Download Mammalian multidrug‑resistance proteins (MRPs)

© The Authors Journal compilation © 2011 Biochemical Society
Essays Biochem. (2011) 50, 179–207; doi:10.1042/BSE0500179
10
Mammalian
multidrug‑resistance
proteins (MRPs)
Andrew J. Slot, Steven V. Molinski and
Susan P.C. Cole1
Division of Cancer Biology and Genetics, Queen’s University Cancer
Research Institute, Kingston, ON, Canada, K7L 3N6
Abstract
Subfamily C of the human ABC (ATP‑binding cassette) superfamily contains
nine proteins that are often referred to as the MRPs (multidrug‑resistance
proteins). The ‘short’ MRP/ABCC transporters (MRP4, MRP5, MRP8
and ABCC12) have a typical ABC structure with four domains comprising
two membrane‑spanning domains (MSD1 and MSD2) each followed by a
nucleotide‑binding domain (NBD1 and NBD2). The ‘long’ MRP/ABCCs
(MRP1, MRP2, MRP3, ABCC6 and MRP7) have five domains with the
extra domain, MSD0, at the N‑terminus. The proteins encoded by the
ABCC6 and ABCC12 genes are not known to transport drugs and are
therefore referred to as ABCC6 and ABCC12 (rather than MRP6 and
MRP9) respectively. A large number of molecules are transported across
the plasma membrane by the MRPs. Many are organic anions derived
from exogenous sources such as conjugated drug metabolites. Others are
endogenous metabolites such as the cysteinyl leukotrienes and prostaglandins
which have important signalling functions in the cell. Some MRPs share a
degree of overlap in substrate specificity (at least in vitro), but differences in
transport kinetics are often substantial. In some cases, the in vivo substrates
1To
whom correspondence should be addressed (email [email protected]).
179
180
Essays in Biochemistry volume 50 2011
for some MRPs have been discovered aided by studies in gene‑knockout
mice. However, the molecules that are transported in vivo by others,
including MRP5, MRP7, ABCC6 and ABCC12, still remain unknown.
Important differences in the tissue distribution of the MRPs and their
membrane localization (apical in contrast with basolateral) in polarized
cells also exist. Together, these differences are responsible for the unique
pharmacological and physiological functions of each of the nine ABCC
transporters known as the MRPs.
Introduction
Cellular efflux of xenobiotics is often mediated by members of the ABC
(ATP‑binding cassette) superfamily of transmembrane proteins which use
the energy of ATP binding and hydrolysis to perform their functions. Three
mammalian ABC proteins, P‑glycoprotein (ABCB1), ABCG2 [also known
as BCRP (breast cancer‑resistance protein)] and MRP1 (multidrug‑resistance
protein 1) (ABCC1), were originally discovered because of their ability to
confer resistance to anti‑neoplastic agents in cultured tumour cells [1]. In
recent years, however, these and other xenobiotic‑transporting ABC proteins
have also been shown to be important determinants of the disposition (tissue
distribution) and elimination of many clinically relevant drugs. Thus the ABC
drug‑efflux proteins contribute to the pharmacokinetic profiles, efficacy and
toxicity (side effects) of a large number of therapeutic and diagnostic agents,
as well as chemicals found in the environment and diet. Equally, if not more
importantly, many of these drug‑transporting ABC proteins also mediate the
cellular efflux of a broad range of physiological metabolites with pivotal roles
in signalling pathways in the cell.
Phylogenetic analyses have divided the 48 human ABC genes into seven
subfamilies: A–G. Subfamily C contains nine drug transporters that are often
referred to as the MRPs or ABCC proteins (Figure 1A and Table 1) [2]. The
MRP/ABCC proteins are found throughout Nature, including in plants,
marine organisms and unicellular eukaryotes where they carry out many
important functions. However, this chapter focuses on human MRPs with
reference to their mammalian orthologues where relevant.
The MRPs
Of the nine MRP/ABCC proteins, four of them, i.e. ABCC4, ABCC5,
ABCC11 and ABCC12 (the ‘short’ MRP4, MRP5, MRP8 and ABCC12
respectively), have a typical ABC structure with four domains comprising
two MSDs (membrane‑spanning domains) (MSD1 and MSD2), also
known as TMDs (transmembrane domains), each followed by an NBD
(nucleotide‑binding domain) (NBD1 and NBD2) (Figure 1B). ABCC1,
ABCC2, ABCC3, ABCC6 and ABCC10 (the ‘long’ MRP1, MRP2, MRP3,
ABCC6 and MRP7 respectively) have five domains with the extra domain,
© The Authors Journal compilation © 2011 Biochemical Society
A.J. Slot, S.V. Molinski and S.P.C. Cole
181
Figure 1. Relatedness and structures of the human MRP/ABCC transport proteins
(A) Dendrogram illustrating the relatedness (degree of homology) of the human MRP/ABCC
proteins. The dendrogram is based on ClustalW alignments and was generated using Phylip’s
Drawgram (http://workbench.sdsc.edu). The proteins encoded by the ABCC6 and ABCC12 genes
are not known to transport drugs and therefore are referred to as ABCC6 and ABCC12 (rather
than MRP6 and MRP9 respectively) throughout the text. (B) Predicted topology of the long
(MRP1, MRP2, MRP3, ABCC6 and MRP7) and short (MRP4, MRP5, MRP8 and ABCC12) MRP/
ABCC transporters. The long MRPs contain three MSDs (with 17 TMHs) and the N‑terminus
is extracellular; the short MRPs contain just two MSDs (with 12 TMHs) and the N‑terminus is
intracellular. The two cytoplasmic NBDs (NBD1 and NBD2) found in all ABC transporters are
shown with their three characteristic motifs indicated (WA, Walker A; WB, Walker B; C, ABC
signature sequence) [4]. (C) A homology model of the core region (MSD1–NBD1–MSD2–
NBD2) of MRP1 that forms the substrate translocation pathway through the membrane. The
model is based on the crystal structure of S. aureus Sav1866 [3] and comprises the two NBDs
and the second (MSD1; TMHs 6–11) and third (MSD2; TMHs 11–17) MSDs [18]. S. aureus does
not contain a third MSD comparable with MSD0 of MRP1 and therefore this domain could not
be modelled. Shown is the α‑carbon backbone in ribbon representation, as viewed from the
plane perpendicular to the membrane bilayer.
© The Authors Journal compilation © 2011 Biochemical Society
1503
16p13.12
6p21.1
ABCC6
ABCC10
© The Authors Journal compilation © 2011 Biochemical Society
16q12.1
16q12.1
ABCC11
ABCC12
1359
1382
1437
1325
1492
ABCC12†
MRP8
MRP5
MRP4
MRP7
ABCC6†
MRP3
28
26
30
31
30
45
56
27
26
30
31
29
37
45
100
MRP2
26
26
27
27
28
100
26
26
28
28
100
MRP7
31
30
31
100
MRP4
42
39
100
MRP5
46
100
MRP8
Short
100
ABCC12†
MRP6 and MRP9 respectively) throughout the text.
proteins encoded by the ABCC6 and ABCC12 genes are not known to transport drugs and are therefore referred to as ABCC6 and ABCC12 (rather than
pl?page=/NPSA/npsa_server.html for alignment by Clustal W using the default parameters.
