily as P-gp/
MDR1 and, as a consequence, was considered a possible
cause of the multidrug resistance characteristics of H69AR
cells. However, the computer-predicted topology of the
MSDs of MRP1 was unusual for an ABC transporter. Furthermore, the two putative NBDs, which in many ABC proteins are identical or very similar, were relatively divergent,
even with respect to highly conserved elements such as the
Walker B motif and the C motif or signature sequence. The
NBDs of MRP1 were in fact much more similar to those of
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FIG. 2. Subcellular localization and
substrate specificity of the MRPs. A cartoon is shown of two polarized cells, one
expressing long MRPs (left) and the
other, short MRPs (right). The subcellular location(s) of each protein on the apical (upper) or basolateral membranes is
shown. The presence of an individual
protein in both locations indicates cell
type specific differences in subcellular
distribution. In the case of MRPs 7, 8, and
9, the question mark indicates that their
subcellular localization is not known.
Some of the major classes of substrates
for each protein are indicated, as well as
specific examples of substrates chosen to
illustrate the overlap in substrate profiles
among the MRPs. Additional details are
provided in sections V–VII.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
B. MRP2 (ABCC2)
Discovery of the second member of the MRP family,
MRP2 (ABCC2), occurred ⬃4 years after the discovery of
MRP1 (39, 381, 494). By this time, considerable information concerning the substrate profile and tissue distribution of MRP1 had accumulated, and it was known that the
protein was capable of transporting a wide range of conjugated organic anions. The substrate profile of MRP1 was
very similar to that of a functionally characterized bile
canalicular transporter, the canalicular multispecific organic anion transporter (cMOAT), which was defective in
certain strains of mutant rats and sheep and in individuals
suffering from a mild, inherited form of conjugated hyperbilirubinemia known as Dubin-Johnson syndrome (110,
430, 476). The similarity in substrate profiles was such
that MRP1 was considered a possible candidate for the
canalicular transporter known as cMOAT. However, the
protein responsible was subsequently shown to be an
MRP1 homolog (224, 230, 382). MRP2 was first cloned in
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1996 from normal rat liver using cDNA probes corresponding to predicted highly conserved regions of human
MRP1 (49, 381).
C. MRPs 3– 6 (ABCC3– 6)
Among all MRP family members, MRP2 displays the
highest similarity to MRP1 with respect to substrate specificity (60, 85, 119, 187, 384). However, from an evolutionary standpoint, MRP1’s closest relative is MRP3, whose
partial sequence was first reported in 1997, together with
MRP4 and MRP5 (251). These three MRPs were identified
by mining of EST databases. Discovery of an additional
member of the family was facilitated by sequencing of
chromosome 16, which revealed an MRP-related gene,
MRP6, the 3⬘-end of which is located within 9 kb of the
3⬘-end of MRP1 (251). Despite the likely origin of MRP1
and MRP6 by gene duplication and the possibility for gene
homogenization, as is thought to have occurred with
some mammalian isoforms of P-gp, MRP6/ABCC6 is less
similar to MRP1 than either MRP2 or MRP3. One of the
most intriguing discoveries involving MRP6 is the striking
association between mutations in the gene and the rare
connective tissue disorder pseudoxanthoma elasticum
(PXE) (36, 41, 416). Why defects in MRP6 result in the
disease is presently unknown.
D. MRPs 7–9 (ABCC10 –12) and MRP10 (ABCC13)
In the last 2–3 years, three, possibly four, additional
MRP-related genes have been identified (34, 191, 493),
bringing the total number of potential family members to
10. MRPs 1– 8 have all been shown to encode functional
ATP-dependent transporters and their combined substrate profiles encompass a wide array of endo- and xenobiotics and/or their conjugated metabolites. Whether
MRP9 encodes a functional protein is not yet known, but
there has been a report of high levels of a truncated MRP9
mRNA in breast cancer (35), and in the mouse, Mrp9
mRNA has been detected at high levels in seminiferous
tubules (465). The most recently identified “MRP,”
MRP10? (ABCC13) (546), represents an interesting example of a gene that has degenerated to varying extents
among mammals (12). In all species examined, with the
possible exception of the rhesus monkey, the ABCC13
gene appears incapable of encoding a functional transporter (see below).
III. EVOLUTION OF THE “C” BRANCH OF THE
ABC SUPERFAMILY
The ”C“ branch is one of the largest of the 7 branches
of ABC superfamily, and in humans, it is composed of 13
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CFTR which, as indicated above, functions as a chloride
channel rather than a drug pump (72).
The lack of structural identity, only ⬃19% overall,
between MRP1 and P-gp/MDR1 was unexpected given the
similarity between the phenotypes of H69AR cells and
cells overexpressing P-gp/MDR1 (72). Nevertheless, gene
transfer experiments established that MRP1 was capable
of conferring resistance to several major families of natural product drugs, including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (73, 146). In transfected
cells, overexpression of MRP1 also resulted in lower intracellular levels of drug and increased rates of efflux,
indicating that, like P-gp/MDR1, MRP1 functioned as a
drug efflux pump (73, 553). However, these early studies
with MRP1 transfected cells revealed some significant
differences between the drug resistance profiles conferred by the two proteins. In addition to conferring resistance to many natural product drugs included in the
P-gp/MDR1 drug resistance profile, MRP1 and its subsequently cloned murine ortholog conferred resistance to
heavy metal oxyanions such as sodium arsenite, sodium
arsenate, and antimony potassium tartrate (73, 482). Although mammalian P-gp/MDR1 does not confer resistance
to these compounds, a previously identified ABC transporter
in Leischmania tarentoliae (ltpgpA), originally thought to
be a P-gp/MDR1 homolog, had been shown to increase
resistance to arsenicals (379). Sequence comparisons indicate that ltpgpA is a homolog of MRP1 rather than P-gp/
MDR1 (72). With respect to clinically important cancer
drugs, P-gp/MDR1 and MRP1 also differ in their ability to
confer resistance to taxanes. Although these compounds are
very good P-gp/MDR1 substrates (340), MRP1 confers only
low levels of taxane resistance (73, 553).
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proteins. In addition to 10 MRPs, the C branch proteins
include CFTR and the sulfonylurea receptors SUR1 and
SUR2A/B mentioned previously. Thus MRPs 1– 6 and
MRPs 7–10 have been designated ABCC1– 6 and ABCC10 –
13, respectively, while CFTR, SUR1, and SUR2A/2B are
designated ABCC7, ABCC8, and ABCC9, respectively (94).
A. Long and Short ABCC Proteins
FIG. 3. Dendrograms of the structural similarities of the full-length ABCC proteins, their nucleotide binding domains (NBDs) and membrane
spanning domains (MSDs) of the long ABCC proteins. Dendrograms are based on ClustalW alignments and were generated using Tree View (65).
All three panels are drawn to the same scale. A: dendrogram of the human ABCC proteins based on their entire sequence. B: dendrogram of the
individual NBDs of the human ABCC proteins together with E. coli HlyB and the NBDs of human MDR1/ABCB1. C: dendrogram of MSD0s of the
“long” ABCC proteins.
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Based on the predicted topology of their membranespanning regions, the ABCC proteins (with the exception
of MRP9) fall into two groups. CFTR (ABC7), MRPs 4, 5,
8, and ABCC13 have a “typical” ABC transporter structure
with two MSDs (MSD1 and MSD2), each of which is
predicted to contains six TM helices (12, 18, 34, 177, 191,
227, 493, 500), while the remaining MRPs and the SURs
(ABCC8 and ABCC9) have an additional NH2-terminal
region comprised of ⬃200 amino acids (Fig. 1). The NH2terminal extensions of the long ABCC proteins are relatively poorly conserved. However, they are all hydrophobic and predicted to contain from four to six transmembrane helices (MSD0). The longer, five-domain MRPs are,
for the most part, relatively closely related to MRP1 (Fig.
3A) (amino acid identity: 45, 56, and 44% for MRPs 2, 3,
and 6, respectively). In contrast, those lacking an NH2terminal extension share almost equivalent amino acid
identity with CFTR (34, 250, 493). The exceptions to this
generalization are MRP7 and ABCC13. MRP7 contains an
NH2-terminal extension, but its overall identity with
MRP1 is only slightly higher (31%) than its identity with
the SURs (28 and 26% with SUR1 and SUR2, respectively)
(191). ABCC13, on the other hand, lacks an extended
NH2-terminal region but has a higher amino acid identity
with the core region of MRP1 than MRP6 (546) (Fig. 3, A
and C). As indicated above, the ABCC13 gene appears to
have undergone a process of gradual pseudogenization in
mammals, and presently, the only species found to contain an ABCC13 gene potentially capable of encoding a
full-length protein is the rhesus macaque (12). Thus it
appears likely that one of the earliest events in this degenerative process may have been deletion of exons encoding the NH2-terminal region of a progenitor relatively
closely related to MRP1.
Comparison of the amino acid sequences of the long
and short ABCC proteins reveals that the NH2-termini of
CFTR and the shorter MRPs align with a relatively conserved sequence that appears to define the COOH-terminal boundary of the additional regions present in MRPs 1,
2, 3, 6, and 7 and the SURs. In the MRP1 gene, this
sequence is found at the 5⬘-end of exon 6, strongly suggesting that the first five exons were acquired as the result
of a gene fusion event (147). Because putative orthologs
of MRP1 exist in plants and yeast, this event must be quite
ancient. To date, six ABCC-like proteins from Streptomyces cerevisiae have been identified that have NH2-terminal
extensions: Ycf1, Bpt1, Ybt1, YKR103w/YKR104w, YHL035c,
and Yor1p (86, 216, 292, 331, 375, 389, 528). Like the
human proteins, these domains are poorly conserved, but
with the exception of Yor1p, they are all predicted to
contain several TM helices. The function of MSD0 in the
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
“long” MRPs remains poorly defined (see sect. IVB). However, in SUR1, the comparable domain is involved in an
interaction with the potassium channel Kir6.2 required for
the trafficking of SUR1 to the plasma membrane and for
the gating function of the KATP channel (16, 54, 378).
B. ABCC Proteins Are Characterized
by an Atypical NH2-Proximal NBD
motifs, as well as a copy of the C sequence that is the
signature of ABC NBDs (170). However, in the ABCC proteins, these elements deviate in some cases from the form
commonly found in other ABC proteins (72). For example,
the amino acid following the Walker B motif in most ABC
proteins is glutamate, and this residue is critical for cleavage
of the ␤-␥ phosphodiester bond of ATP (345, 472, 515).
Although this residue is present at the appropriate location
in NBD2 of the ABCC proteins, it is not present in NBD1
(72). In MRP1 and other ABCC proteins, the residue is an
aspartate, with the exception of CFTR which has a Ser
residue at this location (Fig. 4). Despite the conservative
nature of the substitution in NBD1 of MRP1, the lack of
glutamate has a profound effect on the ATP binding and
hydrolysis characteristics of the NBD and as a consequence,
on the catalytic cycle of MRP1 and the other ABCC proteins
(386) (see sect. IX). Similarly, the ABC signature sequence in
FIG. 4. Sequence alignment of the NBDs of ABCC proteins and selected more distantly related ABC transporters. Multiple sequence alignment
of the NBDs of MRP1 with those of the protein’s presumed yeast ortholog, YCF1, human cystic fibrosis transmembrane conductance regulator
(CFTR), the human antigen processing protein TAP1, and the bacterial transporters HlyB, MsBA, and MJ0796. The alignment was performed using
ClustalW (65). The figure was colored according to the ClustalW color scheme and formatted using Jalview (67).
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Regardless of whether the MRPs have two or three
MSDs, they all share certain highly conserved features in
their NBDs, particularly in NBD1, that are hallmarks of the
entire family. These features are also conserved in CFTR
and to a lesser extent the SURs and provide the most compelling evidence for a common ancestor of all C branch
members (Figs. 3B and 4) (94, 95). Like other ABC proteins,
the NBDs of the C branch proteins contain Walker A and B
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IV. TOPOLOGY OF THE MRP1-LIKE
MULTIDRUG RESISTANCE PROTEINS
A. Experimental Evidence for 3 MSDs, 17
Transmembrane Helices, and Extracellular
NH2 Termini
Some, but not all, computer-assisted hydropathy algorithms predict that the NH2-terminal extensions of the
MRP1-like MRPs and the SURs contain five TM helices and
that the NH2 termini of the proteins are extracellular (18, 74,
177, 398, 500). Thus these proteins are predicted to have 3
MSDs (MSD0, MSD1, and MSD2) and a total of 17 TM helices
arranged in a 5 (MSD0) ⫹6 (MSD1) ⫹6 (MSD2) configuration. Considerable experimental evidence supports the predicted topology of MRP1 (Fig. 5), as well as the extracellular
locations of the NH2 termini of both MRP1 and MRP2 (18,
177, 178, 180, 227, 228, 234, 243, 245, 350). The first evidence
that the NH2 terminus of MRP1 was extracellular came from
mutational studies that revealed the presence of two functional N-linked glycosylation sites very close to the start of
the protein at Asn19 and Asn23 (177). The fact that a third
potential site at Asn71 was not used and presumed to be
intracellular provided additional support for the existence of
five TM helices in MSD0. The extracellular location of the
NH2 termini of MRP1 and MRP2 was subsequently confirmed by epitope insertion studies and the use of an antiPhysiol Rev • VOL
body to the first 25 amino acids of rat MRP2, respectively
(176, 178, 180, 243). The presence of a third N-linked glycosylation site at Asn1006 in MRP1 confirms that the loop
connecting predicted TM12 and TM13 is extracellular (177).
Subsequent epitope mapping studies with the MRP1 monoclonal antibodies (MAbs) MRPr1, QCRL-1, MRPm5, and
MRPm6, determined that amino acids residues 238 –247,
918 –924, 1063–1072, and 1511–1520 were intracellular (176,
178, 180, 243). The MRPr1 epitope is located in CL3, the
intracellular loop connecting TM helices 5 and 6, while MAb
QCRL-1 detects a region beginning at 124 residues downstream of the NBD1 Walker B element in the linker region
preceding MSD2. The epitope for MAb MRPm5 is localized
to CL6, which connects TMs 13 and 14, while MAb MRPm6
recognizes the COOH-terminal region.
Another approach used to determine the topology of
MRP1 has involved expressing mutant proteins containing inserted hemagglutinin (HA) epitopes in predicted
extracellular and cytosolic regions (227, 228). Immunocytochemical detection of HA peptide inserts in intact and
permeabilized cells confirmed the orientation of the NH2
terminus CL3, the region linking NBD1 to MSD2 and the
position of the extracellular loop connecting TM12–13
(Fig. 5). In addition, HA tags at position 163 and 574 were
detected outside the cell, while the region containing
amino acid 653 was located on the cytosolic face of the
membrane. Together, these results support the five and
six TM arrangement of MSD0 and MSD1, respectively.
Some topology algorithms predict that MSD2, most notably in MRP2, contains four rather than six TM helices (179,
247). No experimental data supporting one topology or the
other are presently available for MRP2, but epitope insertion
studies of this domain in MRP1 favor the presence of six
rather than four TM helices (228). However, the evidence
rests on detection of an epitope inserted at amino acid 1222,
which only became detectable extracellularly when two
copies of the HA epitope were inserted in tandem. Whether
this region can adopt more than one topology, as demonstrated recently for certain TM helices of voltage-dependent
ion channels (169), remains an intriguing possibility.
B. What Are the Functions of MSD0 and the
Cytoplasmic MSD0-MSD1 Linker?
As the defining structural characteristic of the longer
MRPs and the SURs, the function of MSD0 in these proteins has been the subject of considerable interest and
speculation. As mentioned previously, only MSD0 of
SUR1 has been shown to fulfill a well-defined functional
role. Interaction between MSD0 of SUR1 and the Kir6.2
potassium channel masks an endoplasmic reticulum (ER)
retention signal and, in so doing, allows the protein complex to exit the ER (16, 54). This mechanism serves to
ensure that the stoichiometry of SUR1 and Kir6.2 in the
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NBD2 of the ABCC proteins differs from that typically found
in other members of the superfamily (72), but the functional
consequences of this deviation are less well defined (387, 490).
In addition to the variations in conserved motifs described above, alignment of the sequences of the ABCC
NBDs reveals additional structural features that distinguish these proteins from most other ABC transporters
(Fig. 4). The NBDs of most prokaryotic ABC transporters
are identical (171). In many eukaryotic ABC proteins, the
NBDs are structurally very similar (59), and in P-gp/
MDR1, the positions of the NBDs in P-gp/MDR1 can be
exchanged with little or no effect on function (26, 199). In
contrast, the NH2-proximal NBDs of ABCC proteins are
quite divergent from their COOH-proximal NBDs, which
have a more typical ABC NBD structure (72). The most
obvious distinguishing feature of the NH2-proximal NBDs
of the MRPs and CFTR is a decrease in the spacing
between the Walker A and ABC signature motifs relative
to their COOH-terminal NBDs and the NBDs of proteins
such as P-gp. The decrease in spacing is attributable to an
apparent deletion at the same location that has eliminated
13 amino acids, the only exception being MRP7/ABCC10
which lacks just 10 amino acids (191). As might be expected from these structural differences, the two NBDs
play distinct functional roles in the catalytic cycle of the
ABCC proteins (54, 136, 194, 195, 386) (see sect. IX).
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
857
plasma membrane is 1:1. Once in the plasma membrane,
MSD0-mediated interactions between the two proteins
are required for nucleotide-dependent gating of the channel. The NH2-terminal MSDs of MRP2 and the yeast MRP1
ortholog Ycf1 have been shown to be required for apical
membrane and vacuolar localization, respectively. At
present, no additional function has been ascribed to them,
and it is unclear whether they are required simply for
correct folding of the protein or contain specific targeting
signals (120, 330). Until very recently, no function had
been ascribed to MSD0 of MRP1.
