Function, evolution and structure of multidrug resistance protein (MRP)

seminars in C A N C E R B I OLOG Y, Vol 8, 1997: pp 193]204

Function, evolution and structure of multidrug resistance protein (MRP) Roger G. DeeleyU and Susan P.C. Cole Multidrug Resistance Protein (MRP) confers resistance to natural product drugs when overexpressed in cultured cells. It has also been detected in human tumors and in some cases, expression has been correlated with a poor response to chemotherapy. MRP is present in normal tissues where it probably functions as an active transporter of amphiphilic anions. It is also presumed to transport the drugs to which it confers resistance, but how and in what form has not been resolved. Unlike other members of the ATP Binding Cassette superfamily, MRP and several related proteins have three potential membrane spanning domains. The additional NH2-proximal domain in MRP contains five membrane spanning helices with an extracytosolic NH2-terminus and is essential for transport. Conserved features of gene organization and protein structure suggest that MRP and its related proteins share their ancestry with the cystic fibrosis conductance regulator.

pression of P-gp was the only mechanism known to be capable of simultaneously conferring resistance to such a broad range of cytotoxic xenobiotics. However, it is now apparent that a similar form of multidrug resistance can be caused by at least one other protein.3 Approximately 10 years ago, several drugselected cell lines were described with resistance phenotypes similar to that conferred by P-gp, but with no alteration in expression of this protein.4 ] 8 In 1992, an abundantly expressed mRNA encoding another high molecular weight membrane protein was cloned from one of these cell lines, the doxorubicin selected, multidrug resistant human small cell lung cancer ŽSCLC. cell line, H69AR.3,4 The level of the mRNA also decreased markedly in drug sensitive revertants of H69AR cells suggesting that elevated expression of the encoded protein could be associated with multidrug resistance.3 Accordingly, the protein was designated Multidrug Resistance Protein ŽMRP.. The open reading frame of the mRNA encoded a 1531 amino acid polypeptide that contained two potential nucleotide binding domains ŽNBDs. and multiple potential membrane spanning helices.3 The predicted topology of the protein and the presence of characteristic sequence motifs in both NBDs indicated that, like P-gp, it was a member of the ATP Binding Cassette ŽABC. superfamily of polytopic, integral membrane transport proteins.9 However, primary structure similarity to the P-gps was very low and restricted primarily to generally conserved regions of the two NBDs.9 When MRP was initially cloned, its closest known relative in the ABC superfamily was ltpgpa, a protein encoded by the H-circles of Leishmania tarentolae that confers low level resistance to arsenite and trivalent antimonials.10 A number of ABC transporters have now been characterized that are more closely related to MRP than ltpgpa. They include: the Multispecific Organic Anion Transporter ŽMOAT., the Epithelial Basolateral Conductance Regulator ŽEBCR. which is a probable rabbit orthologue of MOAT, the yeast cadmium resistance factor, YCF-1, the yeast oligomycin resistance factor, Yor1rYrs1, and the sulfonylurea receptor, SUR, which

Key words: chemotherapy r conjugates r leukotrienes r resistance r transport Q1997 Academic Press Ltd

Introduction APPROXIMATELY 20 YEARS AGO, Juliano and Ling first noted a correlation between decreased drug permeability in colchicine selected multidrug resistant Chinese hamster ovary cells and increased expression of the high molecular weight membrane glycoprotein, subsequently known as P-glycoprotein ŽP-gp ..1 P-gp functions as an ATP-dependent transporter, or efflux pump, for structurally diverse natural product chemotherapeutic agents that exert their cytotoxic effects by interacting with several different subcellular targets.2 Until relatively recently, increased ex-

From the Cancer Research Laboratories, Queen’s University, Kingston, Ontario, Canada K7L 3N6 U Corresponding author. Q1997 Academic Press Ltd 1044-579Xr 97r 030193q 12$25.00r 0r se970070

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Figure 1. Unrooted dendrogram illustrating primary structure similarity between several members of the MRP branch of the ABC-superfamily. Protein sequence similarities were determined using CLUSTAL W to carry out an alignment of all possible combinations of pairs of proteins. An unrooted tree in which similarity between two proteins is inversely proportional to the total length of the lines joining them was generated using TREEVIEW software. The scale bar indicates a unit length corresponding to 10% lack of similarity. Similarity scores between MRP and other proteins are: human cMOAT Ž46%., rat cMOAT Ž45%., rabbit EBCR Ž45%., C. elegans MRP1 Ž44%., S. cerevisiae YCF1 Ž38%., human and rat SUR Ž30%., S. cerevisiae YOR1 Ž27%. and L. tarentolae PGPA Ž26%..