†The
27
29
28
29
31
43
100
MRP3 ABCC6†
Long
were obtained from http://www.uniprot.org and amino acid identity was determined by compiling data from http://npsa‑pbil.ibcp.fr/cgi‑bin/npsa_automat.
3q27.1
ABCC5
*Sequences
13q32.1
ABCC4
Short
1527
17q21.33
46
ABCC3
MRP2
MRP1
100
1545
10q24.2
1531
16p13.11
ABCC2
Protein
name
ABCC1
Amino
acids
MRP1
Chromosome location
Long
Gene
symbol
Identity (%)*
Table 1. Chromosome location and sequence identity of human ABCC/MRP transporters
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Essays in Biochemistry volume 50 2011
A.J. Slot, S.V. Molinski and S.P.C. Cole
183
MSD0, located at the N‑terminus of these transporters. Current evidence
supports a topology model where MSD1 and MSD2 [which each contain six
TMHs (transmembrane helices)] form the translocation pathway through
which substrates cross the membrane (Figure 1C). It also supports a model
where the two NBD proteins associate in a head‑to‑tail orientation to form
a ‘sandwich dimer’ that comprises two composite NBSs (nucleotide‑binding
sites) [3,4] (Figure 1C). These NBSs are each composed of the highly
conserved Walker A and Walker B motifs from one NBD and the ABC active
transport signature motif from the other NBD, and together are responsible
for the binding and hydrolysis of two molecules of ATP. The NBDs of ABC
proteins are generally very highly conserved; however, the NBDs of the
ABCC/MRP‑related proteins contain some sequence variations that are likely
to be responsible for some of the differences in how members of this subfamily
of transporters interact with ATP and the products of its hydrolysis [5,6].
Substrate translocation for the MRPs, as for all mammalian ABC proteins,
is a multistep process which begins with substrate recognition and binding to
a high‑affinity conformation of the transporter on one side of the membrane,
followed by conformational changes in the transporter that allow substrate
translocation across the membrane and then substrate release on the opposite
side of the membrane (Figure 2A). The transporter then once again assumes its
high‑affinity conformation so that another round of transport can take place.
These processes are obligatorily coupled to the binding and hydrolysis of
ATP, as well as the release of ADP/Pi, although the precise details of this cou‑
pling have not yet been fully elucidated [4].
A large number of molecules are transported across membranes by the
MRPs (Figure 2B and Table 2). Some of the MRPs share a limited degree of
overlap in substrate specificity (at least in vitro), but differences in transport
kinetics are often substantial. Important differences in the tissue distribution of
the MRPs and their membrane localization (apical in contrast with basolateral)
in polarized epithelial and endothelial cells also exist (Table 3). Together, these
differences are responsible for the unique pharmacological and physiological
functions of each of the individual MRP transporters.
The long MRPs
MRP1
MRP1 (gene symbol ABCC1) was the first of the drug‑transporting ABCC
proteins to be cloned during investigations into the cause of multidrug
resistance in human lung carcinoma cells [5]. The ABCC1 mRNA expressed
in these lung cancer cells was predicted to encode a 1531‑amino‑acid 170 kDa
protein. However, in mammalian cells, the protein is both N‑glycosylated
and phosphorylated, and thus the mature transporter typically exhibits an
electrophoretic mobility of ~190 kDa. Depending on the cell type in which
MRP1 is expressed, its corresponding protein band in gels is often quite
diffuse, owing to heterogeneity of glycosylation.
© The Authors Journal compilation © 2011 Biochemical Society
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Essays in Biochemistry volume 50 2011
Figure 2. Transmembrane transport of organic anions by MRP/ABCC proteins
(A) Transmembrane transport of MRP/ABCC substrates is a multistep process. 1. ‘Substrate’ or
ligand binds to an accessible high‑affinity site on the transporter that faces one side of the plasma
membrane. 2. The conformation of the transporter changes during translocation of the substrate
such that the substrate‑binding site now faces the opposite side of the membrane. The affinity
of the binding site must also decrease to allow the substrate to be released. 3. The MRP/ABCC
transporter protein undergoes another conformational change such that it reverts back to its
original high‑affinity state to begin another round of transport. 4. The three steps of transport
are obligatorily coupled to the binding and hydrolysis of ATP, as well as the release of ADP and
Pi, but the precise details of how this is accomplished are not yet known. However, it is known
that significant changes in the relative conformations of the two NBSs take place. WA, Walker
A; WB, Walker B; C, ABC signature sequence. (B) Chemical structures of several organic anion
substrates of different ABCC/MRPs illustrating the diversity of molecules recognized by these
transporters, as described in the text and Table 2.
© The Authors Journal compilation © 2011 Biochemical Society
Oestrone 3‑sulfate
DHEA (dehydroepiandrosterone) sulfate
Cyclic nucleotides
cAMP
cGMP
Other
Folic acid
GSSG
Endogenous organic anions
Arachidonic acid derivatives
LTC4
Prostaglandin E2/E1
Prostaglandin A2‑SG
Steroids and conjugates
Bilirubin glucuronide
Glycocholic acid
Oestradiol glucuronide
Substrates
demonstrating that it is a functional transporter [73].
P
nt
P
P
P
100
7.2
2.5
0.7*
5*
1
1
MRP2
2
P
0.1
MRP1
P
P
P
P
248
30
5.3
MRP3
Long
2
P
P
0.6
ABCC6
P
58
P
MRP7
P
P
P
(Continued)
P
P
45
170
P
63
P
nt
MRP8
P
17
379
2
P
MRP5
nt
2
26*
30
P
3.4/2.1
MRP4
Short
a given organic anion is transported by a given MRP/ABCC protein, as this may not yet have been tested. ABCC12 is not included since there are no data yet
GSH (required). In some cases, substrate affinities [apparent Km (μM)] are indicated. The absence of a symbol or number does not exclude the possibility that
The Table is not intended to be comprehensive. Key: P, substrate for this MRP/ABCC protein; nt, substrate not transported; *, transported in the presence of
Organic anions listed are selected from data obtained in in vitro transport assays using membrane vesicles enriched with recombinant human MRP/ABCC proteins.
Table 2. Selected organic anion substrates of human MRP/ABCC drug transporters
A.J. Slot, S.V. Molinski and S.P.C. Cole
185
© The Authors Journal compilation © 2011 Biochemical Society
© The Authors Journal compilation © 2011 Biochemical Society
700
‡Small‑molecule 4‑anilinoquinazoline‑based tyrosine kinase inhibitors (e.g. erlotinib, imatinib, nilotinib, lapatinib).
†Monophosphorylated metabolite(s) transported.