Initial studies suggested that MSD0 was necessary for
the function of MRP1 (135). This suggestion stemmed
from the demonstration that removal of part of MSD0, or
the whole MSD0 and various segments of CL3, resulted in
the loss of leukotriene C4 (LTC4) transport, a high-affinity
physiological substrate of MRP1 (see sect. VIA). Furthermore, coexpression of MRP1 lacking MSD0 and most of
CL3 with a polypeptide corresponding to the missing
Physiol Rev • VOL
region restored activity indicating that the NH2-terminal
fragment containing MSD0 formed stable interactions
with the core of the protein. However, the loss of activity
was subsequently attributed to partial deletion of CL3,
rather than the elimination of MSD0 (20). Thus MRP1
lacking the first 203 amino acids MRP1204 –1531, which
removes MSD0 but leaves most of CL3 intact, traffics to
the plasma membrane and transports at least some substrates with little or no loss in efficiency (20, 394, 409,
529). Removal of as few as six additional amino acids
(MRP1210 –1531) from CL3 causes retention in the ER of
mammalian cells. When expressed in insect cells, the truncated protein remains able to traffic to the plasma membrane, but its LTC4 transport activity is severely diminished
(529). The regions of CL3 necessary for trafficking and activity of MRP1 have now been defined as lying between
Cys208 and Lys270. Although the primary sequence of this
region of CL3 is not particularly conserved among ABCC
proteins, its predicted secondary structure is, even among
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FIG. 5. Predicted topology of MRP1 showing the locations of mutations shown to affect overall activity, substrate specificity, and protein
processing. Molecular dynamic modeling of MRP1 MSD1 and MSD2 was used to predict the positions of helical elements (shown as spirals of amino
acids) and their locations relative to a lipid bilayer of 40 Å (52). Helical elements in MSD0 were predicted using HMMTOP. Mutations shown to affect
the overall activity of MRP1 are colored yellow; those affecting only certain substrates are shown in green. The conserved Walker A (red), signature
C (orange), and Walker B (yellow) are shown in colored boxes. Glycosylation sites at Asn-19, -23, and -1006 are indicated (Y).
858
DEELEY, WESTLAKE, AND COLE
more, mutations of conserved dileucine and phenylalanine residues in the COOH-terminal regions of other
ABCC proteins, such as SUR1, that cause ER retention
(462), do not impede trafficking of full-length MRP1 (530),
but do result in ER retention in the absence of MSD0
(531). Coexpression of MSD0 with these mutated proteins
rescues trafficking, indicating that the NH2-terminal domain must interact with the core of the protein in the ER.
Detailed comparative analyses of the subcellular distribution and internalization of full-length and MSD0-less
MRP1 have also revealed that MSD0 promotes retention
in, or recycling to, the plasma membrane (531). Thus
⬃50% of MSD0-less MRP1 can be detected in recycling
endosomes compared with ⬃10% of the full-length protein. In general, the observations are compatible with
evolution of the MRP1-like ABCC proteins by fusion of a
four-domain ancestor with another integral membrane
protein capable of trafficking independently to the plasma
membrane (147), followed by the subsequent loss to varying extents of duplicated and redundant processing and
trafficking signals.
V. EXPRESSION AND MEMBRANE
LOCALIZATION OF THE MULTIDRUG
RESISTANCE PROTEINS
A. MRP1
The tissue distribution and subcellular targeting of
the MRPs is quite variable. MRP1 is widely expressed,
with high levels reported in the lung, testis, kidney, skeletal and cardiac muscles, and the placenta (72, 129, 479).
Notably, MRP1 is barely detectable in adult human liver,
but in proliferating hepatocytes and liver cancer cell lines,
such as HepG2, expression is considerably higher (421).
FIG. 6. Alignment of the NH2-terminal and cytoplasmic loop 3 (CL3) regions of human ABCC proteins and probable MRP1 orthologs from
Drosophila, yeast, and nematode. The alignment was generated using ClustalW, and two secondary-structure algorithms were used to predict
␣-helical elements. Regions shown in red were predicted by PSIPRED, and those in bold face by PhD (217). The CFTR NH2-terminal ␣-helix boxed
in green was determined by NMR (81). Disease-causing CFTR mutations are indicated with a blue circle (source: CFTR mutation database,
http://www.genet.sickkids.on.ca/cgi-bin/WebObjects/MUTATION).
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more distantly related orthologs and homologs (Fig. 6).
When this 60-amino acid region of MRP1 is expressed alone,
it associates strongly with membranes and can at least partially rescue trafficking of an NH2-terminally truncated protein lacking MSD0 and most of CL3 (21, 529).
Although there is no discernible primary structure homology, the NH2-terminal region of the recently crystallized
bacterial vitamin B12 transporter subunit BtuC contains a
relatively long ␣-helix (residues 2–32) that appears to be
laterally associated with, or embedded in, the inner leaflet of
the lipid bilayer (297). The essential segment of CL3 in MRP1
contains two predicted amphipathic ␣-helices that have
counterparts in all ABCC proteins, even those that do not
contain the third NH2-terminal MSD (147, 529). In CFTR, 19
disease-associated mutations have been identified in this
region of the protein, underlining its functional importance
(CFTR mutation database, http://www.genet.sickkids.on.ca/
cgi-bin/WebObjects/MUTATION). One of these mutations
involves a highly conserved Pro residue corresponding to
209
Pro in MRP1 that defines the NH2-terminal boundary of the
essential region of CL3. Nevertheless, mutation of Pro209
either alone or in combination with two nearby Pro residues
had little or no effect on expression of MRP1, or its transport
activity (210). However, as described below, the mutational studies were carried out in full-length MRP1, and it is
possible that the presence of MSD0 may attenuate the effects of these mutations on the trafficking and activity of the
protein (531).
A probable explanation for the apparent lack of a
functional requirement for MSD0 of MRP1 has been provided by the recent demonstration that the protein contains at least partially redundant trafficking signals (531).
Thus MRP1 lacking either MSD0 or its COOH-terminal 30
amino acids remains competent to traffic to the basolateral membrane (530, 531). However, in the absence of
both regions, the protein cannot exit the ER. Further-
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
859
MRP2 clearly requires the presence of MSD0, but it has
not been established that this region of the protein contains functional apical targeting signals, or whether it is
required for correct folding of the remainder of the protein (120). For example, MSD0 of MRP2 when fused to the
core region of MRP1 does not alter the basolateral localization of the protein (531). Unlike MRP1, MRP2 contains
a PDZ-domain located at its COOH terminus (237). It has
been proposed that this domain may interact with scaffolding proteins such as radixin that could link MRP2 to
the F-actin cytoskeleton in a manner analogous to that
proposed for CFTR (234, 244, 348). In support of this
suggestion, the hepatocanalicular localization of MRP2 is
disrupted in radixin knock-out mice (234). However, it
has been reported that the PDZ motif of MRP2 can be
deleted without loss of apical localization (358). Other
regions of the protein may also contain signals that are
involved in the apical targeting of MRP2 (164, 249, 358),
but attempts to locate them by making various MRP1/
MRP2 hybrid proteins have generated inconsistent results.
B. MRP2
D. MRPs 4 and 5
MRP2 has a more restricted tissue distribution than
MRP1. The protein is expressed in the liver, kidney, small
intestine, colon, gallbladder, placenta, and lung (131, 224,
247, 287, 445). MRP2 is the only MRP that is consistently
found in apical membranes. Thus, in the liver, it is present
in canalicular membranes (224) and on apical membranes
in renal proximal tubules, placental syncytiotrophoblasts,
and intestinal epithelium (118, 131, 229, 384, 445). Its
highest expression in the gut is in the villi of the proximal
jejunum (347). Why MRP2 traffics exclusively to the apical membrane is poorly understood, and there are conflicting reports in the literature. Efficient trafficking of
MRP4 mRNA is expressed at low to moderate levels
in ovary, testis, adrenals, lung, and intestine and at somewhat higher levels in the prostate (267). Like MRP1, expression of MRP4 in normal liver is very low. Also as
observed with MRP1, in some cell types MRP4 is targeted
to apical rather than basolateral membranes (43). For
example, apical localization of MRP4 occurs in the kidney
proximal tubule and endothelial cells of the brain capillaries (270, 508), but the protein is found basolaterally in
prostate tubuloacinar cells, choroid plexus, and HepG2
cells (268, 270, 419). MRP5 is more widely expressed than
MRP4, with the highest levels of MRP5 mRNA being de-
Physiol Rev • VOL
C. MRP3
MRP3 is expressed in the adrenal gland, pancreas,
gut, gall bladder, and placenta, with lower levels being
found in liver, kidney, and prostate (30, 236, 246, 250, 447,
479). In the liver, MRP3 is present in basolateral membranes of hepatocytes close to bile ducts, as well as in
cholangiocytes lining the ducts themselves (252, 474). The
levels of MRP3 increase under any conditions that result
in cholestasis (107, 181, 246) while the presence of MRP2
in the canalicular membrane markedly decreases (497).
Thus there appears to be a reciprocal relationship between the two transporters that is presumed to protect
the liver from accumulation of potentially toxic bile constituents (107, 474, 497). In the intestine, MRP3 is present
in enterocytes in the ileum and the colon, and in the
kidney it is found in the distal tubules (428).
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Although MRP1 expression is widespread in the body, it is
nevertheless found primarily in specific cell types, including bronchial epithelial cells and hyperplastic type II
pneumocytes in the lung; proliferating Paneth cells in the
colon; Leydig and Sertoli cells in the testis; placental
syncytiotrophoblasts and epithelial cells of the endoplacental yolk sac; and mast cells, eosinophils, helper T cells,
and erythrocytes in the circulatory system (15, 46, 287,
304, 352, 380, 388, 480, 481, 498, 535, 539). MRP1 is also
expressed in a cell specific manner in the brain, as well as
the blood-brain barrier and the choroid plexus of the
blood cerebrospinal fluid barrier (99, 339, 536).
MRP1 typically localizes predominantly to the
plasma membrane and traffics selectively to the basolateral component in polarized cells (116, 175, 287, 421, 539).
This contrasts with the apical membrane localization of
other efflux pumps such as P-gp/MDR1 (495), BCRP (318),
and MRP2 (224). However, in some cell types, such as
placental syncytiotrophoblasts and brain microvessel endothelial cells, MRP1 localizes to apical membranes (479,
571). What determines this cell-type specific targeting to
different membrane compartments is not known. The
proportion of MRP1 targeted to cell surface membranes in
cultured cells also varies. For example, 80 –90% of MRP1
localizes to the plasma membrane in transfected HeLa
and HEK293 cells (8, 146, 175, 531) compared with only
⬃50% in multidrug resistant H69AR cells (8, 72). Significant intracellular accumulation of MRP1, possibly in the
Golgi, has also been reported in the drug-selected human
small cell lung carcinoma cell line GLC4-Adr (72, 511). In
GLC4-Adr and H69AR cells, intracellular MRP1 colocalized with vesicles that accumulated fluorescent substrates (96, 511). Similarly, exposure of cells transiently
transfected with MRP1 fused to green fluorescent protein
(GFP) to the fluorescent anthracycline doxorubicin resulted in accumulation of drug in vesicles bounded by
membranes containing the fusion protein (401). Consequently, MRP1 is believed to be functional on the plasma
membrane and in intracellular compartments.
860
DEELEY, WESTLAKE, AND COLE
tected in skeletal muscle and cardiac and cardiovascular
myocytes (30, 43, 93, 186, 250, 337, 571). In the brain,
MRP5 colocalizes with MRP1 and MRP4 on the luminal
(apical) side of capillary endothelial cells and is also
present in astrocytes and pyramidal neurons (186, 360).
Despite the apical location of MRP5 in microcapillary
endothelial cells, the protein is found on the basolateral
membrane of polarized epithelial cells (537, 571).
E. MRP6
F. MRPs 7–9 and ABCC13
MRP7 transcripts have been detected in many tissues
by RT-PCR, but expression levels appear to be relatively
low (19, 223). In the mouse, the highest levels of mRNA
were found in heart, skeletal muscle, and kidney (223).
These studies also revealed two splice variants of Mrp7
mRNA, designated Mrp7A and Mrp7B, the latter of which
contains two, short additional 5⬘-exons capable of encoding 41 amino acids. The relative levels of the two variants
differ among tissues, but the possible functional implications of the observation are not known. RT-PCR indicates
that human MRP8 mRNA is widely expressed, with the
exception of kidney, spleen, and colon (34, 493, 545).
Finally, there is some uncertainty surrounding the tissue
distribution of MRP9, although transcripts have been consistently detected in the testis and in breast (35, 93, 493).
However, even the longest MRP9 transcripts detected
(⬃4.5 kb) do not encode a full-length protein, as predicted
from the exon structure of the gene (35, 493). In contrast,
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VI. SUBSTRATE SPECIFICITIES AND
PHYSIOLOGICAL FUNCTIONS OF THE
MULTIDRUG RESISTANCE PROTEINS
Defining the substrate specificities of the MRPs has
been, and remains, an area of considerable activity. Many
studies have focused on xenobiotic substrates because of
the potential role of the MRPs in clinical drug resistance
and in protection against a wide range of environmental
toxicants (42, 78, 96, 161, 247, 287). Others have sought to
identify potential endogenous substrates to gain insight
into possible physiological functions of the proteins. Identification of exogenous substrates of the MRPs has frequently been based on an assessment of their ability to
confer resistance to candidate cytotoxic drugs and xenobiotics. In some cases, such studies have also incorporated cellular accumulation and efflux assays of radiolabeled or fluorescent xeno- and endobiotics. Identification
of potential physiological substrates has relied heavily on
the use of inside-out membrane vesicles from transfected
cells to directly measure ATP-dependent transport of candidate compounds, or their ability to inhibit transport of
an established substrate (22, 183, 213, 273, 298, 299, 300,
301). For several of the MRPs, information obtained from
in vitro substrate specificity studies has been augmented
by examining the consequences of knocking-out the gene
in mice or in cell lines (311, 312, 405, 534). In the case of
MRP2, much of the work defining substrate specificity has
been done by examining biliary transport defects in
MRP2-deficient GY/TR⫺ or EHBR rats (140).
A. MRP1 and MRP2
The MDR phenotype of the H69AR cells from which
MRP1 was cloned was well characterized before identification of the protein (68, 69, 70, 72, 342). Consequently,
some of MRP1’s potential drug substrates were readily
predictable and confirmed using transfected cells (73,
146, 482, 553). These included primarily natural product
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The tissue distribution of MRP6 is of particular interest because of the association between mutations in this
protein and the degenerative connective tissue disease
PXE (36, 277, 416). Individuals with this disease develop
calcified elastic fibers and abnormal collagen fibers in
elastic tissues of the skin, retina, and arteries. The mechanism by which mutations of MRP6 cause this degeneration is not known. Earlier reports indicated that expression of MRP6 may be restricted to kidney and liver,
prompting the suggestion that the protein might be involved in elimination of a metabolite responsible for the
degeneration observed in affected tissues (31, 251, 504).
This hypothesis remains a possibility, but more recent in
situ hybridization and immunohistochemical studies have
detected murine Mrp6 mRNA and protein in epithelial
cells from many tissues (27). In addition to parenchymal
cells in the liver and the proximal tubules in the kidney,
relatively high levels of the protein and/or mRNA were
found in keratinocytes of the skin, tracheal and bronchial
epithelium, intestinal mucosa and corneal epithelium, as
well as endothelial and smooth muscle cells of the cardiovascular system.
a full-length transcript of the orthologous mouse gene has
been identified, and expression appears to be restricted to
the testis (465). Furthermore, the protein encoded by
murine Mrp9 mRNA displays high homology with the
predicted open reading frame of a full-length transcript of
the human gene. Relatively high levels of expression of
the apparently degenerate human ABCC13 have been
detected in colon, bone marrow, salivary gland, and fetal
liver, but the major transcript is only ⬃1 kb (12, 212). In
the macaque, where the gene may be functionally intact,
a mRNA of ⬃5.0 kb can be found with high expression in
colon and small intestine (12). Currently, the tissue and
subcellular distributions of ABCC10 –13 proteins are not
known.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
Physiol Rev • VOL
the inability to transport cholestatic estrogen conjugates
such as E217␤G, another well-characterized substrate of
both proteins (85, 213, 298).
B. Transport and the Role of GSH
The lack of transport of unmodified drugs, combined
with the demonstrated ability of MRP1 to transport organic anion conjugates, suggested that the protein might
confer resistance by effluxing drug conjugates (213, 299).
MRP1 is indeed capable of transporting the glucuronide
conjugate of etoposide and a GSH conjugate of doxorubicin (213, 393, 433). However, while the transport of drug
or xenobiotic conjugates may contribute the resistance
profiles conferred by MRP1 and MRP2 in some situations,
it is now recognized that other mechanisms are of more
general relevance. The transport of a number of unmodified drugs to which both MRP1 and MRP2 confer resistance, such as vincristine and doxorubicin, is dependent
on or stimulated by GSH (85, 299, 301, 413). The first
indication of a requirement for GSH came from the demonstration that GSH depletion decreased drug efflux by
cells overexpressing MRP1 (451, 510, 554). Vesicle transport assays confirmed that GSH enhanced the ability of
unmodified drugs to inhibit transport of the known MRP1
substrates, E217␤G and LTC4, and that it was possible to
detect ATP-dependent transport of vincristine when GSH
was included in the transport assays (299, 301, 302). Similarly, GSH stimulates transport of unmodified drugs by
MRP2, including etoposide and vinblastine (119, 507).