regulates insulin release.11 ] 15 A family of four MRPrelated transporters has also been described recently in the nematode, C.elegans, one member of which has been shown to contribute to heavy metal resistance.16 The similarity relationships among these proteins are shown in Figure 1. In addition, several human MRPrelated members of the superfamily, identified in various databases of expressed DNA sequences, are currently being characterized Ždescribed in more detail on pages 205]213 of this issue.. Despite the lack of similarity of their primary structures, P-gp and MRP confer similar drug resistance phenotypes. Cells transfected with human MRP typically display increased resistance to Vinca alkaloids, epipodophyllotoxins and certain anthracyclines. The increase in resistance is also associated with decreases in drug accumulation and increases in drug efflux.17 ] 20 However, MRP is notably less effective than P-gp at conferring resistance to taxol and mitoxantrone, and unlike P-gp, increased expression of MRP results in moderate resistance to arsenical and antimonial oxyanions. More recently, the ability of

MRP to cause multidrug resistance has been further substantiated by using antisense oligonucleotides to decrease the levels of MRP mRNA and protein in transfected cells and to reverse drug resistance.21,22

Occurrence of MRP overexpression in cell lines and in human tumours Elevated levels of MRP or its mRNA have been found in cell lines derived from a range of human tumours following selection in a variety of natural product drugs.23 Relatively high levels of MRP or its mRNA have also been found in non-selected cell lines derived from intrinsically drug-resistant tumours.23 These observations, coupled with the demonstration of the protein’s ability to cause multidrug resistance in vitro, have generated considerable interest in examining MRP expression in human malignancies. To date, most clinical studies have relied upon bulk analyses of MRP mRNA levels or, less frequently, levels of MRP itself. However, with the development 194

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Physiological function of MRP

of MRP-specific monoclonal antibodies suitable for immunohistological studies this situation is changing rapidly.24 ] 30 MRP mRNA or protein has been detected in a wide range of solid and hematological tumours.23 There has been limited opportunity to correlate MRP expression with outcome although such data are beginning to emerge.31 ] 33 In one pilot study, deletion of part of an MRP allele was observed in a subset of acute myeloid leukemia ŽAML. patients. This deletion occurs because MRP is located in the vicinity of one of the breakpoints involved in the pericentric chromosome 16 inversion characteristic of the AML subtype, M4Eo. In those patients whose leukemic cells had lost one functional copy of the gene, the median time from diagnosis to failure was 4]5 times longer than that of patients in which the inversion did not affect MRP.34 In addition, expression of MRP mRNA and protein was found to be as strong a negative prognostic indicator in neuroblastoma as amplification of the oncogene, N-myc. 33 In this case, the clinical correlation is supported by in vitro studies of non-selected neuroblastoma cell lines which also indicate that MRP expression is strongly associated with drug resistance and the levels of N-myc protein.35 Very recently, expression of both MRP and P-gp has been documented in cases of retinoblastoma that failed chemotherapy.36 Of particular interest is the fact that only MRP expression was found when patients were treated with a combination of chemotherapy and the P-gp reversing agent, cyclosporin A. In addition, expression of either MRP or P-gp before treatment correlated with the failure of chemotherapy while the absence of both proteins correlated with long-term survival. Some of the most striking clinical data concern the prevalent expression of MRP in non-small cell lung cancer ŽNSCLC., a malignancy which is characteristically inherently multidrug-resistant.37 Several studies have reported frequent and relatively high levels of expression of MRP mRNA in lung adeno- and squamous cell carcinomas.28 ] 30,32,38 In one of these studies, MRP expression was negatively correlated with outcome in squamous cell carcinoma patients.32 Immunohistological analyses of MRP expression in archival lung tumour samples from our own laboratories have confirmed frequent, moderate to high levels of MRP expression in NSCLC Ž83 of 110 cases. with less frequent, focal expression in SCLC Ž8 of 18 cases. Žunpublished observations..