P
P
P
>500
5.7
P
P
P
nt
P
P
2200
P
37*
P
P
6.5
0.2
3.6
28
P
MRP7
(adefovir)
Other
Tyrosine kinase inhibitors‡
ABCC6
GSH
GSH (+ apigenin)
Exogenous metabolites
GSH conjugates
Aflatoxin B1‑SG
Dinitrophenyl‑SG
Ethacrynic acid‑SG
Glucuronide conjugates
NNAL‑O‑glucuronide
Morphine 6‑glucuronide
Morphine 3‑glucuronide
Acetaminophen glucuronide
Antimetabolites
Methotrexate
5‑FU†
6‑Mercaptopurine†
6‑Thioguanine†
Azathioprine†
PMEA [9-(2-phosphonylmethoxyethyl)-adenine]
nt
>5000
>1000
116
MRP3
MRP2
MRP1
Long
Substrates
Table 2. (Continued)
P
P
P
P
220
P
P
MRP4
P
P
P
P
960
P
P
P
P
MRP8
1200
P
P
MRP5
Short
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A.J. Slot, S.V. Molinski and S.P.C. Cole
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Table 3. Expression of MRP/ABCC proteins in human tissues
Relative expression levels represent whole‑tissue analysis, and thus do not reflect cell‑type‑
specific expression within these tissues. It should be noted that variability among studies as well
as inconsistencies in the literature exist. Key: +, low to moderate expression; ++, moderate to
high expression; ap, apical membrane; bsl, basolateral membrane; * only in blood–brain barrier.
Long
Tissue
MRP1
MRP2
Adrenals
Blood–brain barrier
++
Brain
+
Breasts
+
Colon
++
Heart
+
Kidney
++
Liver
Short
MRP3
ABCC6
++
+
+
+
+
MRP4
+
++
+
+
+
+
++
+
+
+
+
++
++
++
++
++
++
++
+
+
+
Lung
++
Ovary
+
Pancreas
+
+
++
Placenta
++
++
+
+
Prostate
+
+
+
Skeletal muscle
++
+
+
Skin
++
+
+
Small intestine
++
+
+
+
++
++
+
Testis
++
Membrane
+
+
+
ap*, bsl
+
++
Smooth muscle
Spleen
MRP5
+
ap
bsl
+
+
+
+
+
+
bsl
ap, bsl
ap*, bsl
localization
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Pharmacological and physiological roles of MRP1
It is well established that, in vitro, MRP1 can confer resistance to many widely
used anti‑neoplastic drugs. These include conventional cytotoxic agents such
as doxorubicin, vincristine, etoposide and methotrexate, as well as some of
the newer ‘targeted’ agents that modify various signal transduction pathways
(e.g. tyrosine kinase inhibitors) [6,7]. MRP1 mRNA and/or protein has also
frequently been detected in tumour samples from patients, but its overall
contribution to clinical resistance is still not well defined. This is also true of
the related P‑glycoprotein and ABCG2 transporters, and is largely due to the
well‑documented difficulties of accurately measuring the amount and activity
of the transporters in tumour compared with normal tissues in rigorously
designed clinical trials. Nevertheless, MRP1 is currently considered to be the
most clinically relevant of the MRPs with respect to drug resistance in cancer,
a major obstacle to successful chemotherapy. In addition to tumour cells,
MRP1 contributes to drug and xenobiotic disposition in normal cells and thus
one of its important roles is that of tissue defence [1].
MRP1 mRNA and protein is found in all organs, but at relatively higher
levels in certain tissues (e.g. testes or lung) (Table 3). The tissue distribution of
MRP1 is consistent with its role in limiting the penetration of certain cytotoxic
agents at a number of blood–organ interfaces and thus MRP1 contributes to
so‑called pharmacological sanctuary sites in the body, such as the blood–brain
barrier and the blood–testis barrier [1]. The most direct evidence for a protec‑
tive role for MRP1 has come from studies of mice in which the Abcc1 gene
has been disrupted. Although these knockout mice are viable and fertile, they
display increased chemosensitivity in certain tissues such as the seminiferous
tubules, the intestine, the oropharyngeal mucosa and the choroid plexus [8,9].
Shortly after the demonstration that MRP1 could confer multidrug resis‑
tance, it was discovered that MRP1 was also a high‑affinity transporter of
the pro‑inflammatory cytokine LTC4 (leukotriene C4) [10]. LTC4 is an ara‑
chidonic acid derivative conjugated to glutathione (GSH) that is involved in
asthmatic and allergic reactions. It is exported from cells after its synthesis and,
together with its metabolites leukotriene D4 and leukotriene E4, it exerts its
biological effects by acting on CysLT (cysteinyl leukotriene) receptors present
on the surface of various target cells. Abcc1−/− mice display a diminished
inflammatory response consistent with a defect in LTC4 efflux, providing con‑
firmatory in vivo evidence that a key physiological role of MRP1 is to mediate
LTC4 export [8].
In addition to LTC4, MRP1 transports many other structurally diverse
GSH‑conjugated organic anions, some of which are endogenous metabo‑
lites and others which are the products of Phase II xenobiotic metabolism
[1,7] (Table 2). Thus it seemed that MRP1 was (at least one of) the ubiqui‑
tous ATP‑dependent GSH conjugate efflux pumps proposed previously by
Ishikawa [11]. However, MRP1 is much more versatile than this because
it can also transport organic anions conjugated to glucuronate and sulfate.
© The Authors Journal compilation © 2011 Biochemical Society
A.J. Slot, S.V. Molinski and S.P.C. Cole
189
Again, some of these are endogenous metabolites (e.g. oestradiol glucuronide,
oestrone sulfate), whereas others are conjugates of xenotoxins such as the glu‑
curonide conjugate of the tobacco‑specific carcinogen NNAL [4‑(methylnitro
soamino)‑1‑(3‑pyridyl)‑1‑butanol] [1]. Despite its ability to transport a range
of important physiological metabolites, there are no known human diseases
associated with a lack of functional MRP1.
Many compounds have been identified as substrates of MRP1 (and other
MRPs) using an in vitro assay that measures the ATP‑dependent uptake of a
radiolabelled form of the compound into inside‑out membrane vesicles pre‑
pared from cells overexpressing MRP1 [10] (Figure 3A). Such vesicular uptake
assays avoid the technical difficulties of quantifying ATP‑dependent efflux
of hydrophilic molecules (the preferred substrates of the MRPs) from intact
cells. Nevertheless, it should be noted that the in vivo relevance of vesicular
transport of many molecules shown in vitro to be MRP substrates has not yet
been established. The Abcc1−/− mice provide an extremely important, but not
ideal, model for in vivo substrate testing because of the marked differences in
the substrate specificity of MRP1 from primates and non‑primates [6]. Thus,
whereas MRP1 from humans and macaque monkeys can transport anthracy‑
cline drugs (e.g. doxorubicin) and oestradiol glucuronide, this is not the case
for MRP1 from rat, mouse, dog or cow, despite the fact that the amino acid
sequence similarity is >90% for all species.
Finally, an unusual aspect of MRP1 is its complex interactions with the
reducing tripeptide GSH (γ‑Glu‑Cys‑Gly) [7]. Among the many functions
of this cellular antioxidant is its critical role in protecting cells from the del‑
eterious effects of oxidative stress. GSH is also required for Phase II xenobi‑
otic metabolism to form hydrophilic GSH conjugates which are then exported
by MRP1 (or MRP2). GSH itself is a low‑affinity substrate of MRP1 (and
MRP2), whereas the pro‑oxidant glutathione disulfide (GSSG) is a relatively
higher‑affinity substrate [12], although the consequences of these transport
activities on redox homoeostasis are still not fully understood. Of relevance,
however, is the observation that GSH levels in some tissues of Abcc1−/− mice
are elevated 2‑fold [13]. Recent reports also suggest that GSH efflux in res‑
ponse to cell damage as part of an apoptotic signalling pathway may be medi‑
ated by MRP1 [14].