Although it was initially presumed that GSH stimulation
was limited to unmodified substrates, this is not the case
and GSH stimulated transport of physiologically relevant
sulfated steroids, such as estrone-3-sulfate and DHEAS, as
well as of the tobacco smoke carcinogen, NNAL-O-glucuronide, and etoposide glucuronide by MRP1 has now
been described (281, 395, 433, 558). Interestingly, MRP2 is
also able to transport NNAL-O-glucuronide, but transport
is not stimulated by GSH and indeed is inhibited by GSH
(281). This probably reflects the fact that NNAL-O-glucuronide establishes different interactions with each protein despite their considerable structural and functional
conservation (see sect. IX). On the other hand, none of the
three known nicotine glucuronide metabolites appears to
be a substrate of MRP1 or MRP2 (288). MRP2 also does
not transport estrone-3-sulfate even in the presence of
GSH (440) but does transport other sulfate conjugates
such as acetaminophen sulfate (556).
The mechanism by which GSH stimulates transport is
complex and not fully understood. GSH itself is a relatively poor substrate for both MRP1 and MRP2 with a Km
of ⬎1 mM (280, 301, 384, 434). In contrast, GSSG is
transported by MRP1 with a much higher Vmax and a Km
of ⬃100 ␮M (274). However, in the presence of some
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cytotoxic agents, such as anthracyclines, epipodophyllotoxins, and Vinca alkaloids, as well as certain heavy metal
oxyanions. MRP1-transfected cells also displayed decreased drug accumulation and increased drug efflux,
strongly suggesting that the drugs were directly transported by MRP1 (73, 553, 554). However, initial attempts
to confirm that MRP1 was a primary, active transporter of
these compounds using vesicle transport assays were
unsuccessful. The first MRP1 substrate to be identified by
in vitro transport studies was the proinflammatory mediator LTC4, which remains the most well-characterized,
confirmed physiological substrate of the protein (272, 303,
349, 534). The discovery that LTC4 was a high-affinity
substrate arose from investigation of the possibility that
MRP1 might be a broad specificity transporter known as
the MOAT that had been identified functionally in hepatocanalicular membranes and membranes from mast cells
(273, 444). Although nothing was known of the primary
structure of MOAT at the time, a great deal was known
about its substrate specificity. As described above, naturally occurring MOAT mutations in rats and sheep with
biliary transport defects similar to human Dubin-Johnson
syndrome (110, 430, 476) enabled extensive profiling of
MOAT substrates by comparative analysis of the bile constituents of mutant and wild-type animals. The profiles
indicated that MOAT transported a diverse array of conjugated organic anions, as well as possibly free glutathione (GSH) and heavy metal GSH complexes. Fortunately,
the substrate specificities of MRP1 and MOAT, now
known to be MRP2, overlap extensively. Thus, although
the original premise was a case of mistaken identity, it
proved to be of tremendous assistance in identifying substrates for MRP1.
In vitro studies using membrane vesicles have confirmed that MRP1 and MRP2 can transport a vast array of
organic conjugates (97, 231, 287). The existence of transporters capable of effluxing GSH conjugates, collectively
termed GS-X pumps, was postulated (205) well before the
MRPs were identified. However, the substrate specificities of MRP1 and MRP2 are broader and encompass not
only GSH conjugates, but also many glucuronide and
sulfate conjugates of both xeno- and endobiotics (Fig. 2).
Consequently, MRP1 and MRP2 are important contributors to cellular extrusion and elimination of the relatively
hydrophilic products of phase II conjugation reactions
that are frequently involved in detoxification of hydrophobic xenobiotics, such as the potent fungal carcinogen
aflatoxin B1 (300). Both MRP1 and MRP2 transport conjugated leukotrienes such as LTC4 and its metabolites
LTD4 and LTE4 (85, 272, 299). Steroid and bile salt conjugates are also among the physiological substrates of
both proteins (66, 85, 212, 214, 298, 395, 558). Thus the
jaundice observed in Dubin-Johnson patients is attributable to a loss of MRP2-mediated canalicular efflux of
bilirubin glucuronides (222, 224, 230, 382), and possibly
861
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DEELEY, WESTLAKE, AND COLE
C. Physiological Roles of MRP1 and MRP2
The substrate diversity and widespread expression of
MRP1 suggest numerous possible physiological functions,
Physiol Rev • VOL
many of which have yet to be confirmed. However, some
anticipated, as well as unexpected, physiological functions of the protein have been revealed from studies of
Mrp1⫺/⫺ mice (139, 312, 313, 420, 455, 513, 534 –536). As
mentioned previously, LTC4 is the highest affinity substrate known for MRP1. LTA4 is the parental compound of
both conjugated and nonconjugated leukotrienes and is
formed from arachidonic acid by 5-lipoxygenase (436).
LTA4 is converted to LTB4, an important chemotaxin in
mediating inflammatory responses, by LTA4 hydrolase
(356), and to LTC4 by LTC4 synthase, which conjugates
the parental leukotriene with GSH at the C6 position
(549). LTC4 is produced in mast cells, basophils, eosinophils, dendritic cells, macrophages, neutrophils, platelets,
kidney, and brain (336, 456). In addition, LTC4 can be
synthesized in liver microsomes and endothelial cells by
LTC4 synthase (452, 464). Once effluxed from the cell,
LTC4 is rapidly converted to LTD4 and LTE4 by ␥-glutamyltranspeptidase and dipeptidase, respectively (266).
Collectively, the cysteinyl leukotrienes are referred to as
the slow-reacting substance of anaphylaxis. LTD4 and
LTE4 bind to G protein-coupled CysLT1 and CysLT2 receptors on target cells and are associated among other
things with mediating asthma pathologies (165). Consistent with a physiological role for Mrp1 as an LTC4 transporter, leukotriene release from eosinophils and mast
cells in response to IgE-mediated inflammation is reduced
in Mrp1⫺/⫺ mice (534). The mice also display an impaired
immune response to contact sensitization. This defect led
to the discovery of LTC4-dependent recruitment of dendritic cells to lymph nodes during responses to inflammation (420). Unexpectedly, the mice are also more resistant
to Streptococcus pneumoniae infections (455). This has
been attributed to accumulation of intracellular LTC4 in
alveolar macrophages, resulting in product inhibition of
LTC4 synthase and a consequential diversion of LTA4 to
LTB4 production. The increased resistance to infection is
likely attributable to the fact that LTB4 is a potent stimulator of phagocytic macrophages (17, 100).
Tissues that normally express relatively high levels of
Mrp1, such as the testis, kidney, and oropharyngeal mucosa, are hypersensitive to etoposide in Mrp1⫺/⫺ mice
(534, 535). The presence of MRP1 in blood-tissue barriers
such as the choroid plexus (404) also suggests that the
transporter contributes to protection of sanctuary sites in
the body. Consistent with this suggestion, Mrp1⫺/⫺ mice
display increased passage of MRP1 substrates from the
blood to the cerebrospinal fluid (536). Similarly, MRP1 in
the placenta may protect the developing fetus from xenobiotics and help prevent the fetal accumulation of endobiotics, such as E217␤G (287, 479). Similarly, the efflux of
estrone sulfate from Leydig cells by MRP1 may help to
protect the testis from the feminizing effects of estrogen
(395). MRP1 expression is also high in hyperplastic reactive type II pneumocytes, which proliferate in response to
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GSH-dependent substrates or modulators of MRP1, the
Km for GSH decreases to approximately the same range
as GSSG (284, 299, 301). Reciprocal stimulation of transport of GSH and second substrate (e.g., vincristine and
aflatoxin B1) has also been observed with both MRP1 and
MRP2, indicative of positive cooperativity between binding of the two substrates (119, 300, 301, 326). The estimated stoichiometry of transport of GSH and a drug such
as vincristine is compatible with the possibility that transport of both substrates is coupled, but this has not been
demonstrated formally. In other cases, such as estrone-3sulfate and NNAL-glucuronide, no stimulation of GSH
transport can be detected (281, 395). In addition, a number of examples exist of compounds that strongly stimulate GSH transport, also by decreasing the Km for GSH by
⬃10-fold, without themselves being transported. These
include drugs such as verapamil and bioflavonoids, such
as apigenin (282, 284, 302).
The GSH-dependent stimulation of transport of organic substrates by MRP1 is not dependent on the reducing potential of the peptide, since short-chain S-alkyl tripeptides, such as S-methyl-GSH, and non-sulfur-containing tripeptides such as ophthalmic acid are also effective
(281, 283, 301, 395). However, the effectiveness of S-alkylGSH analogs tends to decrease with increasing alkyl chain
length, possibly because the larger alkyl derivatives begin
to compete for transport of the second substrate. GSH
derivatives in which the cysteine residue has been replaced by a number of different amino acids are also able
to stimulate transport (283). The effectiveness of these
derivatives increases with the hydrophobicity of the
amino acid side chain, suggesting that the GSH cysteine
may be in a relatively hydrophobic region of the protein’s
binding pocket. The transport of heavy metal oxyanions
to which MRP1 and MRP2 confer resistance may also be
GSH dependent, and it has been suggested that it occurs
via a cotransport mechanism analogous to that proposed
for organic substrates (435). However, the major forms of
arsenic identified by analysis of bile from normal Wistar
rats are arsenic triglutathione and methyl arsenic diglutathione (221, 383). These conjugates are not found in bile
from mutant TR⫺ Wistar rats lacking MRP2, strongly suggesting that they are substrates of the protein (149). Very
recent in vitro transport studies of MRP1 have confirmed
that arsenic triglutathione is a high-affinity substrate (Km
⬍1 ␮M) and that it can be formed enzymatically by
GSTP1–1 (286). Whether other heavy metals are transported as conjugates or via some type of cotransport
mechanism with GSH is not yet known.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
D. MRP3
One of the most remarkable differences between the
substrate specificity of MRP3 and those of MRP1 and
MRP2 is its lack of transport of GSH and the poor ability
of MRP3 to transport GSH conjugates (5, 182, 184, 252,
374, 557, 562, 569). Unlike MRP1 and MRP2, MRP3 shows
a marked preference for glucuronidated compounds (66,
182, 184, 374, 557, 562, 569), consistent with its proposed
role in protecting the liver from accumulation of bile salts
and other potentially toxic conjugated compounds. Moreover, in addition to conjugated bile salts, MRP3 transports
monovalent bile salts such as cholate, taurocholate, and
glycocholate that are not substrates of either MRP1 or
MRP2 (66, 181, 184, 559, 562). This has prompted the
suggestion that MRP3 contributes to the enterohepatic
circulation of bile salts (107, 181, 252, 474). However,
Mrp3⫺/⫺ mice show no symptoms attributable to such a
Physiol Rev • VOL
defect (33, 560). In addition to being responsive to cholestatic conditions, expression of MRP3 may be coordinated with induction of phase I enzymes by xenobiotics
that activate a number of different signal transduction
pathways (317). For example, the induction of Mrp3 by
microsomal enzyme inducers has been associated with
increased hepatic efflux of conjugates of common hepatotoxic drugs, such as acetominophen, into blood (469).
More recently, Mrp3 has been shown to be almost totally
responsible for the basolateral efflux of acetaminophen
glucuronide from the liver (319). Studies with knock-out
mice have also revealed that inactivation of Mrp3 has a
major effect on the efflux of morphine glucuronides from
the liver via sinusoidal membranes resulting in a major
decrease in circulating levels of morphine-3-glucuronide
(319). Overall, the regulation and substrate specificity of
Mrp3 suggests that one of its likely physiological functions is to protect the liver from accumulation of a variety
of hepatotoxic xeno- and endobiotics (66, 107, 183, 184,
252, 447, 474, 557, 561–563, 569).
E. MRPs 4 and 5
Like MRP1, -2 and -3, MRP4 and MRP5 are organic
anion transporters (61, 62, 337, 508, 537, 558). However,
they differ from the MRP1-like proteins in their ability to
transport nucleoside and nucleotide analogs, as well as
cyclic nucleotides (61, 93, 215, 392, 407, 508, 532, 537).
The ability of MRP4 and MRP5 to transport cyclic nucleotides has prompted speculation that they contribute to
modulation of intracellular cAMP and cGMP levels (43,
61, 215, 418). However, some controversy exists concerning their affinities for these nucleotides, and several lines
of evidence suggest that they do not have a major influence over intracellular cAMP and cGMP concentrations
(261, 532). Nevertheless, the colocalization of MRP5 and
phosphodiesterase 5 in smooth muscle cells of the genitourinary tract is intriguing, particularly when coupled
with the observation that the phosphodiesterase inhibitors, such as silfenadil and trequinsin, also inhibit MRP5mediated cGMP efflux (359). This raises the possibility
that the protein may contribute to the efficacy of drugs
such as sildenafil and trequinsin in raising cGMP levels in
genitourinary smooth muscle cells.
It has also been suggested that MRP4 and MRP5 are
involved in determining extracellular levels of cyclic nucleotides. MRP4 is expressed at relatively high levels in
the kidney proximal tubules where it could contribute to
urinary excretion of cAMP and cGMP (508). More recent
studies suggest a broader role for the protein in organic
anion excretion and that MRP4 may be responsible for the
excretion of some organic anions formerly attributed to
MRP2. MRP4 transports p-aminohippurate (PAH), which
is typically used to assess organic anion transport in renal
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airborne cytotoxins and lung insults (539). Finally, there
is evidence that Mrp1 expression is higher in cholestasic
rats (521), suggesting that it may participate in protecting
the liver from the accumulation of toxic levels of bilirubin
conjugates (214, 521).
The ability of MRP1 to transport both GSH and GSSG
raises the possibility that the protein contributes to maintenance of the redox state of the cell. Studies using MRP1
overexpressing cell lines and determination of GSH levels
in tissues from Mrp⫺/⫺ mice support this suggestion. The
levels of free GSH are decreased in cells overexpressing
the protein and increased in tissues from Mrp1⫺/⫺ mice
that normally express high levels of the protein (69, 312,
517, 518). The levels of MRP1 have also been shown to
increase two- to threefold after exposure to agents that
induce oxidative stress, possibly as a result of activation
of transcription by a mechanism involving the antioxidant
responsive transcription factor Nrf2 (166). Thus MRP1
may also contribute to GSSG efflux under conditions
where GSSG production is increased (2, 96). Consistent
with this proposal, Mrp1 protects rat neural astrocytes
from exposure to H2O2 (185). Additionally, MRP1 has
been implicated in the transport of toxic lipid oxidation
products, such as 4-hydroxynonenal-GS, that are formed
during periods of oxidative stress (287, 414).
As indicated above, MRP2 is responsible for the
hepatobiliary excretion of organic anions into bile, including bilirubin and bile salt conjugates (4, 49, 224, 230, 382).
Because of its broad substrate specificity, MRP2 is also
expected to be a major contributor to the biliary disposition of many drugs and xenobiotics, as well as their
conjugates (4, 105, 140, 157, 161, 231, 281, 440, 458, 487,
492, 543, 544). Because MRP2, like MRP1, transports GSH
and GSSG, it has also been suggested that it plays a role
in protecting cells from oxidative stress (384).
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DEELEY, WESTLAKE, AND COLE
uronidated substrates of the other transporters, such as
E217␤G, appear not to be MRP6 substrates (32). In addition, MRP6 transports the cyclic peptide endothelin receptor antagonist BQ-123 but not endothelin-1 itself (32,
316) and has been reported to confer low-level resistance
to various epipodophyllotoxins and anthracyclines (32).
The majority of PXE-causing defects map to NBD2 of the
protein, suggesting that the disease is associated with loss
of its transport function rather than altered substrate
specificity (277–279). The prevalent expression of the protein in the liver and kidney suggests the possibility of a
defect in systemic disposition of an as yet unidentified
substrate. A potentially interesting association between
decreases in sulfated glycosaminoglycans (GAGs) in the
urine has been observed in PXE patients and to a lesser
extent in healthy carriers of the disease (315). Whether
the decrease in urinary GAGs is attributable to loss of
MRP6 function and is in some way linked causatively to
PXE has not been established.
G. MRPs 7 and 8
Little is known of the specificity of MRP7 for potential endogenous substrates. At present, the protein has
been shown to transport E217␤G in vitro, but with relatively low affinity compared with MRP1, MRP2, MRP3,
and LTC4 but also poorly compared with the other three
proteins (63). Among the shorter MRPs, MRP8 appears to
have an exceptionally broad substrate specificity. In addition to the ability to transport cyclic nucleotides, as
shown with MRP4 and MRP5, MRP8 is also able to transport glutathione, glucuronide, and sulfate-conjugated substrates such as LTC4, E217␤G, and estrone-3-sulfate as
shown for MRP1 and MRP2. In addition, MRP8 shares
with MRP3 the ability to transport monoanionic bile acids, such as glycocholate and taurocholate, and like MRP3
is expressed in liver, raising the possibility that it might
contribute to bile acid homeostasis (64).
VII. IN VITRO DRUG RESISTANCE PROFILES
AND INHIBITORS OF THE MULTIDRUG
RESISTANCE PROTEINS
A. Drug Resistance Profiles
F. MRP6
MRP6 has been shown to transport a number of
GS-conjugated organic anions in vitro that are also transported by other MRP1-like MRPs, including LTC4, S-(2,4dinitrophenyl)glutathione, and N-ethylmaleimide S-glutathione (NEM-GS), and the protein is also inhibited by
relatively nonspecific anion transport inhibitors, such as
probenecid and indomethacin (32, 202). However, glucPhysiol Rev • VOL
The drug resistance profiles of the MRPs have been
described extensively in recent reviews, and only a brief
overview is provided here (78, 96, 161, 256, 257, 287).