There has been considerable speculation about whether or not the drug resistance-conferring P-gp isoforms evolved solely to protect the organism from cytotoxic endo- or xenobiotics, or whether this function was an advantageous consequence of their ability to transport other, non- toxic physiological substrates.39 ] 43 Consistent with a role as a drug efflux pump, in vitro transport systems have demonstrated that P-gp can function as a primary active transporter of the chemotherapeutic agents to which it confers resistance.44 ] 46 Direct binding of these compounds to P-gp can also be inferred from the ability of photoactivatable analogs of certain drugs and reversing agents to label the protein.47,48 More recently, studies in ‘knock-out’ mice have provided convincing evidence that major in vivo roles of mdr1a P-gp include the prevention of cytotoxic substances from crossing blood-tissue barriers and the elimination of these compounds through the gut.49,50 In contrast to studies with P-gp, a large number of possible physiological substrates of MRP have been identified, but attempts to label MRP with photoactivatable drug analogs have been unsuccessful. In addition, studies using membrane vesicles from MRP transfected and overexpressing cells have failed to provide convincing evidence that the protein can actively transport unmodified chemotherapeutic agents.51 ] 53 A possible explanation for these observations, and a clue to one of MRPs potential physiological roles, was provided by the demonstration that MRP can function as a high affinity, primary active transporter of the cysteinyl leukotriene, LTC 4 .51,53 ] 55 Since the original finding that this glutathione conjugated eicosanoid was an MRP substrate, the protein has been shown to transport a spectrum of structurally diverse glutathione conjugates ranging from oxidised glutathione itself to the glutathione conjugates of ethacrynic acid and activated aflatoxin B 1.52,56 ] 58 Other anionic Žsulphate and glucuronide. conjugates, some of which were previously proposed to be possible physiological substrates of P-gp Že.g. 17b-estradiol 17b ŽD-glucuronide . and some conjugated bile salts. are also transported by MRP.42,52,59 In addition, MRP has been shown to complement a STE6 mutation in yeast indicating that it is able to transport the farnesylated, a-peptide mating pheromone.60 Interestingly, although MRP does not increase resistance to heavy metal cations, such as Cd 2q,

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when transfected into mammalian cells, it does complement a YCF-1 mutation when expressed in yeast.18,61 Vacuolar accumulation of some glutathione conjugates is also decreased in YCF-1 mutants and is restored by expression of MRP, supporting the suggestion that the two proteins are orthologues. ATP-dependent transport of a variety of anionic conjugates had been previously attributed to the functionally defined, but physically uncharacterized, ‘multispecific organic anion transporter’ ŽMOAT..62,63 MOAT activity is present in membranes from a variety of cell types, most notably in hepatocanaliculi, sarcolemma and plasma membranes of mast cells.64 ] 66 However, its substrate specificity was largely deduced by studying bile composition in congenitally hyperbilirubinemic TRy rats. These rats lack hepatocanalicular MOAT activity but in non-hepatic tissues transport activity is normal.67 It is now apparent that MOAT activity is attributable to at least two Žor possibly more. proteins with overlapping substrate specificities, but different tissue distributions and subcellular localizations.13,68,69 MRP is at least partially responsible for MOAT activity in non-hepatic tissues, while the more recently cloned MRP-related protein, cMOATŽcMRP., Ždescribed in more detail on pages 205]213 of this issue. is defective in TRy rats and people who suffer from a form of congenital conjugated hyperbilirubinemia known as Dubin ] Johnson syndrome.63,70,71 Immunohistological studies indicate that in rat and human liver, cMOAT is confined to hepatocanalicular membranes while the relatively small amounts of MRP normally present are predominantly on basolateral membranes.72 MRP has also been shown to localize to basolateral membranes when transfected into polarized kidney cells.73 Data derived primarily from studies in the rat, suggest that cMOAT may be an hepatocanalicularspecific homologue of MRP.70 Another MRP-related protein, EBCR, has been reported to be present at relatively high levels in rabbit liver, as well as kidney and intestine.14 This protein is almost certainly the rabbit orthologue of cMOAT and it remains to be confirmed whether there are species differences in the tissue distribution of the protein. EBCR was suggested to be a cAMP-activated chloride channel or channel regulator.14 Whether MRP can fulfil a similar function has not been firmly established, but both hypotonically activated Cly and inwardly rectifying Kq channel activities are elevated in H69AR cells relative to parental H69 cells.74,75 Increases in hypotonicity induced anion fluxes have also been detected