Some xenobiotics, including verapamil or bioflavonoids such as apigenin,
stimulate MRP1‑mediated GSH efflux without being transported them‑
selves [7,15]. In contrast, the Vinca alkaloid vincristine markedly enhances
the transport of GSH (and vice versa), and thus, in this case, GSH appears
to be co‑transported with (or cross‑stimulates transport of) the drug with‑
out conjugate formation. MRP1‑mediated transport of the conjugates
NNAL‑O‑glucuronide and oestrone sulfate is also enhanced by GSH; how‑
ever, in contrast with vincristine transport, GSH only stimulates the process
and is not itself transported [1,7]. The biological activities of GSH are typically
attributed to the proton‑donating properties of the thiol (SH) group of its
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Essays in Biochemistry volume 50 2011
Figure 3. Methods used to measure transport activity of MRP/ABCC proteins
(A) Vesicular transport assay. The vesicular transport assay typically measures MRP/
ABCC‑mediated ATP‑dependent uptake of a radiolabelled ligand into inside‑out membrane
vesicles which are then filtered and the amount of radiolabel inside the vesicles quantified by
liquid‑scintillation counting [10]. The membrane vesicles can be prepared from a number of
different cell types, but transfected mammalian or insect cells expressing human recombinant
ABCC/MRP proteins are usually preferred. The commercial availability of radiolabelled substrates
of sufficiently high specific activity can be limiting. However, improvements in the detection of
small quantities of substrate by other analytical methods (e.g. HPLC) are extending the usefulness
of this assay. (B) Vectorial (transcellular) transport assay. This assay uses cells that can grow in a
polarized fashion with distinct apical and basolateral domains [e.g. MDCK (Madin–Darby canine
kidney) cells]. When cultured to confluence on an appropriate semi‑permeable membrane sup‑
port in a transwell plate, tight junctions between cells are established as illustrated. Substrate
(frequently radiolabelled) can be added to the basolateral compartment and, after a prescribed
time period, the medium in the apical compartment can be collected and the amount of sub‑
strate quantified to determine vectorial transport in the basolateral‑to‑apical (B>A) direction.
Transport in the apical‑to‑basolateral (A>B) direction can also be measured using this system.
© The Authors Journal compilation © 2011 Biochemical Society
A.J. Slot, S.V. Molinski and S.P.C. Cole
191
central cysteine residue. However, this is not the case for its stimulatory effects
on MRP1 activity because non‑reducing analogues such as ophthalmic acid
or S‑methyl‑GSH can functionally substitute for GSH [15]. GSH (and some
analogues) can cause changes in the conformation of MRP1, but how these
changes relate to its transport‑stimulating activity is not known [16].
Structure of MRP1
Most evidence supports a topology of MRP1 comprising 17 TMHs divided
among its three MSDs (MSD0, MSD1 and MSD2) (Figure 1B), but the
precise arrangement of these domains is not yet known. Structural analyses
of MRP1 and other mammalian ABC proteins by X‑ray crystallography or
other biophysical methods pose a significant challenge, mainly due to the
large size of these polytopic membrane proteins which makes it difficult to
isolate the large amounts of purified active protein typically required for
such studies. Recent moderate‑resolution electron microscopy structural
studies have been able to resolve the TMHs of MRP1, although their precise
arrangement with respect to one another is still unclear [17]. Current models
of MRP1 are based on a relatively high‑resolution structure of the bacterial
(Staphylococcus aureus) ABC transporter Sav1866 in its nucleotide‑bound
form [3,18]. These models are limited in that they represent only the
four‑domain core structure of MRP1 because Sav1866 (or any other
bacterial transporter) does not contain a domain corresponding to MSD0.
In addition, these models reflect only a ‘snapshot’ of one conformation
(nucleotide‑bound, substrate‑free) of the many assumed by MRP1 during its
complex transport process.
Molecular determinants of MRP1 substrate transport and protein
expression
The technique of site‑directed mutagenesis has been used strategically to
discover specific regions and amino acids of MRP1 which are critical for:
(i) substrate specificity, (ii) proper folding and assembly ensuring stable
expression at the plasma membrane, and (iii) coupling of substrate transport to
the binding and hydrolysis of ATP [1,7,19].
As might be expected, amino acids important for the substrate specificity
of MRP1 are frequently located in the TMHs, particularly those in MSD1 and
MSD2 which form the substrate‑translocation pathway through the mem‑
brane. A notable example of such a mutation‑sensitive residue is Lys332 found
in the first TMH of MSD1, TMH6 [20]. When this basic amino acid is replaced
by either an oppositely charged or neutral amino acid, binding and transport
of LTC4 is essentially eliminated, whereas oestradiol glucuronide transport
remains unchanged [20]. The amphipathic C‑terminal TMH17 also contains a
number of polar amino acids important for substrate specificity. For example,
substitutions of Trp1246 eliminate oestradiol glucuronide transport and drug
resistance, but have little effect on LTC4 transport [21].
© The Authors Journal compilation © 2011 Biochemical Society
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Essays in Biochemistry volume 50 2011
Differences in the substrate and inhibitor selectivity of various MRP1
mutants has led to the conclusion that MRP1 contains at least three classes
of substrate/modulator‑binding sites: one that requires TMH17‑Trp1246 (and
probably other polar TMH17 residues), one that requires TMH6‑Lys332
(and other ionizable TMH6 residues), and a third that requires neither of
these amino acids [22]. It appears likely that that each substrate (or modu‑
lator) establishes its own unique set of atomic contacts in a multipartite
substrate‑binding pocket of MRP1, although it is clear that certain substrates
share at least some common binding determinants [21]. Unequivocal identi‑
fication of these atomic contacts should be forthcoming as the methods for
high‑resolution structural analyses (including MS) of large membrane proteins
continue to improve [23].
MRP2
MRP2 was first cloned from rat liver in 1996 using a strategy that took
advantage of its sequence similarity to human MRP1 (reviewed in [24]).
Before this, MRP2 was known as cMOAT (canalicular multispecific organic
anion transporter), largely based on functional studies of mutant rat strains
deficient in the biliary efflux of organic anions such as bilirubin glucuronide
[24]. These mutant rats were determined to harbour disabling mutations in
Abcc2. The human, rabbit, mouse and canine orthologues were cloned soon
thereafter, and, as for all of the MRPs, they show a high degree of amino acid
identity with one another (77–83%) [24]. Although MRP2 is similar in size
and topology (and presumably structure) to MRP1, it contains additional
sequence motifs in the cytoplasmic region linking MSD0 to MSD1, and at the
C‑terminus of the transporter that confer distinct properties on the protein
with respect to its plasma membrane trafficking and its ability to participate
in functional interactions with other proteins in the cell [25]. Like MRP1,
MRP2 functions as an ATP‑dependent organic anion efflux pump. However,
MRP2 is distinct from MRP1 and most other MRPs in that it is found
exclusively on the apical membrane of polarized epithelial and endothelial
cells, predominantly those in the liver, kidney and intestine, where it is
particularly well situated to play a role in the elimination, as well as in the oral
bioavailability of drugs, xenotoxins and their metabolites [24] (Table 3).