Historically, human ABC drug efflux pumps, typified
by P-gp/MDR1, have been associated with resistance to
natural product-type cytotoxic agents (10, 219). Although
initial characterization of MRP1 revealed a considerable
overlap between the drug resistance profiles of the two
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proximal tubular cells, and does so with considerably
higher affinity than MRP2 (471). Furthermore, PAH excretion is normal in mutant rats lacking MRP2, suggesting
that MRP4 may be the primary physiological transporter
responsible (471). Whether or not GSH is required for
transport of cyclic nucleotides and possibly other substrates by MRP4 and MRP5 remains unresolved, although
it has been reported that MRP4 cotransports GSH with
bile salts (419). Efflux of cAMP by MRP4 was initially
reported to be inhibited by depletion of intracellular GSH
with agents such as buthionine sulfoximine (BSO), and
overexpression of the protein was associated with a decrease in GSH levels (261). Polarized MRP5 transfected
cells were also reported to efflux GSH (537). However,
subsequent studies using transfected, nonpolarized HEK
cells failed to confirm a GSH requirement for cAMP transport by either protein (532).
MRP4 can also transport prostaglandins PGE1 and
PGE2, neither of which are transported by MRP1, MRP2,
MRP3, and MRP5, with relatively high affinity (408). The
transport of PGE1 and PGE2 is also potently inhibited by
other prostaglandins and thromboxane B2, as well as
several nonsteroidal anti-inflammatory drugs (NSAIDS),
suggesting that the release, as well as the synthesis of
prostaglandins, may be blocked by at least some of these
widely used drugs.
Given the initial characterization of the substrate
profile of MRP4, it was unexpected to find that hepatic
expression of the protein is upregulated with that of
MRP3 during cholestasis (14, 102). Furthermore, the levels of MRP4 are even more profoundly affected than those
of MRP3 when expression of the bile salt export pump
(BSEP/ABCB11) is severely diminished (454). The decrease in BSEP expression is also accompanied by an
increase in both serum and urinary bile acids, suggesting
that MRP4 might be involved in the efflux of these compounds in the liver and the kidney. Based on the ability of
various bile salts to inhibit the transport of E217␤G by
MRP4, the protein appears to have a relatively high affinity for sulfated bile salts and steroids (558). Consequently,
it is possible that MRP4, together with MRP3, contributes
to a shift to urinary excretion of bile salts and other
organic anions during conditions when biliary excretion
is compromised.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
Physiol Rev • VOL
small changes in levels of the protein to confer detectable
resistance to some drugs may be important contributing
factors.
As with the MRP1-like MRPs, the shorter MRPs that
have been characterized (MRP4, MRP5, and MRP8) share
overlapping, but not identical, drug resistance profiles
(61, 154, 268, 337, 407, 453, 537). Among these proteins,
only MRP4 has been shown to be overexpressed in response to drug selection (453). The overexpression of
MRP4 in drug-selected human T-lymphoid CEM cells provided the first example of a human ABC transporter capable of conferring resistance to nucleoside analogs, such
as azidothymidine monophosphate (AZT), 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and Lamivudine (453).
In addition to these anti-HIV drugs, MRP4 has been shown
to confer resistance to the antiviral drug Gancyclovir (1).
MRP5 also confers resistance to PMEA, and both proteins
increase resistance to the thiopurines thioguanine and
6-mercaptopurine (537). However, neither MRP4 nor
MRP5 appears to confer substantial resistance to anticancer nucleoside drugs, such as gemcitabine and cytarabine
(407). MRP5 confers resistance to 5-fluorouracil (392),
whereas MRP4 does not (407). MRP8 also confers resistance to 5-fluorouracil as well as other fluoropyrimidines
(154). Like MRPs 4 and 5, MRP8 also confers resistance to
PMEA (154). Thus, while the MRP1-like MRPs are associated with resistance to natural product drugs, the
shorter MRPs primarily confer resistance to nucleoside
and nucleotide analogs. Nevertheless, members of the
two subsets of proteins share the ability to confer resistance to the antimetabolite methotrexate and camptothecin-like topoisomerase I inhibitors, such as irinotecan.
MRP1, -2, -3 , -4, and -8 all confer resistance to short-term
exposure to methotrexate, and all have been shown to
transport the drug in its unmodified nonglutamylated
form (187, 252, 558). MRP5 has also been shown to confer
resistance to methotrexate and to be capable of transporting not only the unmodified drug but also methotrexate dibut not triglutamate (533). In addition, MRP1, MRP2, and
MRP4 have been found to increase resistance to irinotecan and its active metabolite SN-38 (193, 370). Studies
with Mrp4⫺/⫺ mice indicate that Mrp4 may protect the
brain from accumulation of topotecan (270).
Although most commonly associated with resistance
to “traditional” cytotoxic drugs, the MRPs, notably MRP1,
may confer resistance to more recently developed cytotoxic peptides, antimetabolites, and immunoconjugates.
Thus MRP1 has been shown to confer resistance to the
cytotoxic peptide N-acetyl-Leu-Leu-norleucinal (ALLN)
and a toxic peptide derivative, which is based on a ThrHis-Thr-Nle-Glu-Gly backbone conjugated to butyl and
benzyl groups (4A6) (98). In addition, MRP1 also appears
to confer resistance to the naturally occurring bicyclic
peptide depsipeptide FK228 that has shown promise in
phase II clinical trials of cutaneous T-cell lymphoma.
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proteins, some important distinctions were also observed
(96, 161, 256, 287). Notable among these differences was
the ability of MRP1 to confer resistance to certain arsenical and antimonial oxyanions and its relatively poor ability to confer resistance to taxanes (73, 482, 553).
The MRP1-like MRPs all share to a greater or lesser
extent the ability to confer resistance to natural producttype drugs, presumably reflecting their evolution as a
protective mechanism against xenobiotics encountered in
the diet and the environment (161, 256, 287). However,
studies using transfected cell lines have revealed that
some interesting examples of substrate overlap and
complementarily exist among these proteins (32, 73, 85,
118, 146, 187, 192, 252, 268, 337, 482, 533, 557, 569). The
drug resistance profiles of MRP1 and MRP2 are most
similar with respect to natural product type drugs and are
relatively broad (231). Both proteins confer resistance to
anthracyclines, Vinca alkaloids, and epipodophyllotoxins,
and both confer only very low levels of resistance to
taxanes (73, 85, 118, 146, 198, 482, 553). Transport of
several of the natural product drugs by both MRP1 and
MRP2 is also stimulated by GSH (85, 301, 413). A major
distinction between MRP1 and MRP2 is the ability of the
latter to confer low-level resistance to platinum-based
drugs (85, 240). In addition to anticancer drugs, MRP2 but
not MRP1 is able to transport HIV protease inhibitors
such as saquinivir and indinavir (197).
MRP3 appears to have a narrower drug profile than
either MRP1 or MRP2 and confers resistance to epipodophyllotoxins, but not anthracyclines or Vinca alkaloids
(252, 557, 569). MRP3 also differs from MRP1 and MRP2
in that its ability to confer resistance to epipodophyllotoxins is not GSH dependent (557). As indicated above,
MRP3 is a very poor transporter of GS-conjugates and
does not transport GSH, which may explain its inability to
confer resistance to other classes of natural product type
drugs (252). The drug resistance profiles of MRP6 and
MRP7 have been less extensively characterized (32, 192,
316). However, MRP6 has been reported to confer modest
levels of resistance to etoposide and teniposide (3- to
4-fold) and low levels of resistance to anthracyclines and
cisplatin (32). Like MRP3, MRP6 does not confer resistance to Vinca alkaloids (32). MRP7 appears unique
among the MRPs in conferring resistance preferentially to
taxanes, such as docetaxel and paclitaxel (192). The protein also confers low levels of resistance to Vinca alkaloids, but not anthracyclines or epipodophyollotoxins
(192). Despite the overlapping resistance profiles of the
MRP1-like MRPs, selection in natural product drugs to
which several of these proteins can confer resistance
appears overwhelmingly to result in increased expression
of either MRP1 or P-gp/MDR1. Why among the MRPs,
MRP1 is preferentially overexpressed is not fully understood. However, the widespread basal expression of
MRP1 in numerous cell types and the ability of relatively
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DEELEY, WESTLAKE, AND COLE
B. Inhibitors and Reversing Agents
Given the broad substrate specificity of the MRPs,
the definition of an inhibitor becomes to some extent
application dependent, since although there are a growing
number of examples of compounds that bind to these
proteins without being transported, many substrates also
compete reciprocally for transport. Furthermore, in many
cases it is not known whether the “inhibitor” is a substrate
or not. Experimentally, inhibitors have been used as a
means of implicating MRPs in resistance to a variety of
drugs, or in the transport of a wide range of xeno- and
endobiotics. To date, the inhibitors in general use have
been of relatively low affinity and specificity. In some
cases, they were originally designed for extracellular targets and, as a consequence, may have relatively low membrane permeability. One example is MK571, which was
designed as an LTD4 receptor antagonist. MK571 is an
effective inhibitor of MRP1, MRP2, and MRP4, but its use
with intact cells requires concentrations in excess of 5 ␮M
(60, 138, 419, 507). Other compounds have been previously characterized as inhibitors of other ABC transporters, such as the sulfonylurea glibenclamide. Glibenclamide is an inhibitor of SUR1/ABCC8, but it also inhibits
MRP1 and some other ABC transporters that are not
members of the C branch of the superfamily, such as
ABCA1 (29, 48, 77, 163, 385). Some compounds that inhibit MRPs, for example, probenecid, are even less specific and are general inhibitors of organic anion transport
(22, 145, 517). There has also been considerable interest
in the potential of dietary flavonoids such as genistein and
quercetin, as well as synthetic flavonoids such as flavopiridol, to inhibit both MRP1 and MRP2 (106, 188, 211,
282, 284, 516). This is both because of their potential as
reversing agents and also because of possible drug interPhysiol Rev • VOL
actions that may occur as a result of dietary intake of
these compounds or their use as alternative therapies
involving these compounds.
In addition to inhibitors that have been used for
experimental purposes, there has been considerable interest in developing novel compounds and treatment regimens that may prevent or reverse clinical multidrug resistance mediated by MRP1 (reviewed recently by Boumendjel et al., Ref. 44). Some strategies designed to
specifically inhibit MRPs as opposed to other drug transporters exploit the GSH dependence of transport of some
cytotoxic drugs and the fact that GSH conjugates are not
in general substrates for proteins such as P-gp/MDR1 and
BCRP (161, 287). One approach has been to block GSH
synthesis with the ␥-glutamylcysteine synthetase inhibitor
BSO (138, 150, 338, 518, 554). In addition, a number of
GSH peptidomimetics, some of which were developed as
glutathione S-transferase (GST) inhibitors, also inhibit
transport by MRP1, as do various protease-resistant GSH
derivatives (50, 372). GS-conjugates, some of which are
GST inhibitors, such as GS-ethacrynic acid, are also good
substrates for MRP1 and potential competitive inhibitors
of the transport of other pharmacologically active compounds (44, 50, 51, 555).
Several compounds that were developed originally
as inhibitors of P-gp/MDR1 have also been shown to
inhibit MRP1. These include the quinoline derivative
MS-209 (354) and the pipecolinate derivative VX-710
(biricodar) (141, 142). The latter compound appears to
inhibit not only P-gp/MDR1 and MRP1, but also BCRP
(341). The naturally occurring polyhydroxylated sterol
Agosterol A can also inhibit both P-gp/MDR1 and
MRP1, and an azido-derivatized analog of the compound has been used to photolabel both proteins (see
also sect. IXB) (13, 343, 409 – 411). Although multifunctional agents such as VX-710 hold the appeal that they
may simultaneously inhibit multiple drug transporters,
they may also increase the potential for changes is
pharmacokinetics that potentially limit tolerable drug
dosages, and their IC50 values for the individual transporters may vary considerably. In part because of these
considerations, several attempts have been made to
develop high-affinity and high-specificity MRP1 inhibitors (44). One such series of inhibitors, comprised of
pyrrolpyrimidine analogs, has been described, some of
which show considerable specificity for MRP1 as opposed to other drug transporters, with IC50 values in the
submicromolar range (526, 527). However, these compounds display some ability to inhibit drug metabolizing cytochrome P-450s such as CYP3A (527). At
present, the most highly specific MRP1 inhibitors described are based on tricyclic isoxazoles, and these
were discovered by high-throughput screening. LY465803
and a closely related photoactivatable derivative,
LY475776, inhibit MRP1 with EC50 values in the range of
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FK228 interferes with several transduction pathways and
has most recently been demonstrated to be a potent inhibitor of histone deacetylase (344). Interestingly, FK228
is a prodrug that is activated intracellularly by glutathionedependent reduction of a disulfide bond. Whether it is the
parental compound or its reduced form that may be transported by MRP1 is not presently known (542). MRP1 has
also been implicated in resistance to the immunoconjugate gentuzumab-ozogamicin, a promising agent in the
treatment of acute myeloid leukemia (523). The conjugate
consists of an antibody targeted to CD33 that is conjugated via an acid-hydrolyzable linker to a derivative of the
antitumor antibiotic calicheamicin-␤1, which is released
following internalization and entry into lysosomes (269).
Finally, there is some evidence that MRPs’ capability of
transporting methotrexate may limit the uptake of recently developed antifolates, such as 10-deazaminopterin
(233, 467, 533).
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
10 – 80 nM (327, 366, 367, 396, 463). These compounds do
not inhibit either MRP2 or BCRP, and their affinity for
P-gp/MDR1 appears to be ⬃100-fold lower than that for
MRP1 (88, 463). Their ability to bind to MRP1 is also
dependent on GSH or certain of its analogs (see sect. IXB)
(88, 327, 396).
VIII. CLINICAL RELEVANCE OF THE
MULTIDRUG RESISTANCE PROTEINS
below). The possible involvement of MRPs and other
transporters in clinical drug resistance has been the
subject of recent reviews (75, 275, 276). Consequently,
we have focused primarily on studies that either indicate a significant association between transporter expression and negative disease outcome and/or that are
supported by relevant biological studies.
B. MRP1 and Solid Tumors
A. Background
The widespread expression of MRP1 in normal tissues provides an additional challenge to assessing the
implications of its presence in clinical samples. This is a
particular problem when using approaches such as RTPCR and immunoblotting with tumor samples that may
contain varying amounts of normal tissue (28, 168). Nevertheless, these approaches, combined with immunohistochemistry using well-characterized MRP1-specific
monoclonal antibodies (128, 129, 175, 176, 178, 243, 446,
539), have provided convincing evidence of elevated expression of MRP1 in a variety of solid tumors, including
such common cancers as lung, breast, and prostate. The
strongest case for a functional role of MRP1 in the clinical
resistance of solid tumors is provided by several consistent studies of lung cancer.
1. Lung cancer
Frequent expression of high levels of MRP1 has
been found in non-small cell lung cancer (NSCLC),
which accounts for ⬎75% of lung cancer cases (363,
484, 539). NSCLC, unlike small cell lung cancer (SCLC),
is inherently multidrug resistant, and moderate to high
levels of expression of MRP1 have been found in a high
proportion of both untreated adenocarcinoma and
squamous cell carcinomas, the two major forms of
NSCLC (267). High levels of MRP1 have been correlated
with a higher grade of differentiation of NSCLC, particularly adenocarcinoma (363, 484, 539). Although this
would suggest that the protein is more prevalent in less
aggressive tumors that might be expected to have a
better prognosis, two early studies concluded that elevated MRP1 levels are a predictor of poor response to
treatment with drugs known to be substrates of the
protein (376, 377). A recent, more extensive study of
NSCLC not only confirmed the expression profiles
found previously, but also found that the level of MRP1
was a highly significant negative indicator of response
to chemotherapy and overall survival (38).
The frequency of MRP1 expression in untreated
SCLC is lower than in NSCLC. When present, the protein
appears to be limited to small foci of cells within the
tumor and the tumor periphery, rather than the more
uniform expression observed in NSCLC (38, 539). Nevertheless, MRP1 positivity in untreated tumors has been
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Despite overwhelming evidence from in vitro and
intact animal studies of the ability of ABC drug transporters to increase resistance to a wide variety of cancer chemotherapeutic agents, with few exceptions their
contribution to clinical drug resistance remains poorly
defined. To a considerable degree, this may be attributable to the challenges of designing and implementing
informative clinical trials. Ideally such trials would 1)
utilize reliable assays to determine the profile and levels of transporter expression in patient tumors before
and after treatment, 2) correlate outcome with transporter expression, and 3) demonstrate improved outcome following confirmed inhibition of individual
transporters. A number of trials of P-gp/MDR1 reversing agents that attempted to meet these goals have had
disappointing results (25, 45, 121, 126, 127, 275). However, interpretation of the outcome is confounded by
the fact that the existence of alternative transporters
was unknown or not assessed in the patient population.
Furthermore, earlier P-gp/MDR1 reversing agents were
of relatively low specificity and affinity and in some
cases were found to have significant pharmacokinetic
effects that required reduction in dosing of the chemotherapeutic agent(s) used. With at least one second
generation P-gp/MDR1 reversing agent, PSC833 (a nonimmunosuppressive derivative of cyclosporine), inhibition of other ABC transporters involved in hepatic
clearance, such as MRP2 and the bile salt transporter
BSEP/ABCB11, as well as cytochrome P-450s, such as
CYP3A, may have been a contributing factor (25, 275,
501). More recently, high-affinity highly specific, P-gp/
MDR1 specific reversing agents have been developed.