in MRP-overexpressing large cell lung cancer cells, COR-L23rR.76 The diversity of potential substrates identified by in vitro studies suggests that the physiological functions of MRP could range from fundamental roles in the regulation of intracellular redox potential and ion flux, to mediation of inflammatory responses to cysteinyl leukotrienes, and the elimination of conjugated endo- and xenobiotics. Thus the in vitro data serve to emphasize the need for in vivo studies in animal models, such as gene knock-out mice, to determine what the actual physiological functionŽs. of MRP may be, as well as the possible consequences of inhibiting the activity or expression of the protein in a clinical context.

Mechanism of MRP-mediated drug resistance The lack of binding or ATP-dependent transport of unmodified drugs by MRP, combined with the characteristics of its demonstrated substrates, prompted the proposal that MRP might transport anionic drug conjugates rather than free drugs.51 ] 53 In support of this suggestion, the glucuronide conjugate of VP-16 has been shown to be a substrate for MRP.52 However, there are several reasons why the hypothesis does not provide a convincing explanation of the protein’s ability to confer resistance to such a wide range of xenobiotics. First, there is no evidence that phase II conjugation plays a significant role in the metabolism of natural product drugs.77 Second, phase II biotransformation reactions occur primarily in the liver and it is unlikely that all cell types which overexpress MRP are competent to carry out such conjugations with the efficiency required to cause resistance.78 Finally, since phase II conjugation is usually regarded as a drug inactivation pathway, it is difficult to rationalize why enhanced efflux of the presumably less toxic conjugated metabolites would have a pronounced effect on drug sensitivity. An alternative possibility is that drug efflux by MRP might occur by a co-transport mechanism involving reduced glutathione ŽGSH. and possibly other anions. Physiological concentrations of GSH have been shown to enhance the ability of Vinca alkaloids to inhibit ATP-dependent transport of LTC 4 into MRP enriched membrane vesicles.51 The addition of GSH to such vesicle preparations also results in significant levels of ATPdependent transport of certain cytotoxic xenobiotics, such as vincristine and aflatoxin B 1 , which alone fail 196

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to behave as MRP substrates51,56 The stimulation of transport by GSH is not simply a consequence of altering the redox state of the protein because it cannot be replaced by other reducing agents such as dithiothreitol, L-cysteine or 2-mercaptoethanol. 51 Furthermore, vincristine transport can be enhanced by the non-reducing GSH methyl conjugate, S-methyl GSH Žunpublished observations.. In several drug-selected cell lines that overexpress MRP, depletion of GSH has been found to increase sensitivity to some drugs, including vincristine, VP-16 and doxorubicin.79 ] 81 However, the effect on resistance to a given drug varies between cell lines and not all MRP substrates are similarly affected.82,83,87 For example, GSH depletion appears to have no effect on the ability of MRP to efflux the free acid forms of fluorescent dyes, such as calcein.81,84 The mechanism by which GSH facilitates transport of some compounds is not known. MRP can bind GSH conjugated substrates such as LTC 4 with high affinity. If GSH and unconjugated substrates such as vincristine and aflatoxin B 1 can bind independently Žalbeit with relatively low affinity., binding by both compounds may permit, or facilitate, conformational changes in the protein that presumably accompany transport. Such a mechanism might result in cotransport of GSH although not necessarily so. Data suggestive of a co-transport mechanism have been obtained from membrane vesicle transport studies, but their interpretation is complicated by the fact that oxidised glutathione is a substrate for the protein Žunpublished observations.. Studies with intact cells have yielded conflicting results. In one case, it was concluded that MRP increased GSH efflux from transfected cells.85 In another, efflux of daunorubicin from two lung tumour cell lines that overexpress MRP had no detectable effect on efflux of GSH.83,86 We have observed that expression of relatively high levels of MRP in transfected HeLa cells is associated with a significant decrease in steady state intracellular GSH levels without the addition of exogenous MRP substrates.82 The concentration of GSH in MRP-overexpressing H69AR cells is also sixfold lower than in parental H69 cells.87 One interpretation of these data is that the transport of some endogenous substrates by MRP is accompanied by efflux of GSH from the cell and that when highly overexpressed, the transport activity of the protein is sufficient to decrease GSH levels. However, this seems not to be the case in all cell types since there are several examples of MRPoverexpressing cell lines whose intracellular GSH lev-

els are similar or even higher than in their corresponding parental cell lines.82