MRP2 and Dubin–Johnson syndrome
Dubin–Johnson syndrome is an autosomal recessive disorder caused by
mutations in the ABCC2 gene. Both nonsense and missense mutations have
been described and, in many cases, these result in severely diminished levels or
absence of the protein. Individuals with Dubin–Johnson syndrome frequently
have increased serum‑conjugated bilirubin levels [24], consistent with the fact
that biliary elimination of bilirubin glucuronides (which are MRP2 substrates)
is impaired. Although those with Dubin–Johnson syndrome are mostly
asymptomatic, neonates can present with cholestasis, and, during pregnancy,
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overt hyperbilirubinaemia can lead to jaundice in women. The mutant Abcc2
rat strains mentioned above have served as models for the study of this human
disease.
Physiological and pharmacological roles of MRP2
In the liver, MRP2 primarily functions to mediate the efflux of bile acids
and GSH, which helps in biliary homoeostasis. Many GSH, sulfate and
glucuronide conjugates have been identified as MRP2 substrates, some of
which are listed in Table 2. Prominent among these are a variety of oestrogen
conjugates which suggests a role for MRP2 in sex steroid homoeostasis and/
or cytoprotection. Whereas MRP1 and MRP2 both transport oestradiol
glucuronide, the kinetics differ substantially [26]. Thus it appears that MRP2
contains two similar, but non‑identical, ligand‑binding sites: one site from
which substrate is transported and a second site (allosteric) that regulates
the affinity of the transport site for the substrate. Oestradiol glucuronide
appears to bind to both sites, but this is not the case for all ligands that bind
to MRP2.
In vitro, MRP2 is similar to MRP1 in its ability to confer resistance to a
spectrum of natural product anticancer drugs. Distinct from MRP1, however,
MRP2 can confer resistance to platinum‑containing drugs [1,6,24,27]. The
clinical relevance of these in vitro observations, however, is debatable since
MRP2 expression in tumour samples has not been associated with response
to chemotherapy or clinical outcome. In contrast with its questionable role in
drug‑resistant tumours, MRP2 has a well‑established role in the biliary elim‑
ination of conjugated metabolites of numerous drugs and other xenobiotics
(Table 2). Glucuronide conjugates of acetaminophen and diclofenac are sub‑
strates of MRP2, as are glucuronide conjugates of several carcinogens, at least
in vitro [1,24,28,29].
Structure–function studies of MRP2
As shown for MRP1, mutational analyses of MRP2 point to a particularly
crucial role for charged amino acids in the TMHs of MSD1 and MSD2 as
determinants of substrate specificity, although the number of such studies
is relatively limited [30]. Two highly conserved uncharged residues, Trp 1254
at the cytoplasmic interface of TMH17 and Pro1158 in the cytoplasmic loop
connecting TMH15 to TMH16, are also important for activity [31,32].
However, the consequences of mutating these amino acids in MRP2 compared
with the analogous Trp1246 and Pro1150 in MRP1 (and Trp1242 and Pro1147
in MRP3) are dramatically different [31–33]. Substantial differences in the
molecular environments of these residues in the two transporters probably
account for the marked differences in the substrate specificities of the mutants.
These studies demonstrate that correlation of the structure to the function of
the MRPs is complex and cannot be predicted on the basis of primary amino
acid sequence alone.
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Vectorial (transcellular) transport
As noted for MRP1, most quantitative information regarding the substrate
specificity and affinity of MRP2 has been obtained using vesicular transport
assays (Figure 3A). Some interspecies differences exist with respect to the
substrate specificity and substrate affinity of MRP2 and its non‑primate
orthologues [34], and current evidence suggests that animal (at least rat)
pharmacokinetic data are often poor predictors of pharmacokinetics in
humans. This limitation provided the impetus to develop cell culture systems
that more closely approximate polarized cells directly involved in drug
disposition (found in intestine, kidney and liver) and restricted distribution
to tissue ‘sanctuaries’ (blood–tissue barriers). Such polarized cells have apical
and basolateral membranes separated by a tight junction. Drug transporters
tend to be asymmetrically localized in these two membranes which facilitates
transcellular (i.e. vectorial) transport of a drug or metabolite from the apical
to basolateral (and basolateral to apical) side of a cell. Thus double‑ and
multiple‑transfected polarized cell lines have been generated which express one
or more human uptake transporters [e.g. OATP1B1 (organic anion transporter
polypeptide 1B1); gene symbol SLCO1B1] in the basolateral membrane, and
efflux transporters human MRP2 (and/or MRP4) in the apical membrane
(although uptake transporters can be present here as well) [29,35] (Figure 3B).
Such cell lines are proving useful as models of hepatobiliary and renal drug
transport.
MRP3
MRP3 (gene symbol ABCC3) and MRP1 have the highest degree of
sequence similarity among the MRPs (Table 1), and, like MRP1, MRP3 is
expressed on the basolateral membranes of polarized cells. MRP3 is expressed
predominantly in the small intestine, the kidney (distal tubules) and the
pancreas (Table 3) [36]. It is also highly expressed in two of the three zones of
the adrenal cortex where its function remains unknown. Despite its sequence
similarity to MRP1 (and MRP2), MRP3 has a very different (and more limited)
substrate profile. The most striking difference is the very low affinity and
capacity of MRP3 to transport GSH [37]. Unlike MRP1 (and MRP2), MRP3
also does not require this tripeptide for efflux of the anticancer drug etoposide.
Like MRP2, MRP3 displays complex transport kinetics (in vitro) with some
of its conjugated substrates. This suggests that the protein contains both a
transport‑binding site and a modulatory site, although these sites have not yet
been defined at the molecular level [36].
There are no diseases associated with mutations in the human ABCC3
gene and, consistent with this, Abcc3−/− mice are healthy and show no appar‑
ent phenotype until challenged with certain pharmacological agents [36,38,39].
Although a helpful model for pharmacological studies (see below), these mice
have not yet provided any major insights into the physiological role of MRP3.
However, because MRP3 expression on the hepatic sinusoidal membrane is
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often up‑regulated under cholestatic conditions (bile duct ligation in rats and
cholestatic disease in humans), it is a commonly held view that MRP3 serves
as a ‘backup’ system for the removal of toxic liver metabolites via blood when
MRP2‑mediated transport into bile is impaired [36,40]. This concept is well
supported by studies of Abcc2/Abcc3 double‑knockout mice [38]. Thus MRP3
transports its substrates over the basolateral sinusoidal membrane of liver and
gut towards the circulation for subsequent elimination in urine.
In an attempt to identify the physiological substrate(s) of MRP3, Borst
and colleagues developed a targeted metabolomics approach based on the
premise that the abundance of MRP3 substrates (glucuronide conjugates) in
plasma and urine should be reduced in Abcc3−/− compared with wild‑type
mice [41]. Thus plasma and urine from these mice were screened for com‑
pounds containing a glucuronic acid moiety by MS. In this way, glucuronide
conjugates of several plant‑derived dietary phyto‑oestrogens were identified
as substrates of MRP3 [41]. This unbiased methodological approach for sub‑
strate identification may prove useful in studies of the less well characterized
ABCC6 and MRP7 (see below).