One of these, zosuquidar (LY335979), has shown minimal pharmacokinetic effects, combined with confirmed
inhibition of P-gp/MDR1 in recent phase I trials involving solid and hematological malignancies (130, 143,
437). At present, the outcome of phase II and phase III
trials of zosuquidar is not known. To date, there have
been no comparable trials of MRP specific reversing
agents. However, studies of clinical samples have revealed widespread expression of the MRPs in a variety
of tumor types, particularly in the case of MRP1 (see
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DEELEY, WESTLAKE, AND COLE
found to be predictive of poor response to chemotherapy
in two studies (200, 259). The restricted expression of
MRP1 may be correlated with the fact that SCLC is generally responsive to initial chemotherapy but almost always manifests itself as drug-resistant disease upon relapse. For ethical reasons, tumor material is rarely collected from relapsed SCLC patients. Consequently, the
levels of MRP1 in relapsed drug-resistant SCLC have not
been firmly established. However, a small study in which
longitudinal samples were obtained did find a posttreatment increase in MRP1, as well as several other drug
resistance markers (255).
Several independent studies indicate that MRP1 expression is a negative prognostic marker for some types
of breast cancer. Three of them concluded that MRP1
positivity of early-stage breast cancer was associated with
shorter times to relapse after postsurgical adjuvant chemotherapy (364, 365, 431). The results of these investigations have recently been supported by a larger analysis of
MRP1 expression in over 500 premenopausal women with
early-stage breast cancer (124). This latest study also
indicates a strong association between expression of
MRP1 and reduced time to relapse, as well as reduced
overall survival.
The levels of MRP1 in tumors of relapsed breast
cancer patients who received presurgical adjuvant chemotherapy have consistently been found to be higher than
in the initial tumor (122). However, the increases in MRP1
levels were not predictive of response to chemotherapy.
Analysis of MRP1 expression in axillary lymph nodes of
breast cancer patients also indicated that levels of protein
were higher in the metastases than in the primary tumor,
while the reverse was the case for P-gp/MDR1 (573). The
drug regimen used for adjuvant chemotherapy in the
above studies is comprised of cyclophosphamide, 5-fluorouracil, and methotrexate. A number of MRPs, including MRP1, have been shown to confer resistance to methotrexate (62, 187, 252, 557). In addition, increased expression of MRP1 in transfected MCF7 breast cancer cells
results in a moderate increase in resistance to cyclophosphamide (346). To date, only MRP5 and MRP8 have been
shown to confer resistance to 5-fluorouracil (154, 392).
However, MRP5 is only able to transport the metabolized
form 5⬘-fluoro-2⬘-deoxyuridine, and it is not known if this
is also true for MRP8. MRP8 has also been reported to be
expressed in breast cancer (34). However, its expression
was not determined in the studies described.
4. Neuroblastoma
Neuroblastoma is the most common extracranial
solid tumor in children (335). One of the strongest negative molecular indicators of outcome in neuroblastoma is
amplification of the NMYC gene (82). The expression of
MRP1 has been reported to be positively correlated with
NMYC amplification and to be an independent, negative
prognostic indicator (324, 368). Studies of MRP1 expression in neuroblastoma cell lines support a role for NMYC
as a positive regulator of MRP1 transcription. In addition,
modulation of NMYC levels influences resistance to drugs
that are known MRP1 substrates, and VX-710 sensitizes
neuroblastoma cell lines that express MRP1 to a similar
range of drugs (155, 324, 369, 547).
C. MRP1 and Hematological Malignancies
3. Prostate cancer
MRP1 overexpression has been documented as a
resistance mechanism in prostate cancer cell lines sePhysiol Rev • VOL
There have been a number of analyses of MRP1 expression in both acute and chronic leukemias with discordant
results. As with solid tumors, the clinical significance of
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2. Breast cancer
lected by exposure to doxorubicin (552). The protein is
expressed at moderate levels in normal prostatic epithelium and at high levels in prostatic intraepithelial neoplasia, as well as in prostatic adenocarcinoma (75). The
levels of MRP1 have been reported to increase with cancer stage and invasiveness (485) and to be positively
associated with mutant p53 status of the tumors, an association that has also been observed in NSCLC (259,
485). The clinical correlation between high levels of MRP1
expression and p53 mutation is supported by in vitro
studies of the regulation of the human and murine MRP1/
Mrp1 genes that have shown that wild-type p53 is a strong
suppressor of MRP1/Mrp1 transcription (351, 486, 525).
The fact that the commonly used antiandrogen flutamide
and its active metabolite hydroxyflutamide are effectively
effluxed by MRP1 overexpressing cells raises the possibility that MRP1 may contribute to the development of
hormonally refractory forms of the disease for which
there is currently no effective systemic therapy (153). To
date, there has been one phase II trial of the effect of
combining mitoxantrone and prednisone with the dual
P-gp/MDR1 and MRP1 inhibitor VX-710 (Biricodar, Incel)
in the treatment of hormone refractory prostate cancer.
The results of the trial showed little if any effect of the
addition of the inhibitor (400). However, the ability of
MRP1 to confer resistance to mitoxantrone has not been
firmly established. Although some drug-selected cell lines
that overexpress MRP1 have been reported to be crossresistant to mitoxantrone (162, 342, 450), studies with
MRP1 transfected cells have yielded contrary results (73).
Consequently, additional trials with other drug regimens
that include more firmly established MRP1 substrates
might be justified.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
D. Clinical Relevance of Other MRPs
Assessment of the contribution of other MRPs to clinical resistance is at a very early stage, and there is presently
little evidence of an association between expression and
treatment response or disease outcome. A survey of the
presence of MRP2 in various cancers using tissue microarrays found that the protein was expressed at varying frequency and levels in renal, gastric, breast, lung, colon, and
ovarian carcinomas (438). Unlike MRP1, expression of
MRP2 has been causatively linked to cisplatin resistance in
a number of in vitro studies (85, 229, 230, 293, 333), and
increased expression of MRP2 has been associated with
cisplatin resistance in human colorectal carcinoma (174).
Elevated expression of MRP2 and MRP3 has also been reported in hepatocellular carcinoma (40, 357). Together with
other factors, MRP2 expression levels were found to be
predictive of response in ovarian cancer (332), while other
studies of ovarian cancer have found an unfavorable outcome to be linked to MRP1 and MRP3 levels (373). Posttreatment increases in MRP1, MRP2, and MRP3 expression
have also been reported in bladder cancer (491), and increased expression of MRP3, together with MRP1, has been
observed in human glioma (156). Recently, MRP3 was reported to be overexpressed in pancreatic carcinoma (248)
and to be associated with poor outcome and a failure to
respond to chemotherapy in childhood ALL and AML, respectively (477). Among the short MRPs, MRP4 has been
reported to be a negative prognostic factor in neuroblastoma and MRP5 to be overexpressed in pancreatic carcinoma (248, 370), while MRP8 and a mRNA encoding a
possibly truncated form of MRP9 have been detected at high
levels in breast cancer (34, 35).
Physiol Rev • VOL
IX. MECHANISM OF TRANSPORT
A. ATPase Activities of Purified ABCC Proteins
The ATPase activities of purified ABCC proteins such
as MRP1, MRP2, and CFTR are about two orders of
magnitude lower than reported for some prokaryotic ABC
transporters and P-gp/MDR1 (7, 11, 158, 291, 320, 325, 326,
402, 406). Consequently, it has proven difficult to study
the hydrolytic properties of these proteins using crude
membranes. Native human MRP1 has been purified from
H69AR cells, and a histidine-tagged form of the protein
has been purified from transfected baby hamster kidney
cells and in P. pastoris (57, 58, 320, 325, 326, 540). When
reconstituted into proteoliposomes, purified MRP1 has an
ATPase activity of 5–10 nmol/mg protein. In addition to a
much lower Vmax value than P-gp, MRP1 displays a severalfold higher affinity for ATP with a Km of 100 –300 ␮M
(325, 326, 461). A similar Vmax value was obtained for the
ATPase activity of purified reconstituted MRP2 (158).
Possible reasons for the relatively low ATPase activities
of the ABCC proteins when compared with some other
ABC proteins have been provided by studies of the ATP
binding and hydrolysis characteristics of the individual
NBDs of CFTR (6, 7, 24, 132, 488), MRP1 (136, 194 –196,
353, 386, 387, 412, 530, 548, 572), and SUR1 (334, 503). For
the purposes of this review, we have focused on studies of
MRP1 that illustrate functional differences between the
protein’s NH2- and COOH-proximal NBDs. However, the
results of these and other studies reveal extensive similarities among MRP1, CFTR, and SUR1 that can likely be
extrapolated to other C branch proteins.
B. ABC NBDs Functional Cooperatively
A fundamental characteristic of all ABC transporters
studied to date is that their ATPase activity is highly
dependent on cooperative interactions between the two
NBDs. Although there have been reports describing the
ATPase activity of purified, soluble forms of the NBDs
from a number of eukaryotic ABC proteins, including
ABCC proteins, these data are difficult to interpret given
the established interdependence of the two domains in
the intact protein. The reason for this interdependence
was initially suggested by the crystal structure of the
soluble ABC-like protein Rad50, which dimerizes in the
presence of ATP (189, 190). These studies were subsequently supported by elucidation of the structures of bacterial ABC transporters such as MsbA from Vibrio cholera
(vcMsbA) (56) and BtuCD (297). The crystal structures of
Rad50, vcMsbA, and BtuCD indicate that the Walker A
and Walker B motifs of one NBD cooperate with the C
motif of the apposing NBD, to form two composite nucleotide binding sites (NBSs) (Fig. 7A). Thus involvement of
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MRP1 expression in leukemic cells is complicated by the
fact that the protein is expressed in all normal hematopoietic cell lineages. In acute myeloblastic leukemia (AML),
MRP1 expression has generally not been found to be
prognostic of response to chemotherapy (239, 391, 499,
509). However, it may be correlated with stage in AML
(391, 509) and possibly overall survival (123). One of
the challenges to interpretation of the results of studies
such as those cited is the possible presence in leukemic
cells of multiple transporters. For example, one study
of AML found that the combined presence of MRP1 and
P-gp/MDR1 was predictive of resistance to treatment
while each transporter alone was not (271). Similarly,
MRP1 has not been found to be prognostic in acute
lymphocytic leukemia (ALL) (87, 239, 441). In contrast,
expression of MRP1 in chronic lymphocytic leukemia
has generally been found to be high and may be of
clinical significance (79, 220, 362).
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FIG. 7. A: the ATP binding pocket based on the crystal structure of HlyB NBD dimer. The figure shows views of an ATP molecule in one of two
nucleotide binding pockets based on the crystal structure of the HlyB dimer. Views were chosen to illustrate the interaction between the nucleotide
and the conserved Walker A and Walker B motif of one NBD molecule and the signature “C” sequence from its partner (551). The figure was
generated and edited using PyMol to expose the nucleotide binding pocket. B: 2-dimensional schematic of a closed NBD dimer with two bound ATP
molecules. The schematic shows a possible, generic, “head-to-tail” MRP NBD dimer model that is based on conserved structural elements from
known ABC protein crystal structures with ␣-helices and ␤-sheets shown as cylinders and arrows, respectively. NBD1 is shown in blue, and NBD2
is in green. The ␣-helix in NBD2 designated as 7’ corresponds to the 13-amino acid region between the Walker A and ABC signature sequence that
is missing from NBD1. Regions believed to be important for NBD-NBD communication are shown in transparent boxes.
both NBDs in the formation of each NBS provides an
explanation for the cooperativity observed during studies
of the transport cycle of ABC proteins such as P-gp/MDR1
and some bacterial transporters (19, 92, 173, 253, 309,
Physiol Rev • VOL
448). For ease of discussion, we have followed a convention when referring to ATP binding or hydrolysis by a
specific NBD, of ascribing the function to the NBD that
contributes the Walker motifs to the NBS.
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TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
C. Stoichiometry of ATP Hydrolysis and
Substrate Transport
D. Experimental Approaches Used to Study ATP
Binding and Hydrolysis
Much of the evidence of functional differences between the NBDs of MRP1 has come from ATP binding and
Physiol Rev • VOL
ADP trapping experiments with photoactivatable derivatives of ATP such as ␥- or ␣-[32P]8-azido-ATP derivatives
(136, 194, 353). At 4°C, these derivatives can be used to
examine nucleotide binding under conditions that minimize hydrolysis, and the bound nucleotide can be covalently linked to one or the other NBD by ultraviolet
irradiation. Comparison of the results obtained with ␣and ␥-32P-labeled derivatives allows determination of
whether the cross-linked nucleotide is ATP or ADP (125,
136, 194, 353, 387, 530). At physiological temperatures,
labeled azido-ADP generated by hydrolysis can be
trapped in the presence of orthovanadate or beryllium
fluoride, either in a form believed to mimick a posthydrolytic transition state or an ATP-binding ground state, respectively (125, 505). Two approaches have been used to
selectively investigate nucleotide binding and trapping by
each NBD. The first relies on labeling of the protein
followed by cleavage at a protease hypersensitive site in
the cytoplasmic linker connecting the NH2-proximal NBD
to MSD2 (Fig. 5) (176, 194, 353). The two major fragments
produced can then be separated by SDS-PAGE. Alternatively, dual-expression vectors have been used to coexpress stoichiometrically equivalent amounts of two protein fragments, similar to those produced by limited
trypsinolysis, which associate with very high efficiency to
form a functional LTC4 transporter (136).
With the use of the experimental approaches described above, it has been shown that Mg2⫹-dependent
ATP binding at 4°C occurs primarily at NBD1, while trapping of ADP at higher temperatures in the presence of
orthovanadate or beryllium fluoride occurs predominantly at NBD2 (136, 194, 242, 353, 387, 530). These and
other studies have revealed that the affinity of NBD1 for
ATP is two- to threefold higher than that of NBD2 (548)
and that the latter is the major, perhaps only, site of ATP
hydrolysis. Several studies have concluded that NBD1 of
proteins such as MRP1 (136, 194, 353, 572), CFTR (7, 24,
37), and SUR (334) may be incapable of ATP hydrolysis.
However, at least in the case of MRP1, there is also
evidence to the contrary (see below) (386). The nonequivalence of the two NBDs is supported by several
additional observations. Thus, although ATP binding and
hydrolysis at NBD2 is highly dependent on ATP binding to
NBD1, binding of ATP by NBD1 is much less dependent
on the functional state of NBD2. For example, binding of
ATP by NBD1 does not decrease when the ATP binding or
ATPase activity of NBD2 is eliminated by mutation of
essential residues in its Walker A or Walker B motifs (136,
194). Furthermore, the expression of soluble forms of the
two NBDs of MRP1 has shown that NBD1 is able to bind
ATP with relatively high affinity in the absence of NBD2,
while no binding could be detected under similar conditions with soluble NBD2 (136, 538). These observations
indicate that relatively tight binding of ATP can occur at
NBD1 independently of interactions with NBD2 while
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Studies of ABC proteins from prokaryotes where the
NBDs are structurally identical and of P-gp/MDR1 have
supported a general model of the transport cycle in which
NBDs are functionally equivalent and hydrolysis of ATP
occurs alternately at each NBS (262, 361, 443, 459, 460,
512). The fact that the NBDs of P-gp/MDR1 can be exchanged without loss of function provides strong support
for the model (26, 199). However, the stoichiometry of
ATP hydrolysis and substrate transport has not been fully
resolved. In the case of P-gp, considerable evidence exists
to support a model in which hydrolysis of ATP at either
NBS results in transport of one molecule of substrate
(459). A more recent variation of this model proposes that
the binding and hydrolysis of one ATP molecule drives a
“power stroke” in which the protein shifts from a high- to
low-affinity substrate binding state with the concomitant
transport and release of one molecule of substrate (443).
Hydrolysis of a second ATP is then required to reset the
protein in a high-affinity state for the next transport cycle.
In this model, it remains unclear whether binding and
hydrolysis of ATP at each of the two NBSs is dedicated to
a different step in the transport cycle of proteins such as
P-gp/MDR1 and the prokaryotic transporters, or whether
the process is stochastic. Although many studies support
the proposal that the NBDs of these proteins are truly
functionally identical, some evidence suggests that the
position of the NBD may influence its function (26, 199).
Until relatively recently, it had also been commonly assumed that the hydrolysis of ATP drove the conformational changes in the protein required for transport. However, recent studies, including those of ABCC proteins,
have provided strong evidence that it is ATP binding
rather than hydrolysis that converts the protein from a
high- to low-affinity substrate binding state (173, 328, 329,
386, 424). The differences between these two models
notwithstanding, it is clear that C branch proteins do not
fit readily with a model in which the NBDs are functionally equivalent. Unlike P-gp, the two NBDs differ considerably with respect to their ability to bind and hydrolyze
ATP (136, 194, 353). In the case of MRP1, there is considerable evidence that each NBD is responsible for distinct
steps in the transport cycle and that the shift from a highto low-affinity substrate binding state involves the ordered rather than stochastic binding of ATP to the two
NBDs (136, 194, 353).
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DEELEY, WESTLAKE, AND COLE
E. Functionally Important Structural Differences
Between ABCC NBDs
What are the structural features that contribute to the
nonequivalency of the two NBDs? Sequence comparisons
of ABC protein NBDs reveal that, along with the Walker
A/B and signature C elements, there are other motifs,
namely, the Q, D, and H (switch) loops, that contain
highly conserved Glu, Asp, and His residues, respectively
(Fig. 4). NBD crystal structures have been solved for a
number of bacterial ABC proteins besides, including HisP
(201), MJ0796 (550), MalK (104), HlyB (449, 551), GlcV
(514), BtuD (297), and MsbA (55, 56, 415), as well as for
the human proteins TAP1 (137) and, most recently, both
the wild-type CFTR NBD1 and the NBD containing the
common ⌬508 mutation (290). Although there are some
regions of unique architecture among these proteins, a
common configuration of eight ␣-helices (␣1– 8) and nine
␤-sheets (␤1–9) is observed (Fig. 7B) (with the exception
of MJ0796, which lacks the final 2 conserved helices).