Structure of MRP Evolution The level of amino acid identity shared between MRP and other related mammalian ABC transporter homologues, such as MOATrEBCR, SUR and the newly reported MRP3]5, ranges from approximately 30]50%. MRP orthologues themselves are more highly conserved. For example, the human protein is 87% identical to murine mrp and 38% identical to YCF1, MRPs probable yeast orthologue.11,88 Thus the mammalian MRP-related transporters described to date diverged well before the mammalian radiation and, while they may belong to the same branch of the ABC superfamily, they are not members of a closely related family, as exemplified by the P-gps. The most striking structural feature that distinguishes the MRP-related transporters from previously characterized members of the superfamily was revealed when their hydrophobicity profiles were compared with those of other ABC transporters.88,89 In addition to the two polytopic membrane spanning domains ŽMSDs. and two cytosolic NBDs, found in members of the ABC superfamily such as the P-gps and CFTR, MRP and its closer relatives have a third MSD that is comprised of the NH 2-proximal 200]250 amino acids of each protein. With the exception of the additional MSD, the hydrophobicity profiles of the MRP-related transporters resemble that of P-gp ŽMDR1..88,89 This type of analysis combined with comparison of protein primary structure suggests that the MRP-related transporters could have evolved from a common ancestral gene, encoding an ABC-protein with two MSDs and two NBDs, that fused with a gene or genes encoding other integral or membrane associated proteins. ABC-transporters with structures like P-gp are believed to have evolved by the duplication or fusion of genes encoding ‘half’ transporters containing one MSD and one NBD.90 ] 93 At least two independent events of this type appear to have occurred, because phylogenetic analyses indicate that P-gp and CFTR branches of the ABC-superfamily probably evolved from different fused or duplicated ancestors.94 The phylogram in Figure 2 clearly shows that the MRP-related transporters are far more likely to have evolved 197

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from an ancestor they shared with CFTR than with P-gp. Several conserved features of both protein primary structure and gene organization serve to highlight the evolutionary relationship between CFTR and the MRP-like proteins. In the P-gps, the NH 2and COOH-proximal NBDs are very similar to each other. In the MRP-like transporters and CFTR the similarity is considerably lower, partly as a consequence of conserved structural features not found in the P-gp branch of the superfamily.3 The most notable of these is a comparably located ‘deletion’ of 13 amino acids in the NH 2-proximal NBDs of MRP, ltpgpa, MOAT, C. elegans MRP1 and MRP2, yeast YCF-1 and YorrYrs and CFTR, that decreases the spacing between the Walker A and B motifs relative to that in the P-gps. 3,88,95 At present, the structures of genes encoding most of the MRP-related transporters have not been defined. However, the intron-exon organization of MRP was determined very recently and comparison with CFTR also supports the suggestion that they shared a common ancestor.96 For example, alignment of the amino acid sequences of MRP and CFTR with the exon organization of their cognate genes reveals that the splice acceptor and donor sites of MRP exon 28 coincide with those of CFTR exon 20. In both genes, these exons encode a

Figure 3. Alignment of the amino acid sequence encoded by MRP exon 6 with the corresponding regions of murine mrp, human and rat cMOAT, rabbit EBCR, YCF1 and human CFTR. Amino acid residues that are either identical or conservatively substituted in two or more proteins are shown in reverse text. Those that are identical ŽU . or conservatively substituted Žv. in all seven proteins are indicated below the figure. Amino acid residues in CFTR that are identical or conservatively substituted in at least two other proteins are also indicated Ž'.. Although only human CFTR is shown, the choice of species has little effect on the alignment. The NH 2-proximal 17 amino acids of CFTR are highly conserved Žf 90% similar. in species ranging from Xenopus to primates.

region of the COOH-proximal NBD that includes the conserved Walker A motif. Similarly, the donor and acceptor sites of introns 9 and 4 of MRP and CFTR align and the adjacent exons in each gene encode regions that are predicted to be topologically comparable in the two proteins.96 One other observation is consistent with the additional MSD of MRP being acquired as the result of a gene fusion event in which one of the partners encoded a CFTR-related ancestor. The NH 2-proximal MSDs of MRP-related proteins Žwhich in MRP is encoded by exons 1]5. are relatively divergent in comparison with other regions of these proteins. However, alignment of MRP, MOAT and YCF-1 with CFTR reveals a stretch of 21 amino acids, encoded in MRP by exon 6, that is relatively highly conserved. Furthermore, the first amino acid encoded by exon 6 ŽAsn206 . aligns with the second amino acid residue of CFTR ŽGln2 ., suggesting that this location in MRP might correspond to the region that originally encoded the NH 2-terminus of a common ancestor ŽFigure 3.. However, because of the sequence divergence in the NH 2-proximal MSDs it is not possible to predict whether the MRP-like transporters evolved from the product of one or several independent fusion events.