Relevant pharmacological examples of MRP3 substrates include the glu‑
curonide conjugates of morphine and acetaminophen. The case of morphine
is particularly interesting because the two glucuronide conjugates that are
formed during its metabolism in humans differ profoundly in their pharma‑
cological activity. Thus morphine 6‑glucuronide is a potent analgesic like
the parent drug, whereas morphine 3‑glucuronide is not. Relative to their
wild‑type counterparts, Abcc3−/− mice showed increased levels of morphine
3‑glucuronide in liver and bile, whereas plasma levels and excretion via the
urine were decreased [42]. Associated with these pharmacokinetic differences,
the antinociceptive potency of the bioactive morphine 6‑glucuronide was also
decreased in the Abcc3−/− mice. These observations suggest that, because of its
influence on the disposition of morphine and its active metabolite, individual
variations in MRP3 expression could contribute to the differences in morphine
pharmacokinetics and efficacy observed in human populations [42].
ABCC6
The human ABCC6 gene is located within 9 kb of the ABCC1 gene on
chromosome 16, suggesting that the two genes may have arisen from a
gene‑duplication event (Table 1). The structural organization of the two genes
is similar, and the encoded proteins are also comparable in size and topology.
Initial in vitro studies suggested that the 190 kDa ABCC6 protein (which is
found on the basolateral membrane of polarized endothelial and epithelial
cells) was an organic anion transporter with properties similar to those of
MRP1 and MRP3, with the notable exception that it transported glucuronide
conjugates (e.g. oestradiol glucuronide) rather poorly. However, despite
their genetic proximity and the similarities of their in vitro properties, the
in vivo functions of ABCC6 and MRP1 are now known to differ dramatically.
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Thus, quite unexpectedly, it was discovered that recessive mutations in
ABCC6 are responsible for a rare human genetic disorder known as PXE
(pseudoxanthoma elasticum) [43,44].
PXE is a late‑onset progressive disease characterized by aberrant ectopic
mineralization of soft connective tissues such as the skin, as well as in the
eyes and the cardiovascular system, with the most devastating complication
in many patients being the eventual loss of visual acuity in the third or fourth
decade of life. The connective tissue pathology of PXE has been recapitulated
in Abcc6−/− mice, as have the cardiac dysfunction and skin manifestations seen
in humans afflicted with this disease [44,45]. Well over 300 ABCC6 mutations,
including numerous point mutations, deletions and insertions, have been
described in patients with PXE, which is presumed to reflect the instability
of the ABCC6 region of chromosome 16. Unfortunately, only experimental
therapies are presently available to treat this disease.
ABCC6 is expressed predominantly in the liver and kidney, and it is rath‑
er curious that the pathology of PXE does not directly affect these tissues. The
precise role of ABCC6 in mineralization processes remains obscure, although
several intriguing hypotheses are currently being investigated [44]. Most evi‑
dence supports the ‘metabolic hypothesis’ which postulates that, in the absence
of ABCC6 activity, there is a deficiency of circulating factors or metabolites
required to maintain normal mineralization under calcium and phosphate
homoeostatic conditions [44]. Recent skin grafting and other experiments
utilizing Abcc6−/− mice have been particularly valuable in providing mecha‑
nistic insights into the defects underlying PXE [44]. For example, skin from a
Abcc6−/− mouse did not develop mineralization when grafted on to a wild‑type
Abcc6+/+ mouse, but the skin from a wild‑type mouse showed mineralization
after grafting on to a Abcc6−/− mouse, consistent with the conclusion that cir‑
culating factors in the blood of the recipient mouse were regulating the degree
of mineralization of the graft, irrespective of the graft genotype.
MRP7
MRP7 (gene symbol ABCC10) was the last of the long MRPs to be described
and remains the least well characterized. Although its general architecture
appears similar to that of the other long MRPs, MRP7 is distinct primarily
due to substantial sequence divergence in its first MSD (MSD0). As a result,
MRP7 lacks the highly conserved N‑glycosylation sites found in this region of
MRP1–MRP3 and MRP6 [46,47]. Whether or not this difference contributes
to any functional differences is not yet known.
The tissue and membrane localization of MRP7 has not yet been fully
characterized, in part because of the scarcity of specific high‑affinity anti‑
bodies. The physiological role(s) of MRP7 is unknown, and Abcc10−/− mice
have not yet been described. In vitro, MRP7 transports a broad range of pro‑
totypical MRP organic anions (including LTC4 and oestradiol glucuronide),
each with its own set of kinetic parameters (affinity and capacity), as do other
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MRPs. MRP7 can also confer resistance to several classes of natural prod‑
uct anticancer and antiviral drugs, including antimitotic agents (vincristine
or docetaxel) and certain nucleoside analogues [48] (Table 2). The resistance
spectrum associated with MRP7 expression is thus narrower than for MRP1
and MRP2, and more similar to that for MRP3 since it does not include resis‑
tance to anthracyclines (e.g. doxorubicin). There are some recent reports that
certain small‑molecule inhibitors of tyrosine kinases (e.g. imatinib) interact
with MRP7. Despite these interesting laboratory findings, however, there is
still little evidence that supports a role for MRP7 in clinical drug resistance or
sensitivity in cancer patients.
The short MRPs
Because they lack the MSD0 characteristic of the long MRPs, the short MRPs
(MRP4, MRP5, MRP8, ABCC12) have a more typical ABC structure with just
two MSDs and two NBDs (Figure 1B). MRP4 (gene symbol ABCC4) is the
best characterized of the short MRPs, and, curiously, its closest homologue is
the CFTR (cystic fibrosis transmembrane conductance regulator) (ABCC7),
a Cl− channel regulated by cAMP. However, no ion channel activity has ever
been ascribed to MRP4. On the other hand, sequence alignments place MRP5
(gene symbol ABCC5), MRP8 (gene symbol ABCC11) and ABCC12 into a
separate subcluster of the ABCC/MRP subfamily (Figure 1A). Comparatively
little is known about these three ABCC proteins, and, consequently, they are
considered together below.
MRP4
Human ABCC4 is highly polymorphic, although no genetic diseases have been
firmly linked with mutations in this gene. However, the absence of MRP4
protein has recently been associated with a selective defect in ADP storage
in platelet δ‑granules which in turn is associated with prolonged bleeding
times and bleeding diathesis [49]. MRP4 is present at low levels in all normal
tissues, with substantially higher levels found in the prostate. It has also been
implicated in the aggressiveness of some tumours, including prostate tumours
and neuroblastoma [50,51].