These L-shaped domains are functionally separated into
two arms: arm I, an F1-like ATP-binding unit comprised of
the Walker A and Walker B elements, and arm II, containing the C motif (172). Alignments of the ABC NBDs indicate a high level of tertiary structural conservation in the
nucleotide binding pocket. The adenine moiety of ATP
generally stacks against a conserved aromatic residue
near the end of, or following, ␤1, while the side chains of
residues in the linker region between ␤1 and ␤2 stabilize
the ribose group (104, 137, 201, 290, 514, 550). Residues in
the Walker A motif typically hydrogen bond with the ␣-,
␤-, and ␥-phosphates, and the negatively charged residues
Physiol Rev • VOL
in the Walker B motif coordinate Mg2⫹ binding and/or
stabilize ADP in the binding pocket through interactions
with water (Fig. 7A). Similarly, Asp and His in the D and
H loop, respectively, make contacts with water that stabilize the binding of nucleotide, while the conserved Gln
residue in the Q loop interacts with the catalytic Mg2⫹ and
attacking water molecule. Comparison of nucleotide free,
ADP-bound, and ATP-like AMP-PNP· Mg2⫹ bound NBD
structures in ABC NBDs such as GlcV suggests that significant structural changes occur on nucleotide binding
(514). These conformational changes primarily involve
the Walker A motif and the Q loop and are thought to be
transmitted to the MSDs through the sequence adjacent to
the latter (297).
The most obvious difference between the two NBDs
of the MRP-related transporter proteins and CFTR is the
previously mentioned, highly conserved, apparent deletion of 13 amino acids between the Walker A motif and
the Q loop in NBD1. On the basis of the general structure
illustrated in Figure 7B, this would eliminate a ␤-sheet as
well as an ␣-helix located between ␤4 and ␤5 found in
approximately half of the structures of the ABC proteins
determined to date (137, 201, 449, 550). Whether the foreshortening of the region between the Walker A motif and
the Q loop contributes to the relatively high affinity with
which NBD1 of CFTR and MRP1 binds ATP is not clear (7,
136). The insertion of a 13-amino acid sequence from
NBD1 of P-gp/MDR1 that corresponds to the region missing in MRP1 eliminated high-affinity ATP binding and
changed the conformation of the domain, since a conformation-dependent monoclonal antibody (QCRL-3) against
this region no longer recognized the protein (136, 180).
However, the most recent crystal structure of CFTR
NBD1 indicates that the atomic contacts established by
ATP are similar to those found in other ABC NBDs with
known crystal structures (290). Clearly, additional features are important for the observed differences in ATP
binding and hydrolysis exhibited by the two NBDs of the
C branch proteins.
As mentioned earlier, in most ABC NBDs, there is a Glu
residue immediately following the Walker B motif that
serves as a catalytic base for cleavage of the ␥-phosphate
group (345, 472, 515). Although Glu is present at the appropriate location in NBD2 of the ABCC proteins, the corresponding amino acid in NBD1 is Asp with the exception of
CFTR in which it is Ser (72, 386). The consequences of
mutating the Asp and Glu residues in NBD1 and NBD2 of
MRP1 have illustrated the importance of these residues in
the catalytic cycle and have provided useful information
about the probable transport mechanism of the protein.
Studies of other ABC proteins (201, 345, 506) suggest that
conversion of Asp to Glu in MRP1 NBD1 would be expected
to increase the ATPase activity of this domain and, based on
an alternating sites model of catalysis, might enhance the
rate of substrate transport. Paradoxically, such a mutation
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binding of ATP by NBD2 is strongly dependent on the
binding of ATP to NBD1.
Further evidence that the two NBDs of MRP1 are not
functionally equivalent comes from the effect of various
mutations on substrate transport, as determined by in
vitro studies with inside-out membrane vesicles. Typically, mutations of conserved amino acids in the Walker A
and Walker B motifs of NBD1 that are known to eliminate
ATP hydrolysis decrease transport of LTC4 by ⬃50 –70%,
depending on the nature of the mutation, while comparable mutations in NBD2 essentially inactivate the protein
(136, 194, 387). It appears likely that the partial activity of
the NBD1 mutant proteins may be attributable to retention of some level of ATP binding by the mutant NBD that
enables a reduced level of ATP binding and hydrolysis by
NBD2. In support of this suggestion, conversion of the
conserved Walker A Lys residue in NBD1 to a neutral
amino acid or to Arg results in a partially active protein
(136, 194, 387), while substitution with a negatively
charged Asp residue almost completely eliminates MRP1
LTC4 transport activity (387).
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
Physiol Rev • VOL
F. High- and Low-Affinity Substrate Binding States
A number of experimental approaches have demonstrated that ABC transporters such as P-gp/MDR1 and
LmrA shift from a high- to low-affinity substrate-binding
state in the presence of ATP (295, 402, 403, 442, 512, 519,
524). This change in affinity is most apparent in the presence of ATP and orthovanadate or beryllium fluoride,
both of which markedly increase occupancy of the protein by ADP. The decrease in substrate binding is presumed to be a consequence of conformational and positional changes in the NBDs involved in formation of a
closed NBD dimer that are transmitted to the MSDs. The
consequential reorientation of TM helices then results in
the substrate being transferred from the high-affinity site
to which it initially bound, to a low-affinity site from
which it would be released. At present, there is little
evidence to demonstrate that this actually involves physical transfer of substrate from one site in the protein to
another, rather than a decrease in the affinity of the initial
binding site and the release of the substrate into a vestibule open to the extracellular space.
The presence of substrates and inhibitors and, in
some cases, compounds that bind but are not transported,
also influences ATP binding and hydrolysis by proteins
such as P-gp/MDR1 and MRP1 (282, 321, 386, 394, 402,
403, 442, 524). Studies with purified proteins indicate that
the activation of P-gp/MDR1 ATPase activity can be quite
substantial (9, 402, 461). In the case of MRP1, substrates
such as LTC4 appear to have less effect and typically
stimulate ATPase activity 1.5- to 2-fold at most (57, 325,
326). The presence of GSH or GSH-independent substrates such as LTC4 modestly stimulates the binding of
ATP to NBD1 of MRP1, which, in turn, stimulates the
binding and hydrolysis of ATP by NBD2 (136, 194, 282). In
a number of ABC transporters, substrate binding has been
shown to result in changes in accessibility of the NBDs to
proteases and chemical modification (232, 254, 294, 322,
323, 355, 473). Fluorescence spectroscopy has provided
strong evidence that the binding of some substrates to
P-gp/MDR1 results in alterations that are transmitted from
the MSDs to the NBDs (294 –296, 473). In a study of MRP1
using protease accessibility and attenuated total reflection-Fourier transform infrared spectroscopy (ATRFTIR), it was concluded that binding of drugs that are
transported in a GSH-dependent manner did not result in
conformational changes in the cytosolic portions of the
protein (322). However, such changes were elicited by
GSH and resulted in increased nucleotide binding/hydrolysis (322, 520). Precisely how this stimulation occurs has
not been completely defined but presumably involves a
conformational change in the MSDs of the protein that
facilitates initial binding of ATP and subsequent formation of a closed NBD dimer in the presence of ATP.
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decreases LTC4 transport activity by ⬃80% (386). As expected, the Asp to Glu mutation markedly increases cleavage of the ␥-phosphate of ATP by the mutant NBD1. However, it also results in vanadate-independent trapping or
occlusion of nucleotide and a failure to release the ADP
produced. The mutation also results in a major decrease in
ADP trapping at NBD2, suggesting that occupancy of NBD1
by ADP prevents hydrolysis at NBD2. The decrease in hydrolysis at NBD2 under these circumstances would be predicted by the alternating sites model of hydrolysis, since it is
postulated that only one NBS can be occupied by ADP at
any given time (459). The reciprocal Glu to Asp mutation in
NBD2 also inactivates the protein and results in tight binding
of nucleotide by the mutant NBD2 that is also vanadate
independent. In this case, the bound nucleotide was found
to be a mixture of ATP and ADP, suggesting that the mutation had not completely eliminated cleavage of the ␥-phosphate. The NBD2 Glu to Asp mutation also increased ATP
binding and ADP trapping at the associated wild-type NBD1.
This is consistent with other evidence suggesting that the
binding of ATP by NBD2 stimulates ATP binding by NBD1
(136, 194). However, it also reinforces the possibility that the
native NBD1 of MRP1, like the Glu to Asp NBD2 mutant, has
some ability to hydrolyze ATP, albeit low when compared
with the native NBD2 (386).
In addition to the atypical Walker B motif in NBD1,
the C signature in NBD2 of MRP1 and other ABCC proteins is unusual (72, 147, 387). The signature sequence in
NBD1 of ABCC proteins conforms well to the canonical C
signature and contains the conserved core LSGGQ motif.
However, this core motif in NBD2 of MRP1, CFTR, and
SUR1 is LSVGQ, LSHGH, and FSQGQ, respectively (3, 72,
417). Furthermore, the more extended signature sequence
normally contains highly conserved Arg and Ser residues
at positions 8 and 10, relative to the start of the core.
Although these residues are present in the NBD1 signature, the Arg is replaced by Leu and the Ser by Cys in
NBD2 of most of the ABCC proteins (386, 387). In P-gp,
the conserved Ser residue has been shown to be required
for hydrolysis (309, 496). Since the NBD2 signature sequence would be involved in hydrolysis of ATP by NBD1,
it appears highly likely that these variations contribute to
the low or absent ATPase activity of this NBD. As with
several other ABC proteins, studies of the signature sequences in MRP1 indicate that they are not required for
ATP binding (387, 412, 466, 489, 490, 496). However, mutation of conserved Gly residues in the signature sequence
of NBD1 and NBD2 inactivate or partially inactivate LTC4
transport, respectively (387, 412). Although binding of
azido-ATP appears to be unaffected, the C signature mutations prevent the transition to a low-affinity substrate
binding state (387, 490). This is presumed to be a consequence of a failure to form the correct interface in the
NBD closed dimer (387, 390). Similar observations have
been made with P-gp/MDR1 (489, 496).
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DEELEY, WESTLAKE, AND COLE
G. Interactions Between MSDs and NBDs
How conformational changes are transduced between the NBDs and MSDs of the ABC transporters remains poorly understood. There is considerable evidence
that conformational changes of the NBDs both within and
between the two domains upon nucleotide binding and
hydrolysis result in significant reorientation of at least
some of the TM helices in their associated MSDs (136,
194, 308, 327, 386, 394, 425, 429). Similarly, changes in the
orientation of certain TM helices may be capable of influencing the functional interactions between the NBDs that
are required for ATP hydrolysis (218, 242, 310, 468, 570).
It has been suggested based on the crystal structures of
bacterial transporters such as MsbA and BtuCD that intraPhysiol Rev • VOL
cellular domains in contact with, or connected to, both TM
helices and the NBDs may serve as “bridges” that transduce
signals between the NBD and TMDs (55, 56, 297).
BtuCD is a vitamin B12 transporter from Escherichia
coli that consists of two identical MSDs (BtuC) and two
identical NBDs (BtuD) (103, 297). The structures of the
MSDs of BtuCD are unusual in that they appear to contain
10 helices, which complicates extrapolation of the structure to other ABC proteins (297). However, a dihelical
structure in CL3 of BtuC, located between the sixth and
seventh TM helices, makes multiple side-chain contacts
with BtuD residues around the Q loop, and to Met and Gln
residues downstream of the Walker A and the ABC signature motifs, respectively. The lipid A transporter MsbA
from both E. coli and V. cholera has a more typical
structure with six TM helices in each MSD (55, 56). In
these proteins, NBD-MSD contacts occur between CL1,
which connects the second and third TM helices of each
MSD and the Q loops in each NBD. These two CLs each
form a trihelical ”U“-like structure that has been suggested to provide a pivot point on which the associated
NBDs may rotate during hydrolysis (52, 55). Although
there is no extended homology between BtuC CL3 and
MsbA CL1, both loops contain a conserved Gly motif that
is found in other ABC proteins, including MRPs, suggesting that these domains may be functionally conserved
(297). The equivalent regions in MRP1 correspond to CL4
located between TMs 7 and 8 in MSD1 and CL6 between
TMs 13 and 14 in MSD2, respectively (Figs. 1 and 5). There
is some experimental evidence to suggest that MRP1 CL6
communicates with the NBDs, since nonconservative mutation of Asp1084, predicted to be at or near the cytoplasmmembrane interface of TM14, to Gln severely decreases
binding and vanadate-induced trapping of azido-ATP at
NBD2 indicating reduced hydrolysis of ATP (568). This
mutation also prevents the transition of the protein from
high- to low-affinity substrate binding states in the presence of ATP or ATP␥S. However, mutation of Pro1060 and
Pro1068 predicted to disrupt CL6 secondary structure had
little effect on activity, and MAb MRPm5 directed against
a peptide epitope in this loop does not inhibit LTC4 transport (242, 243). On the other hand, mutations of a conserved Pro residue (Pro1150) in CL7 connecting TM15 and
TM16 affects both the substrate specificity and catalytic
activity of MRP1 (242). The substrate specificity of MRP1
is also affected by mutations of two conserved Tyr residues in this region (77).
The cytoplasmic regions directly connecting MSDs
and NBDs are also likely to be important for transmitting
conformation changes between membrane and cytosolic
domains (56). There is strong experimental evidence from
studies of P-gp/MDR1 that the binding of ATP results in a
change in the predicted tilt of TM6, which is linked directly to NBD1 (429). In MRP1, the corresponding TM is
TM11, and it was found recently that Ala substitution of a
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Investigation of the steps involved in shifting MRP1
from high- to low-affinity states has been facilitated by the
availability of photoactivatable, natural substrates, such
as LTC4, and various mutant proteins, such as those described above, that have permitted substrate binding to be
examined with the protein effectively being locked in
various states of nucleotide occupancy. The results of
these studies indicate that conversion of the Walker B
catalytic Glu residue in NBD2 to Asp markedly potentiates the shift from high- to low-affinity LTC4 binding in the
presence of ATP or the poorly hydrolyzable analog adenosine 5⬘-O-(3-thiotriphosphate) (ATP␥S) (386). As indicated above, this mutation results in impaired release of
ATP and ADP from NBD2 and increases ATP binding at
NBD1. Thus formation of the low-affinity state appears to
involve binding of ATP to both NBDs and persists as long
as NBD2 is occupied by either ATP or ADP. Conversely,
mutation of the atypical Walker B Asp residue in NBD1 to
Glu results in stable occupancy of NBD1, primarily by ADP,
and decreases ATP binding and trapping at NBD2 (386). It
also prevents the conversion of the protein from a high- to
low-affinity state in the presence of ATP or ATP plus vanadate. Further evidence that the decrease in substrate affinity
occurs when both NBDs are occupied by ATP is provided
from a double mutant in which the Asp and Glu residues
were switched (386). The mutant protein is completely inactive with respect to LTC4 transport but binds ADP and
ATP avidly at NBD1 and NBD2, respectively. In the presence
of ATP or ATP plus vanadate, this mutant remains in a
high-affinity LTC4 binding state. However, ATP␥S is able to
promote the formation of the low-affinity state, although less
effectively than with the wild-type protein. Thus occupancy
of the mutant NBD1 by ADP prevents formation of the
low-affinity state. It is tempting to speculate that this mimics
the step involved in resetting the wild-type protein in a
high-affinity state, i.e., as a result of the hydrolysis of ATP at
NBD1. However, the possibility remains that this step may
simply involve dissociation of ATP from NBD1.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
H. The Transport Cycle of MRPs
A model of the hypothetical transport cycle of MRP1
is illustrated in Figure. 8. The various steps shown in
Figure 8 can be summarized as follows. Step 1) Binding of
substrate (or GSH plus GSH-dependent substrate) to a
high-affinity site(s) induces conformational changes that
enhance binding of ATP to NBD1. Step 2) The initial
binding of ATP by NBD1 stabilizes the interaction between NBDs by establishing contacts with the C signature
of NBD2, facilitating the binding of a second molecule of
ATP. Step 3) Binding of the second ATP completes formation of the closed NBD dimer and also causes conformational changes in NBD2. The combined positional and
conformational changes resulting from ATP binding are
transmitted to the MSDs resulting in a decrease in the
affinity for substrate. Step 4) The protein maintains a
low-affinity state following hydrolysis of ATP at NBD2, as
long as NBD1 is occupied by ATP and ADP has not been
released by NBD2. Although the steps in the model leading to formation of the low-affinity substrate binding state
are strongly supported by experimental data (57, 136, 386,
387, 394, 572), how the protein resets for another cycle
remains more speculative and hinges on whether or not
NBD1 is catalytically active. Step 5) If NBD1 lacks ATPase
activity, resetting of the protein might occur after the release
of only ADP from NBD2, or require release of ADP from
NBD2 and ATP from NBD1. Step 6) However, it may be
premature to exclude the possibility that hydrolysis of ATP
by NBD1 is required to reset the protein. As mentioned
above, low levels of orthovanadate-dependent trapping of
ADP by wild-type NBD1 can be detected, and mutations that
increase ADP trapping at NBD1 lock MRP1 in a high-affinity
substrate binding state, even when ATP is bound by NBD2
(136, 386). Finally, given that the ATPase activity of purified
MRP1 is ⬃100-fold lower than that of similar preparations of
a protein such as P-gp/MDR1 (320, 325, 326, 402), it remains
possible that the rate-limiting step in the transport cycle is
the hydrolysis of ATP by NBD1.
X. SUBSTRATE RECOGNITION AND BINDING
BY MULTIDRUG RESISTANCE PROTEINS
1, 2, AND 3
A. Experimental Approaches Used to Investigate
Substrate Recognition
Several complementary approaches have been taken
to identify regions and specific amino acids in the MRPs
FIG. 8. Model of the hypothetical
transport cycle of MRP1. Illustrated are
hypothetical steps in the transport cycle
of MRP1 and possibly other MRPs.