Figure 2. Unrooted phylogram of human P-glycoprotein, human CFTR and some members of the MRP-branch of the ABC superfamily. The phylogram illustrates the relative evolutionary distances between the proteins shown. In contrast to the similarity dendrogram of Figure 1, distances are based on a multiple sequence alignment using the Neighbor-Joining method of Saitou and Nei that excludes all positions which contain gaps. In addition, all distances have been corrected for multiple amino replacements at the same site according to Kimura. Because of this correction, lack of similarity may exceed 100%. The scale bar represents a unit length corresponding to replacement of 20% of the amino acid residues scored in the analysis.

Topology Topological models of MRP have been derived primarily by relying on various computer algorithms or 198

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programs that predict the secondary structure of membrane proteins, complemented by comparison with more extensively studied ABC proteins.88,89,97,98 Structures predicted for each of the three MSDs of MRP depend to a large extent on the algorithmrprogram used ŽFigure 4.. In MSD1, the number of TM helices predicted varies from 4 to 6 and in those models with five TM helices, the NH 2-terminus is predicted to be on the extracellular face of the membrane. For MSD2, most programs predict six TM helices but these are not always located in precisely the same positions. Finally, MSD3 is predicted to contain either 4 or 6 helices. Current investigations to distinguish among these different topologies take advantage of the fact that MRP is modified by N-linked glycosylation. Analysis of the primary structure of MRP indicates that the protein contains 14 potential N-glycosylation sites or sequons. Because only those sequons on the extracellular or lumenal side of the membrane can function as glycosylation sites, identification of those that are used can provide experimental support for some topologies while eliminating others.

Initially, partial proteolysis studies, combined with immunoblotting using antibodies directed against various regions of the protein, demonstrated that functional glycosylation sites were located in both halves of the molecule.25,98 In MSD1, asparagine residues at positions 19, 23 and 71 are potential sites of glycosylation and four possible topologies of this region can be distinguished by the disposition of these sites relative to the membrane. Studies of MRP mutated at these glycosylation sites, individually and in combination, have demonstrated that Asn19 and Asn23 , but not Asn71 , are glycosylated.97 The only predicted topology consistent with these data is one in which MSD1 contains five TM helices and the NH 2-terminus of MRP is extracytosolic. This is an unusual topology for an ABC protein but current data suggest that an extracytosolic NH 2-terminus may be a feature of proteins within the MRP branch of the superfamily.97,99 The favoured models of MSD2 contain six TM helices and predict that the first extracellular loop of this domain contains a potential glycosylation site at Asn354 ŽFigure 4.. However, it has now been es-

Figure 4. Membrane topology of MRP predicted by the PredictProtein server. A multiple sequence alignment of human and mouse MRPrmrp, human and rabbit MOATrEBCR, and YCF1 was used as input for profile-based network prediction of the topology of MRP by the PredictProtein server. ŽA. The best model of MRP compatible with the neural network output consists of five TM helices in MSD1, six in MSD2, and four in MSD3. This topology correctly places the MRP glycosylation sequons that are utilized on the extracytoplasmic side of the membrane. Ž B. A model of MSD3 with a more conventional ABC transporter configuration of 6 TM segments is shown.