MRP4 was first functionally identified as a transporter of adefovir, a
nucleoside monophosphate antiviral agent [52]. However, uptake studies in
MRP4‑enriched isolated membrane vesicles and efflux studies using intact
MRP4‑transfected cells, as well as studies in Abcc4−/− mice, have established
that the substrate specificity of this transporter extends well beyond this class
of drugs [6,36,38,53]. In addition to its own unique substrate‑specificity pro‑
file (see below), MRP4 is unusual because of its ability to localize to either
basolateral or apical membranes in polarized cells, depending on the tissue
where it is found. For example, in prostate tubuloacinar cells and hepatocytes,
MRP4 localizes to the basolateral membrane, whereas it is found at the apical
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membrane in renal proximal tubules and the luminal side of brain capillaries
[53,54]. The mechanisms underlying the tissue‑specific localization of MRP4
are only partly understood and appear to involve interactions with different
adaptor and scaffolding proteins in the different cell types [55].
Pharmacological and physiological roles of MRP4
In addition to adefovir and other antiviral agents, MRP4 also confers resistance
to anticancer agents including thiopurine analogues, methotrexate and
topotecan [6,53,56,57]. Thiopurine analogues and most nucleoside‑based
antivirals require intracellular phosphorylation before they can exert their
pharmacological activity. For example, 6‑mercaptopurine is metabolized to
the monophosphate nucleotide 6‑thio‑IMP, which in turn inhibits several
enzymes of de novo purine nucleotide synthesis. Thus MRP4 confers
resistance to thiopurine bases (and by inference nucleoside analogues), by
effluxing the anionic phosphate metabolites rather than the parent compounds.
Much of the evidence demonstrating the importance of MRP4 in drug dis‑
position/elimination has come from studies of Abcc4−/− mice. Although these
mice exhibit no discernible phenotype in the absence of drugs, topotecan (a
topoisomerase I inhibitor) accumulation in both brain tissue and in cerebral
spinal fluid is enhanced in Abcc4−/− mice, reflecting the dual localization of
MRP4 at the basolateral membrane of the choroid plexus epithelium, and at
the apical membrane of the endothelial cells of the brain capillaries [57]. Renal
elimination of many drugs is also reduced in Abcc4−/− mice, and a key pro‑
tective role for MRP4 in the kidney is suggested [53,58]. Common NSAIDs
(non‑steroidal anti‑inflammatory drugs) (e.g. celecoxib) inhibit transport by
MRP4 which may contribute to clinically significant kidney toxicity when
cytotoxic agents such as adefovir or methotrexate are co‑administered with
NSAIDs [53,59]. Abcc4−/− mice are also more sensitive to the haemopoietic
toxicity of thiopurines [56]. In humans, a non‑synonymous polymorphism in
ABCC4 encodes an inactive transporter and it may be that the increased sen‑
sitivity of some Japanese patients to thiopurines reflects the greater frequency
(>18%) of this polymorphism in the Japanese population [56].
Equally important to its drug (and drug metabolite)‑transporting func‑
tion, MRP4 mediates the cellular efflux of several endogenous metabolites
that play key roles in signalling pathways involved in processes such as cell
proliferation, differentiation, cell‑cycle progression and apoptosis. Notably,
MRP4 transports cAMP (and cGMP), molecules that relay external signals to
downstream protein kinases [58,60]. Because of the relatively low affinity of
MRP4 for cAMP (and cGMP), its relevance in regulating intracellular levels
of cyclic nucleotides has been questioned [36]. However, the growing evidence
that cyclic nucleotide signalling is highly compartmentalized suggests that
MRP4 may be more involved in regulating local microdomain levels rather
than whole‑cell concentrations of cAMP [61]. Several eicosanoids, including
PGE2 (prostaglandin E2), are also substrates of MRP4 [36,59]. Best known as
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a mediator of pain and inflammation, PGE2 has also been implicated in the
development of some tumours as well as the stimulation of their growth and
angiogenesis, and response to cytotoxic chemotherapy [62,63].
MRP5, MRP8 and ABCC12
MRP5
There are no known diseases associated with mutations in ABCC5, and
the phenotype of the Abcc5 −/− mouse has not yet been reported in the
literature. Although ABCC5 mRNA is present at low levels in most tissues,
the tissue distribution of the MRP5 protein is only partially characterized,
in part due to the limited availability of suitable antibodies for definitive
immunolocalization studies [36,54,64] (Table 3). Relatively higher levels of
MRP5 have been detected in the brain capillary endothelial cells, pyramidal
neurons and astrocytes, as well as in smooth muscle cells of various tissues
in the human genitourinary system [54,64]. However, the physiological role
of MRP5 in these tissues is unknown. MRP5 has been reported to localize to
the basolateral membrane of some polarized epithelial cells, but, in the brain
capillary endothelial cells, it localizes to the apical membrane [36,54] (Table 3).
It may be that, like MRP4, MRP5 is capable of dual‑membrane localization,
but there are presently too few data to support this conclusion just yet.
There are some discrepancies in the literature with respect to the reported
substrate specificity and affinity of MRP5 which have not yet been resolved
[36]. Xenobiotic substrates identified by in vitro studies include methotrex‑
ate, the antiviral agent adefovir, and various other nucleoside and nucleotide
monophosphate analogues or metabolites [36,65] (Table 2). For example,
MRP5 confers resistance to the important anticancer drug 5‑FU (5‑fluorou‑
racil) by mediating the cellular efflux of its cytotoxic active monophosphate
metabolite, 5‑dUMP, rather than the parent drug itself [66].
Physiological metabolites transported by MRP5, at least in vitro, include
folic acid and glucuronide conjugates (e.g. oestradiol glucuronide), as well as
GSH and GSH conjugates (e.g. LTC4) [36]. Cyclic nucleotides, mainly cGMP,
are also transported by MRP5, a property it shares with MRP4 and MRP8
[67]. It has been speculated that MRP5 may act as a cGMP ‘overflow’ pump
[36]. Thus it is conceivable that, when intracellular levels of cGMP are elevated
either by induction of cGMP biosynthesis or by inhibition of phosphodieste‑
rases, normal cGMP levels could be restored via efflux by MRP5. However,
direct evidence supporting this role for MRP5 is still lacking. Similarly, the
in vivo relevance of the ability of MRP5 to transport the therapeutic agents
mentioned above has not yet been established.
MRP8
Human ABCC11 is located on chromosome 16q12.1 and, whereas multiple
mRNAs are transcribed from this gene, the full‑length MRP8/ABCC11
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transporter is predicted to be 1382 amino acids with a typical four‑domain
ABC structure [68,69]. There is no evidence yet to suggest that any of the
putative polypeptides encoded by the shorter ABCC11 transcripts would
be functional transporters. ABCC11 is unusual among the ABCC family
members in that no orthologous genes have been found in mammals, except
for primates, and thus Abcc11 is clearly not an essential gene. However, the
discovery that a single nucleotide polymorphism in ABCC11, 538G>A,
determines the type of earwax (cerumen) secreted by the ceruminous apocrine
glands has revealed a physiological function for this transporter. The 538G>A
polymorphism causes a non‑conservative arginine substitution of Gly180 in the
first TMH of MRP8 which appears to disrupt the glycosylation and stability
of the protein [70]. The 538A/A genotype corresponds to the dry earwax
type common in East Asian populations, whereas the 538G/A and 538G/G
genotypes correspond to the wet earwax type more common in populations
of European and African origin. Individuals with the 538G/G and 538G/A
genotypes also suffer from axillary osmidrosis (armpit secretions of foetid
sweat), a condition rarely seen in 538A/A individuals [70]. Thus evidence
to date suggests that accumulation of glandular secretions into vacuoles and
granules before their release is a physiological function of MRP8. However,
the molecular identity of these secretions is not yet known.