MSD1-NBD1 and MSD2-NBD2 are shown
in blue and gray, respectively. MSD0 is
not shown both for simplicity and because it is not required for transport of at
least some substrates, including LTC4. A
description of the various steps in the
model and the experimental evidence
supporting it is provided in section IX, E,
F, and H.
Physiol Rev • VOL
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residue, Asn590, located in this helix caused a global decrease in MRP1 transport activity by decreasing ATP binding
to NBD1 (570). Nonconservative mutation of other nearby
residues in TM11, Arg593, Phe594, and Pro595, also causes a
global elimination of transport activity and LTC4 binding (52,
160, 242). Interestingly, Arg593 of MRP1 corresponds to
Arg347 of CFTR, mutation of which has been correlated with
a mild form of cystic fibrosis (83). This mutation has been
shown to decrease the ATPase activity of CFTR three- to
fourfold (238). Given the many functional similarities between the cooperative interactions of the NBDs of MRP1
and CFTR, the CFTR Arg to Glu mutation may also be
affecting ATP binding to NBD1. Thus the regions linking
NBDs and MSDs may transmit conformational changes both
from the NBDs to the MSDs, and vice versa.
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DEELEY, WESTLAKE, AND COLE
B. Photoaffinity Labeling Studies
Partial proteolysis has been used to identify regions
of MRP1 that are cross-linked to several photoactivatable
compounds (89, 90, 225, 327, 394). After cleavage, labeled
fragments can be identified using well-characterized
MRP1 MAbs or antibodies against exogenous epitopes
that have been introduced at specific locations in the
protein. Alternatively, advantage has been taken of the
ability to coexpress various fragments of MRP1 that associate to reconstitute an active transporter and that can
readily be separated by gel electrophoresis (134) (see
sect. VIIID). Three different patterns of labeling have
emerged.
The earliest attempts to localize substrate binding
sites in MRP1 used azido derivatives of the quinoline
N-(hydrocinchonidin-8⬘-yl)-4-azido-2-hydroxybenzamide
(IACI) and iodoaryl azido-rhodamine 123 (IAARh123) (89,
90). Proteolytic digestion and epitope mapping indicate
that IACI and IAARh123 cross-link to a similar extent to
one or more amino acids between Ser542-Arg593 in TMs 10
and 11 and Cys1205-Glu1253 in TMs 16 and 17 (89, 91).
Physiol Rev • VOL
These regions correspond topologically to locations in
P-gp/MDR1 that have been previously implicated in substrate binding (47, 101, 148, 541). Unmodified LTC4 has
also been used to photolabel MRP1 (272, 299, 540). Both
coexpression and partial proteolysis studies indicated
that LTC4 cross-linked strongly to MSD1 and only weakly
to MSD2 (394). This profile of labeling differs significantly
from that obtained recently using an arylazido derivative
of LTC4 that appears to predominantly label MSD2 in a
region that includes TMs 16 and 17 (226, 540). The reason
for the differences between the labeling patterns of LTC4
and the derivative appears likely to be attributable to the
presence of the bulky arylazido group that affects the
affinity of the latter compound for MRP1, since, as indicated below, most mutations in TMs 16 and 17 have little
effect on LTC4 transport and TM17 (1228 –1248) can be
replaced entirely with an alanine helix without loss of
leukotriene transport (23). Recent improvements in
MALDI TOF mass spectrometry methods have allowed
the more complete analysis of integral membrane proteins and identification of their ligand binding sites. For
example, Ecker et al. (111) have shown by MADLI TOF
mass spectrometry that ATP binding increases the accessibility of the fifth TM helix of the bacterial ABC transporter LmrA to labeling by a photoactive ligand while ATP
hydrolysis has an opposite effect. Human P-gp/MDR1 has
also been analyzed by mass spectrometry, and ligandbinding regions have been identified (112). Recently, this
methodology has been applied to MRP1, and comparisons
of the mass spectra of the peptides generated by in-gel
protease digestions of purified unlabeled MRP1 and MRP1
photolabeled with LTC4 have identified six candidate
LTC4-modified fragments that include the COOH-proximal
region of CL3 (amino acids 260 –274), TM6 (amino acids
320 –331), TM7 (amino acids 372–385), TM10 and its cytosolic juxtamembrane region (amino acids 546 –553), and
TM17 and its cytosolic juxtamembrane region (amino
acids 1233–1255 and 1248 –1264) (540). To a significant
extent, the sequence assignments of the LTC4-modified
peptides are consistent with other LTC4 photolabeling
and transport studies of wild-type and mutant MRP1 proteins, but less so with studies using the arylazido derivatized LTC4. However, the apparent binding of LTC4 to the
COOH-proximal region of CL3 (amino acids 260 –274) is
not consistent with earlier findings (529), and further
investigations using more sophisticated mass spectrometry methods for fragmentation studies of the LTC4-labeled
peptides are clearly needed.
A third pattern of photolabeling has been observed
with two compounds that require GSH in order for them
to bind to MRP1. The first is a polyhydroxylated sterol,
agosterol-A, that has been shown to reverse P-gp/MDR1and MRP1-mediated resistance in vitro (13). In contrast to
P-gp/MDR1, the binding of azidoagosterol-A to MRP1 is
GSH dependent, and contrary to the behavior of LTC4, the
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that are important for substrate recognition and transport. To date, these studies have focused on MRP1-like
proteins, primarily MRP1 itself and to a lesser extent
MRP2 and -3. Extensive use has been made of in vitro
transport studies with vesicles prepared from the plasma
membranes of transfected mammalian and insect cells
expressing wild-type and mutant MRPs (134, 161, 274, 287,
299). This experimental approach has been extremely
informative with respect to identification of potential substrates, inhibitors, and modulators of the proteins and has
provided a powerful complement to the use of intact cells
for drug efflux or chemosensitivity assays. In addition,
transport studies with membrane vesicles containing mutant MRPs have allowed identification of regions or amino
acids that are critical for overall activity, as well as substrate selectivity, and stable expression of the transporter.
A major reason why this approach has been so informative is attributable to the relatively hydrophilic characteristics of many MRP substrates compared with those of
drug transporters such as P-gp/MDR1. This has facilitated
kinetic analyses of a substantial number of substrates and
mutant MRPs. The in vitro transport studies have also
been complemented by photoaffinity labeling with several
azido derivatized and intrinsically photolabile compounds
to identify regions of the protein predicted to be in or
close to substrate binding sites (90, 89, 225, 273, 327, 394,
396, 409, 540). Although far from complete, sufficient
information has now been obtained to develop a tertiary
structure model of the disposition of a considerable number of amino acids involved in determining MRP1 substrate specificity and/or transport activity.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
C. Mutagenesis Studies
Regardless of the diverse structures of the photoactivatable compounds used to label MRP1 and the differences in their labeling profiles, competition studies suggest that they bind to mutually exclusive sites on the
protein (89, 90, 327, 396, 409). This observation is consistent with many in vitro vesicular transport studies demonstrating reciprocal competition between structurally
unrelated substrates, with some exceptions (281). Mutational studies have also identified individual amino acids
that are important for the transport of a range of diverse
substrates, supporting the notion of a common binding
site or pocket (52, 76, 77, 159 –161, 241, 242, 281, 289, 410,
468, 565, 567, 568, 570). However, numerous examples
exist of amino acid residues, mutation of which alters
transport of some substrates and not others (76, 77, 159,
160, 207, 241, 242, 285, 289, 565–568). In addition, apparent positive cooperativity has been observed between
certain substrates, or between substrates and compounds
that may bind to the protein but not be transported (281,
282, 284, 299 –303, 413, 518). Thus the experimental evidence, which is reviewed below, suggests that substrates
establish multiple, often but not always, overlapping inPhysiol Rev • VOL
teractions with amino acid residues that collectively form
a relatively large binding pocket. If these interactions
induce conformational changes in MRP1, as appears
likely, it can be envisaged how in some instances the
binding of one compound may facilitate binding of another by unmasking additional contact points. Alternatively, positive cooperativity may result from the binding
of one compound masking residues that disfavor interaction with another substrate or ligand. Finally, it should be
noted that several residues have been identified that,
rather than being important for the activity or substrate
specificity of MRP1, play a critical role in the stable
expression of the transporter in mammalian cell plasma
membranes (84, 160, 241, 242, 468).
To some extent, mutagenesis studies of the MRPs
have been guided by previous investigations of the location of functionally important residues in P-gp/MDR1, as
well as the characterization of naturally occurring mutations in CFTR/ABCC7 and other ABCC proteins that underlie genetic disorders. Major differences in substrate
specificity also exist between human and nonprimate,
mammalian MRP1 orthologs (314, 371, 482), which has
aided identification of amino acids critical for the transport of some substrates (371, 483, 565, 566). Finally, systematic mutation of amino acids with side chains predicted to contribute to substrate binding (e.g., polar, ionizable, and aromatic residues), or to influence ␣-helical
geometry (e.g., Pro) or membrane insertion during biogenesis (e.g., aromatic and basic residues) has illustrated
the importance of these residues with respect to substrate
specificity, overall activity, and stable expression of the
transporters in mammalian cell membranes (52, 76, 77,
159, 160, 207, 241, 242, 281, 285, 289, 410, 468, 565–568,
570).
As discussed in section IXB, studies of P-gp/MDR1
indicate that TM6 and TM12 play major roles in determining its substrate specificity. In MRP1-like MRPs, the corresponding TMs are 11 and 17. TM17 is relatively highly
conserved, exceptionally so between MRP1 and MRP3
(Fig. 9). Mutational studies of TM17 in MRP1, MRP2, and
MRP3 have revealed multiple polar and/or aromatic residues and basic residues that have pronounced effects on
substrate specificity, with respect to various classes of
natural product drugs and conjugated organic anions,
such as E217␤G and LTC4, as well as folic acid analogs
such as methotrexate and leucovorin (206, 207, 374, 432,
468, 565, 567, 569). In MRP1, the majority of these residues are located in a region of TM17 predicted to reside in
the inner leaflet of the membrane and at the membranecytosol interface (Fig. 9). Although conservative or nonconservative substitutions of some residues, such as
Tyr1236 and Thr1241, affect transport of only certain substrates, conservative mutations of others, such as Tyr1243,
Thr1242, and Trp1246, alter transport of multiple, structurally unrelated compounds. Whether this is because the
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predominant site labeled is in MSD2 (409). Mutational
studies have provided evidence that the region to which
cross-linking occurs involves TM helices 16 and 17 (410,
411). A similar profile of GSH-dependent cross-linking is
observed with a highly potent inhibitor of MRP1 (327, 366,
396). The compound LY475776 is a tricyclic isoxazole that
inhibits MRP1-mediated LTC4 transport in a GSH-dependent fashion, with a EC50 in the 50 nM range (88, 327, 366,
396). Inhibition is completely dependent on the presence
of GSH (or certain of its analogs), as is the ability of the
compound to photolabel MRP1, which is adversely affected by mutations in TM17.
The contrast between the labeling profiles obtained
with LTC4 and the two GSH-dependent compounds suggested that the predominant labeling of MSD1 by LTC4
might be attributable to the GSH moiety. Consistent with
this possibility, the labeling of MRP1 by azidophenacylGSH, which has been shown to substitute for GSH in
vesicle transport studies, is very similar to that of LTC4
(396). Although apparently not directly labeled by either
unmodified LTC4 or azidophenacyl-GSH, very weak labeling of CL3 has been reported with a different arylazido
derivative of GSH (225). The significance of this low level
of labeling is unclear, but CL3 residues 204 –280 are
clearly important for transport activity and trafficking of
MRP1 (see sect. IVB) (20, 21, 529). This region has also
been shown to be essential for binding of both unmodified
LTC4, azidophenacyl-GSH, azido AG-A, and LY475776
(394, 396, 409, 529).
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DEELEY, WESTLAKE, AND COLE
latter residues make specific contacts with a wide array of
substrates or because their mutation perturbs the architecture of the drug-binding pocket is not yet known. The
highly conserved Trp1246 in MRP1 may also play a critical
role in defining the position of the helix-cytosol interface,
possibly by forming stabilizing pi-cation interactions with
Arg1249 at position i ⫹ 3 (207, 410, 468). Surprisingly, most
mutations in TM17 of MRP1 (defined as residues 1228 –
1248) have a negligible effect on the transport of LTC4.
For example, mutation of Trp1246 to Cys, Ala, Phe, or Tyr
eliminated E217␤G and NNAL-O-glucuronide transport,
resistance to natural product drugs, and binding of the
GSH-dependent inhibitor LY475776, but had little effect or
no effect on LTC4 transport (207, 281, 327). Thus residues
in TM17 appear to be critically involved in the recognition
and transport of various natural product drugs and glucuronide conjugates, but not for the high-affinity binding or
transport of LTC4. Consistent with this notion, transport
studies have shown that unmodified natural product
drugs compete more effectively for E217␤G transport
than for LTC4 transport, despite the fact that the two
organic anion conjugates compete reciprocally with each
other (298, 299, 349). More direct evidence is provided by
Physiol Rev • VOL
the recent demonstration that TM17 (residues 1228 –1248)
of MRP1 can be replaced by a stretch of Ala residues
without loss of LTC4 transport activity (23). However,
LTC4 transport by the TM17-poly(Ala) mutant is no longer
inhibitable by E217␤G. Nevertheless, it should be noted
that when residues in the COOH-proximal portion of the
TM17 ␣-helix that extends beyond position 1248 into the
cytoplasm (e.g., Arg1249) are mutated, loss of LTC4 binding
and transport is observed (410, 468), consistent with such
residues having a role in maintaining an active conformation of the substrate binding pocket of MRP1 rather than
interacting directly with its substrates.
In contrast to TM17, TM6 of MRP1 is relatively poorly
conserved among ABCC family members. In addition, the
corresponding TM1 in P-gp/MDR1 has not been implicated by mutagenesis studies to be important for the
activity of this drug transporter. However, TM6 of MRP1
contains significantly more polar residues than does TM1
of P-gp/MDR1, and consequently, it was proposed that
this might favor interaction of MRP1 with its more hydrophilic substrates (457). Consistent with this idea, several
charged residues in TM6 of MRP1 have been demonstrated to be critical for overall transport activity in one
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FIG. 9. Comparison of the effects of mutating comparable polar amino acid residues in transmembrane (TM) 17 of human MRP1 and MRP3 on
substrate specificity. Illustrated are the positions of comparable polar residues in TM17 of human MRP1 and MRP3 and the effects of their mutation
on the ability of the proteins to transport or confer resistance to various organic anions and drugs. The model of TM17 of MRP1 is derived from
the energy-minimized model of MSD1 and MSD2 described in text (sect. XIA) and Figure 10 (52). The helix was then “mutated” at four specific
locations (Thr-1242, Tyr-1243, Leu-1247, Val-1248). Triton version 3.0 was used to create an approximate model of MRP3 TM17. PyMol was used to
highlight and color the specific mutations on the two helices. As can be seen from the inset, which shows the predicted position of TM17 in the
plasma membrane, the residues that influence substrate specificity are predominantly in the predicted inner leaflet regions of the helices. Of
particular note are the substantial differences between the two proteins with respect to residues involved in conferring resistance to VP-16
(etoposide) and in transport of E217␤G.
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
Physiol Rev • VOL
TM16 that contribute (depending on the substituting
amino acid) to misfolding of MRP1 and, in the case of the
neutral Glu1204 Leu mutant, disruption of the signaling
between the TMs that comprise the substrate translocation pathway through the membrane and NBD2.
D. Is Amino Acid Sequence Conservation
Predictive of Substrate Specificity?
What is the implication of the sequence conservation
of regions such as TM17 with respect to the ability of
MRP1, -2, and -3 to transport common substrates? Although TM17 of MRP1 and MRP3 are identical in 19 of 21
amino acids, mutation of conserved polar residues has
strikingly different effects on the overall activity and substrate specificity of the two proteins (207, 374, 567, 569).
For example, elimination of the hydrogen bonding capability of polar residues, Tyr1232, Ser1229, Ser1231, and
Ser1233, predicted to be in the outer leaflet region of TM17
in MRP3 had major effects on both overall transport
activity and specificity (569). Despite the complete conservation of this region in MRP1, the only mutation that
affected function was the conversion of Tyr1236 to Phe
that selectively decreased resistance to vincristine (567)
(Fig. 9). Similarly, mutation of the conserved Trp residue
predicted to be at the membrane cytoplasm interface of
TM17 in MRP1, -2, and -3 had different effects on the
transport of the common substrate E217␤G (206, 207,
374). Conservative and nonconservative substitutions of
this amino acid in MRP1 and MRP3 decreased and increased transport of the conjugated estrogen, respectively, while in MRP2, only nonconservative substitutions
had an effect. Thus, even in highly conserved regions that
are clearly important for substrate recognition and transport, the correlation of structure and function is complex
and cannot be predicted on the basis of primary amino
acid sequence.
A number of amino acid residues in TM11 of MRP1
are also involved in determining the overall activity and
substrate specificity of the protein (52, 160, 242, 570). As
found with TM17, most of these mutation-sensitive residues are located within the predicted inner leaflet region
of the helix or at its cytoplasmic interface. However,
unlike TM17, mutations of at least four residues, Asn590,
Arg593, Phe594, and Pro595, affected LTC4 binding and
transport, as well as the transport of other substrates (52,
160, 242, 570). Conservative substitutions of Phe594 with
Tyr or Trp had selective effects on substrate specificity,
suggesting that it may be involved in direct interaction of
MRP1 with its substrates (52). Nonconservative (Glu) and
neutral (Leu) substitutions of Arg593 reduced transport of
multiple substrates, whereas this was not the case for the
Lys-substituted mutant (160), indicating the positive
charge at this position likely plays a role in maintaining
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case (Asp336) and in particular, for LTC4 binding and
transport in two others (Lys332 and His335) (159, 160).
