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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
ABSTRACT 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 1. Lung cancer 2. Breast cancer 3. Prostate cancer 4. Neuroblastoma 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 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 ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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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 energy-dependent 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 (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 general, it appears not to be essential (19, 253, 291, 397, 448, 490, 496).
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II. DISCOVERY OF THE MULTIDRUG RESISTANCE PROTEIN FAMILY OF TRANSPORTERS |
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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 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 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).
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 1996 from normal rat liver using cDNA probes corresponding to predicted highly conserved regions of human MRP1 (49, 381).
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).
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III. EVOLUTION OF THE "C" BRANCH OF THE ABC SUPERFAMILY |
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A. Long and Short ABCC Proteins
Based on the predicted topology of their membrane-spanning 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 NH2-terminal 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 NH2-terminal 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.
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B. ABCC Proteins Are Characterized by an Atypical NH2-Proximal NBD
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 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 
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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 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).
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IV. TOPOLOGY OF THE MRP1-LIKE MULTIDRUG RESISTANCE PROTEINS |
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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 antibody 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.
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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 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 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 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).
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-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 Pro209 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. Furthermore, 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.
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V. EXPRESSION AND MEMBRANE LOCALIZATION OF THE MULTIDRUG RESISTANCE PROTEINS |
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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). 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.
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 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.
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).
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 detected 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).
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.
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, 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.
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VI. SUBSTRATE SPECIFICITIES AND PHYSIOLOGICAL FUNCTIONS OF THE MULTIDRUG RESISTANCE PROTEINS |
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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 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 the inability to transport cholestatic estrogen conjugates such as E217G, 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, E217G 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 µM (274). However, in the presence of some 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-3-sulfate 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-alkyl-GSH 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 µM) 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.
C. Physiological Roles of MRP1 and MRP2
The substrate diversity and widespread expression of MRP1 suggest numerous possible physiological functions, 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 eosino