In contrast with its closest relative MRP5, full‑length MRP8 localizes
to apical membranes in stably transfected polarized epithelial cells. ABCC11
mRNA has been found in a variety of tissues, but the multiplicity of
ABCC11 transcripts makes it difficult to make any correlations with respect
to MRP8 protein expression patterns [68,69]. The MRP8 protein has been
detected in axons of neurons in the human central and peripheral nervous
systems using immunofluorescence microscopy, and it has been proposed that
it may mediate the efflux of neuromodulatory steroids such as dehydroepian‑
drosterone 3‑sulfate [71].
In membrane vesicles prepared from transfected cells, MRP8 can trans‑
port a wide range of compounds including cGMP and cAMP (such as MRP4
and MRP5), as well as conjugated organic anions such as LTC4, and sulfated
and glucuronidated steroids (such as MRP1–MRP3) [6,47,71,72] (Table 2).
Whether or not any of these molecules are physiological substrates of MRP8
is not known. In transfected cell systems, MRP8 can confer resistance to
methotrexate as well as 5‑FU by effluxing its active metabolite 5‑FdUMP
(5‑fluoro‑2′‑deoxyuridine‑5′‑monophosphate, a property it shares with MRP5
[47,66]. However, to date, there is no convincing evidence that MRP8 plays a
role in resistance or sensitivity to these anti‑metabolites in cancer patients.
ABCC12
ABCC12 is located on the same chromosome as ABCC11, and the two
genes are oriented in a tandem fashion just 20 kb apart, suggesting that they
probably arose from a gene‑duplication event [68,69]. Also like ABCC11,
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multiple splicing variants of ABCC12 exist, with the longest full‑length
mRNA transcript predicted to encode a protein of 1359 amino acids [68].
ABCC12 transcripts are most abundant in testes, but only a minor fraction
of these are able to encode a full‑length transporter. This profile is generally
shared with both the rat and mouse Abcc12 orthologues [68,69]. When
full‑length human ABCC12 is ectopically expressed in human embryonic
kidney cells, the 150 kDa protein is not glycosylated, and rather than
localizing to the plasma membrane like other MRPs, it is found predominantly
in the endoplasmic reticulum [73]. The recombinant ABCC12 protein does
not transport any of the organic anions transported by the other MRPs in
vesicular transport assays, nor does it confer resistance to cytotoxic agents
in intact cell assays. Thus the substrate specificity of ABCC12 is unknown,
and, for this reason, it is not referred to here as an ‘MRP’. Because full‑length
ABCC12 appears to be expressed only in testicular germ cells and sperm, it
has been suggested that ABCC12 may play a role during the latter part of
the male meiotic prophase, the development of spermatids and/or possibly in
sperm function [73]. At present, however, there is no experimental evidence
supporting these proposed functions. The generation and characterization of
Abcc12−/− mice may prove useful in determining the physiological role of this
elusive transporter.
Conclusion
When MRP1 was first cloned in 1992, it was difficult to imagine that within
less than 9 years, eight additional homologues of this transporter would be
known. Considerable effort has been expended dissecting out the similarities
and differences among the MRP/ABCC transporters, and, although much
has been learned about some, others remain poorly characterized. MRP1,
MRP2 and MRP4 are currently the best studied MRPs, and both important
pharmacological and physiological functions have been described for them.
MRP1 remains the most likely of the MRPs to be clinically relevant in
drug‑resistant tumours. Its role in mediating inflammatory responses through
its ability to efflux LTC4 is also firmly established. MRP2 and MRP4, on the
other hand, appear to contribute significantly to the elimination of xenobiotics
and their metabolites into the bile and urine respectively. MRP4 is further
noted for its ability to transport important signalling molecules such as cAMP
and PGE2. MRP3 also has a role in drug metabolite elimination, but it seems
to be more secondary. In contrast with MRP1‑4, the in vivo physiological
substrates of MRP5, ABCC6, MRP7, MRP8 and ABCC12 have still not been
identified. It is clear that the connective tissue disorder PXE is caused by
mutations in ABCC6, but, until the physiological substrate of this transporter
is known, the underlying pathogenesis of this disease will remain a puzzle.
Further work is also needed to better understand the substrates and functions
of MRP5, MRP7, MRP8 and ABCC12. Finally, the involvement of GSH in
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the transport mechanisms of a few, but not all, MRPs is a distinctive feature
of the MRP/ABCC subfamily, even if the physiological implications of this
involvement are still unclear.
Differential plasma membrane trafficking (apical in contrast with baso‑
lateral membranes) in polarized epithelial and endothelial cells contributes
to the specialized functions of several MRPs. However, little is known about
the mechanisms that regulate the amount of a given MRP/ABCC on the
plasma membrane, although it seems certain, at least in the case of MRP2 and
MRP4, that interactions with intracellular scaffolding/chaperone proteins are
involved. It is also known that the interindividual variation in basal transport
activity of a particular MRP can be substantial (up to 80‑fold), but, again,
relatively little is known about what determines the actual amounts of MRP/
ABCC proteins expressed in different tissues, or how these levels might be
affected by exposure to xenobiotics, stress or disease conditions.
Finally, high‑resolution crystal structures of both the short and long
MRP/ABCC proteins are needed to better understand their complex trans‑
port mechanism(s), and precisely how substrate binding and translocation is
coupled to ATP binding and hydrolysis. Such structures would also enhance
understanding of how the MRP/ABCC proteins can recognize such a vast yet
MRP‑selective array of endogenous and xenobiotic substrates. In the case of
the long MRPs, such studies should also reveal how their extra MSD0 func‑
tionally interacts with the core four‑domain structure of these transporters.
Summary
•
•
•
•
•
The nine MRP/ABCC transporters comprise a subfamily of the ABC
superfamily of proteins that utilize the power of ATP binding and
hydrolysis to move their physiological and pharmacological substrates
across the plasma membrane
Mutations in two of the MRP/ABCC genes, ABCC2 and ABCC6,
cause hereditary human diseases (Dubin–Johnson syndrome and PXE
respectively)
Some MRP/ABCC transporters are believed to either play a role in
resistance to anticancer and antiviral drugs in patients with resistant
disease, and/or influence drug efficacy and toxicity through their abil‑
ity to modulate disposition or elimination.
Many MRP/ABCC proteins transport organic anions that can be
formed either from endogenous molecules (such as LTC4 or PGE2) or
by conjugation of drug metabolites. However, the in vivo substrates of
some MRP/ABCC transporters are still not known.
The substrate specificities of some MRP/ABCC transporters overlap
to some degree, although differences in transport kinetics are often
substantial. Significant species differences in the substrate specificity of
MRP/ABCC transporters have also been noted.
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•
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Differences in tissue‑expression patterns and membrane localization
(apical in contrast with basolateral) in polarized epithelial cells contri‑
bute to the unique pharmacological and physiological functions of each
MRP/ABCC transporter.
This work has been supported by grants to S.P.C.C. from the Canadian Institutes of
Health Research.
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