Thus both conservative and nonconservative substitutions of Asp336 eliminate binding and transport of a broad
range of organic anions. In contrast, comparable substitutions of Lys332 cause a selective loss of LTC4 transport
by MRP1. GSH transport is also reduced, but transport of
E217␤G, methotrexate, and estrone sulfate is unaffected.
A similar substrate-selective loss of transport activity is
observed when His335 (which is on the same face of the
TM6 helix as Lys332) is mutated to Glu, Gln, or Leu.
Confirming the critical importance of TM6 for binding and
transport of LTC4 by MRP1 is the recent observation by
Bao et al. (23) that replacement of TM6 (amino acids
320 –337) with a poly(Ala) chain, in contrast to a similar
replacement of TM17, eliminates LTC4 transport.
Finally, as mentioned previously, six single amino
acid substitutions have been described thus far which
substantially reduce or eliminate expression of MRP1, at
least in mammalian cells. These residues are located
throughout the primary structure of the protein. They
include nonconservative substitutions of Trp142 predicted
to be in TM4 of MSD0 (241), Asp430 at the membranecytosol interface of TM8 in MSD1 (160), Asp792 in NBD1
(84) and in MSD2, Pro1113 in the extracellular loop connecting TM14 to TM15 (242), and Arg1202 and Glu1204 at
the membrane-cytosol interface of TM16 (468). In some
cases, culturing the transfected cells harboring the mutant MRP1 at reduced temperatures (conditions where
the proofreading machinery for monitoring protein folding is less stringent) restores MRP1 protein expression
levels. This suggests that the mutations may cause misfolding and destabilization of the transporter leading to
enhanced degradation, as observed for some mutant
CFTR proteins (242, 468). In other instances where MRP1
protein expression is abrogated by opposite charge substitutions, expression is unaffected by substitutions with
more neutral or same-charge residues. However, the expressed mutants are not necessarily active. For example,
MRP1 mutants in which Arg1202 in TM16 is replaced with
Lys, Gly, or Leu are expressed but not when the substituting amino acid is Asp. Similarly, the Lys substituted
mutant of the nearby Glu1204 is poorly expressed while
mutants with neutral (Leu) and same-charge (Asp) substitutions at position 1204 are expressed at levels comparable to wild-type MRP1. However, the expressed Arg1202
mutants possess wild-type MRP1 transport activity while
the neutrally substituted Glu1204 mutant is inactive, despite the fact that it retains the ability to bind LTC4. The
Glu1204 mutant also exhibits a dramatic increase in vanadate-induced trapping of ADP at NBD2, indicating that the
catalytic activity of the transport is compromised (468).
The distinct phenotypes associated with mutations of the
highly conserved Arg1202 and Glu1204 are presumably
caused by perturbations in the ␣-helical geometry of
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Physiol Rev • VOL
taurocholate, while introduction of a basic amino acid in
place of Leu in Mrp3 resulted in a loss of both taurocholate transport and transport of the common substrate
E217␤G (209).
Certain types of amino acids have been systematically mutated in MRP1 (and to a lesser extent in MRP2),
because the chemical and physical properties of their side
chains suggest that they are likely to be involved in the
overall architecture and flexibility of the translocation
pathway of the protein and/or in direct interactions of the
transporter with its substrate(s). Pro residues have the
potential to kink ␣-helical elements and, because of their
low intrinsic hydrogen bonding capacity, may increase
the flexibility of TMs (439). As a consequence, Pro hinges
and other features of TM helix geometry may be important in the structural responses to conformational
changes that occur during the binding and transport of
MRP1 substrates (210, 242). When the 12 Pro residues in
MSD0 and CL3 were individually replaced with Ala, relatively few substantial changes in MRP1 organic anion
transport activity were observed (210). In contrast, 14 of
the 18 Pro residues in MSD1 and MSD2 were mutation
sensitive (242). Interestingly, while there are 9 Pro residues in each of the second and third MSDs, their distribution is quite asymmetric. Thus the majority of Pro
residues in MSD1 occur in the predicted TMs, whereas
this is not the case in the third MSD. Moreover, the Pro
residues in the COOH-proximal MSD are significantly
more conserved among ABCC family members than are
those in MSD1.
Consistent with their predicted importance in maintaining the structure of a functional transporter, single Ala
substitutions of seven of nine Pro residues in MSD1 and
five of nine in MSD2 reduced transport of at least some
organic anion substrates by 50% or more (242). The mutation-sensitive Pro residues in MSD1 were all found in
the predicted TMs, whereas this was not the case for
MSD2. One particularly interesting phenotype was observed after Ala substitution of the highly conserved
Pro1150 that is located in the cytoplasmic loop (CL7) connecting TM16 to TM17. This mutant exhibited a substantial increase in E217␤G and methotrexate transport, while
transport of other organic anions was reduced or unchanged. The increase in E217␤G transport by the Pro1150
mutant was not only associated with an increase in uptake affinity (Km) but also with an increase in the apparent affinity of the mutant transporter for ATP. Orthovanadate-induced trapping of ADP by the mutant protein was
also dramatically reduced, indicating the ability of NBD2
to hydrolyze ATP is significantly compromised. Together,
these observations suggest that the structural integrity of
CL7 is important for direct interactions of at least some
substrates with the transporter, as well as coupling transport with conformational changes in NBD2 necessary for
its catalytic activity (242). It is worth noting that muta-
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the architecture of the substrate binding pocket. Similarly, only Ala substitution of Asn590 (a “cavity”-creating
substitution) adversely affected MRP1 activity, while replacing this residue with Asp or Gln had no effect,
suggesting that the polar side chain of Asn590 may be
involved in interhelical interactions that influence the
conformation of the protein in the vicinity of the binding
pocket (570).
In addition to TMs 11 and 17, the substrate specificity
of MRP1 is affected by mutation or naturally occurring
variation of residues in several other TM helices, including TMs 6, 7, 8, 9, 10, 14, 15, and 16 as well as some of the
TMs connecting cytoplasmic loops (most notably CL7
between TM15 and TM16) (76, 77, 160, 169, 241, 242, 411,
468, 565, 566, 568). Mutational studies of MRP2, although
relatively limited, have also identified functionally important residues in TMs 6, 11, 14, and 16 (208, 432) in addition
to TM17 (206). For example, mutation of the Arg586 in
TM11 of MRP2 was found to selectively decrease transport of GSH conjugates (208). However, this was not the
case for the corresponding Arg593 in TM11 of MRP1 where
a comparable mutation affected the transport of a broad
range of organic anion substrates as well as binding of
LTC4 (160).
One of the most striking examples of a major alteration in substrate specificity resulting from single amino
acid variation came from the functional characterization
of mammalian MRP1 orthologs. MRP1 is relatively highly
conserved among mammals, and the human protein exhibits 88, 86, 92, and 98% sequence identity with the
mouse, rat, dog, and macaque proteins, respectively (144,
314, 371, 481). However, with the exception of macaque
MRP1, the other orthologs fail to confer resistance to
anthracyclines and are poor transporters of E217␤G (144,
314, 371, 482). The lack of anthracycline resistance has
been traced to the presence of a Gln rather than Glu
residue in TM14 (Glu1086 in human MRP1), while the poor
E217␤G transport seems attributable in large part to the
presence of Ala rather than Thr in TM17 (Thr1242 in human
MRP1) (565, 566). Surprisingly, the common ability to
confer resistance to vincristine and VP-16 was lost following reciprocal substitution of the Glu or Gln residue in
TM14 of the human and murine proteins, respectively
(565). However, resistance could be rescued by a second
mutation of the Thr or Ala residue in TM17 that restored
the “pairing” of Glu and Thr or Gln and Ala. Thus an
apparently conserved function of the human and mouse
proteins depends on the pairing of two nonconserved
amino acids. Residues in TM14 of MRP2 and MRP3 have
also been implicated in determining the substrate profiles
of both proteins (209). MRP3 differs from both MRP1 and
MRP2 in its ability to transport monovalent bile salts such
as taurocholate (184). Mutation of Arg1096 in TM14 of rat
Mrp2 to Leu, as found at the corresponding position of rat
Mrp3, resulted in acquisition of the ability to transport
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
Physiol Rev • VOL
the three-dimensional structure of the transporter becomes available.
Studies of charged residues in MRP2 and MRP3 are
not as extensive as those of MRP1 and, since many of the
substrates of these transporters are anionic, have focused
primarily on the role of basic amino acids. Mutation of Lys
and Arg residues in TMs 6, 11, 13, 14, 16, and 17 of MRP2
that are conserved either in MRP1 and MRP3, or only in
MRP1, identified Lys325 (TM6) and Arg586 (TM11) as being
important for transport of glutathione conjugates such as
LTC4, but not compounds conjugated with glucuronide or
sulfate (208). Thus, as observed with MRP1, mutations
identified to date that affect transport of GSH conjugates
appear to primarily involve TMs in MSD1. Overall, the
data are consistent with the notion that the binding
pocket contains regions that interact primarily with the
anionic moieties of conjugated substrates (or GSH) and
others are involved in binding the hydrophobic components of the substrate.
XI. HIGHER ORDER STRUCTURE OF
MULTIDRUG RESISTANCE PROTEIN 1
A. Molecular Modeling
In the absence of a high-resolution crystal structure,
it is often challenging to interpret the results of mutational studies. This is because of the difficulty of ascertaining whether a specific mutation directly affects molecular contacts with substrate or alters specificity indirectly, either by changing the architecture of the substrate
binding pocket or affecting the ability to undergo the
conformational changes involved in substrate transport.
In lieu of a high-resolution crystal structure, atomic models of the tertiary structure of the two core MSDs of MRP1
have been derived by homology modeling and molecular
dynamics simulations and used to indicate the possible
disposition of residues in the protein that have been
shown to affect substrate specificity and overall transport
activity (52). These atomic models are based on the crystal structures of the bacterial lipid transporter MsbA from
V. cholera and E. coli, as well as a model of P-gp/MDR1
that has incorporated structural data derived from various
sources, including cysteine-scanning mutagenesis (55, 56,
478). Because MsbA and P-gp/MDR1 share a relatively
high level of homology, P-gp/MDR1 was modelled initially
on the MsbA structure. The derived P-gp/MDR1 model
was then manipulated to accommodate electron microscopic data obtained from two-dimensional crystals and
single particles, as well as disulfide cross-linking data
from cysteine scanning mutagenesis (305–308, 422, 424).
Although the sequence similarity between MsbA and
MRP1 is low by comparison with P-gp/MDR1, it was possible to identify two regions of homology, corresponding
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tions of some of the conserved Pro residues in CFTR/
ABCC7 and MRP6 ABCC6 are associated with cystic fibrosis and PXE, respectively, indicating that the functional importance of these residues is apparently
conserved.
The side chains of aromatic and polar aromatic
amino acids are potentially capable of establishing a variety of bonding interactions with various MRP1 substrates, as well as influencing the position of TM helices in
the membrane through a variety of inter- and intrahelical
interactions and interactions with membrane phospholipids (52, 77, 206, 207, 241, 289, 374). Trp residues occur at
a greater frequency in most ABCC family members than
they do in transporters belonging to other ABC subfamilies, including P-gp/MDR1. Furthermore, mutation of all
11 Trp residues of P-gp/MDR1 revealed that none of them
is essential for the transport function of this protein (260).
In contrast, of the 30 Trp residues in MRP1, mutations of
6 of them, which are moderately to highly conserved and
are found in or near TMs (including Trp1246 in TM17
described earlier), result in substantial changes in transport activity or substrate specificity (241). Thus Ala substitution of Trp445 (TM8), Trp553 (TM10), and Trp1198
(TM16) eliminated or dramatically reduced transport levels of a broad range of organic anion substrates. On the
other hand, Ala substitutions of Trp361 (TM7) and Trp459
(TM9) caused more moderate alterations. More conservative substitutions of Trp445, Trp553, and Trp1198 resulted in
substrate-selective retention of transport in the case of
Trp445 and Trp1198 but not Trp553 in TM10. It would appear
that while these mutation-sensitive Trp residues may play
a role in determining the position of certain TM helices in
MRP1, the side chains of each of these residues also
contribute in their own distinct way to the transport
activity and substrate specificity of the transporter.
Finally, as already alluded to above, systematic substitutions of charged amino acids have implicated a significant number that are important for MRP1 activity.
These mutation-sensitive ionizable residues are distributed throughout the primary structure of the protein, and
the mutant phenotypes observed are quite variable. Thus
the phenotypes associated with nonconservative mutations of basic and acidic residues in MRP1 range from a
total loss of protein expression (e.g., Asp430, Asp792,
Arg1202, Glu1204) (84, 160, 468) to a substrate-selective loss
(e.g., Lys332, Arg433) (76, 159, 160) or general loss of
substrate binding and transport activity (e.g., Asp336,
Arg1197, Arg1249) (159, 160, 410, 468) to a loss of transport
activity but retention of substrate binding (e.g., Asp1084,
Glu1204) (468, 568). At present, it is not possible to predict
the phenotype that will ensue from mutation of a charged
residue based on its location in the primary structure of
MRP1 or its degree of conservation among other ABC
proteins. This should change as more information about
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DEELEY, WESTLAKE, AND COLE
four different substrates tested and eliminated photolabeling by LTC4 (52). Conservative substitutions with Trp
or Tyr on the other hand differentially affected transport
of specific substrates. Thus mutation to Trp decreased
transport of E217␤G and methotrexate but not estrone
sulfate and GSH, while the Tyr mutation had the opposite
effect. The selective alterations in transport resulting
from the two conservative mutations suggest that Phe594
may interact directly with at least some substrates and are
consistent with the possibility that the entrance to the
translocation pore may be defined by a ring of aromatic
residues. The model also predicts that the majority of
residues in TMs 6, 11, 14, 16 and 17, shown by mutational
studies to influence substrate specificity, are located on
faces of their respective helices that form the lining of the
putative translocation pathway through the membrane.
Thus the model is consistent with the possibility that
substrates establish interactions primarily with amino
acid side chains lining the inner leaflet region of the
translocation pathway. Although in agreement with data
derived from a number of mutagenesis studies, the models remain to be validated by other experimental approaches such as cysteine-scanning mutagenesis and
high-resolution studies of the three-dimensional structure
of the protein in nucleotide and substrate-bound states.
B. Electron Crystallographic Studies
FIG. 10. Three-dimensional cartoon of MRP1. The figure is based in
part on the energy minimized model of MSD1 (blue) and MSD2 (green)
of MRP1 described in Reference 52. NBD1 (blue) was modeled by
threading onto the crystal structure of NBD1 of CFTR (290), and NBD2
(green) was threaded onto HlyB (449). The threaded NBDs were then
aligned with the crystal structures of the NBD dimer of MJ0796 and the
structure of MsbA from Vibrio cholerae (472). Walker A, Walker B, and
the C signature are shown in red, yellow, and orange, respectively. The
5 TM helices of MSD0 are depicted in red. However, the tertiary structure of MSD0 and the manner in which it interacts with the remainder of
the protein is completely unknown.
Physiol Rev • VOL
Electron crystallographic studies of P-gp/MDR1,
CFTR, and MRP1 have provided three-dimensional structures with resolution limits of 8, 20, and ⬃22 Å, respectively (423, 426, 427). All three proteins display some
twofold pseudosymmetry with a ring of protein surrounding an electron-dense cavity or barrel of variable size and
shape, which is presumed to correspond to the translocation pore. The structure of MRP1 is presently less refined
than those of the other two proteins, but analysis of single
particles revealed some deviation from twofold pseudosymmetry in the form of two small electron-dense regions
on the periphery of the ringlike structure (423). One of
these regions may be attributable to the third NH2-terminal MSD. However, individual TM helices are not resolvable, and the current images of MRP1 provide no information about the manner in which the TM helices of
MSD0 may be packed, or how they might interact with the
core of the protein. Unlike P-gp/MDR1 and CFTR, the
two-dimensional crystals of MRP1 had a unit cell that
consisted of a protein dimer (423). However, this arrangement may be a crystallization artifact, and although some
previous biophysical and biochemical studies of MRP1
and CFTR have suggested that their native, functional unit
is a dimer (115, 399, 475, 564), the evidence remains
inconclusive for both proteins.
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to residues 325–596 in MSD1 and 1019 –1249 in MSD2 (52).
These regions were used to derive energy-minimized
models of MSDs 1 and 2 of MRP1 (Fig. 10). Thus the
structure lacks MSD0 and CL3, as well as some other CL
loop regions and the NBDs and the COOH-terminal region
of the protein.
The best models predict that several polar aromatic
residues previously shown by mutational studies to influence substrate specificity (Trp553 TM10, Trp1198 TM16,
and Trp1246 TM17 ) (207, 241, 567) are located close to the
membrane cytosol interface with their side chains projecting toward a chamber formed by the TM helices of the
two MSDs. Based on the model, Phe594 (TM6) was predicted to be located in the inner leaflet region with its side
chain also projecting into the chamber (52). The predicted
position of the Phe residue suggested that it might be a
component of an “aromatic basket” formed together with
the previously identified Trp and Tyr residues. Indeed,
mutation of Phe594 to Ala drastically reduced transport of
TRANSMEMBRANE TRANSPORT BY MRP-RELATED PROTEINS
XII. CONCLUSION AND PERSPECTIVES
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence:
R. G. Deeley, Cancer Research Institute, 10 Stuart St., Suite 300,
Queen’s University, Kingston, Ontario, Canada, K7L 3N6 (e-mail:
[email protected]).
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