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tablished that this site is not glycosylated.97 Close examination of the sequence surrounding Asn354 revealed that it is not an optimal glycosylation site;100 in addition, it is predicted to be very close to the beginning of a TM helix which further reduces the probablity that it will be functional.101 Consequently, the absence of glycosylation at this site does not infer that the model is incorrect. Site-directed mutagenesis studies have also demonstrated that Asn1006 is the only site of glycosylation in MSD3.97 Several MRP-related proteins and CFTR, contain glycosylation sequons at a similar location. Thus, although of no known function, glycosylation of the first extracellular loop of the COOH-proximal MSD appears to be an ancient and highly conserved feature of these proteins. In MRP Asn1006 is predicted to be extracellular in models of the domain that contain either 4 or 6 TM helices. Consequently, glycosylation at this site does not provide evidence in favour of one or the other model, but it does support the predicted location and orientation of the first pair of TM helices in MSD3. If MSD3 contains four rather than six TM helices, Asn1156 is a second possible site of glycosylation. The fact that the site is not used is consistent with the presence of six TM helices, but the sequon at this location, like that surrounding Asn 354 , is of the type that is inefficiently glycosylated.97,100 Consequently, direct experimental evidence in favour of one or the other conformation of MSD3 is still lacking.

nal a-factor peptide transporter.102 Similar, studies of P-gp have provided evidence of direct interaction between the two NBDs, the two MSDs and the NBD and MSD in each half of the protein.103 The ability to reconstitute a functional transporter by co-expression of its individual domains provides an opportunity to identify sites of interaction that are essential for formation of a correct higher order structure and to search for regions of the protein involved in substrate binding. MRP is a particularly interesting candidate for this type of study both because of its third MSD and because of the availability of the high affinity substrate LTC 4 that can also be used for photoaffinity labelling of the reconstituted transporter. Intact MRP and various combinations of its predicted structural domains have been produced in Sf21 insect cells using baculovirus expression vectors.104 Although poorly glycosylated, the transport characteristics of MRP appear essentially identical to those of the protein produced in transfected mammalian cells.98,104 In addition, MRPs five structural domains can be efficiently expressed individually and, with few exceptions, as single polypeptides containing various domain combinations Žunpublished results.. Those containing predicted MSDs accumulate to high levels in membrane fractions that can be recovered for vesicle transport studies. Using this approach, it has been possible to demonstrate that when co-expressed, MRP fragments generated by dividing the protein at a site in the ‘linker’ region predicted to connect NBD1 to MSD2 ŽFigure 4. can associate to form a transporter with kinetic characteristics very similar to the intact protein.104 The linker region of MRP, like that of P-gp, can be phosphorylated and it contains potential recognition sites for several serinerthreonine protein kinases, suggesting that it could have some regulatory role.105,106 However, removal of most of the linker has no significant effect on the ability to reconstitute a functional transporter Žunpublished observations.. Thus, while not excluding a regulatory function for this region, the linker is clearly not essential for interaction between the two halves of the molecule, or for the ability of the protein to transport substrates such as LTC 4 . The possible evolution of the MRP- related transporters from an ancestor with a domain structure more similar to that of CFTR and the P-gps raises the question of whether or not their additional NH 2-terminal MSDs are essential for all aspects of function. Membrane vesicle transport studies have established that baculovirus encoded MRP fragments

Higher order structure of MRP Although the MSDs and NBDs of ABC transporters, such as the P-gps, Ste6, CFTR and MRP, are contained within a single polypeptide, this is not true of many members of the superfamily. Some transporters are formed by association of four different polypeptides each corresponding to an individual domain, while others consist of two subunits, each containing a MSD and a NBD. Examples also exist of transporters formed by association of three polypeptides, two of which may correspond to single domains while the third may be comprised of two fused MSDs or NBDs wreviewed in Ref Ž9.x. The existence of these various quaternary structures strongly suggests that covalent linkage of the individual domains of transporters such as P-gp and MRP may not be essential for function. In some cases, this has been confirmed. For example, co-expression of polypeptides corresponding to the NH 2- and COOH-proximal halves of STE6 restores mating efficiency in yeast lacking a functio200

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lacking MSD1, retain no detectable transport activity with several known substrates. However, activity can be restored by co-expression of fragments that extend from the NH 2-terminus of the protein to sites within the cytoplasmic loop predicted to link MSD1 and MSD2 Žunpublished observations.. Thus at least in the case of MRP, the additional MSD is essential for transport activity, rather than contributing to some alternative function, such as an ion channel or regulator. The results of the co-expression experiments also demonstrate that the domain is not simply ‘tethered’ to the rest of the molecule via its linker region but can fold independently and form stable interactions with the rest of the protein. Knowledge of the regions involved in interactions between the NH 2-proximal MSD and other domains in the protein may begin to place some constraints on the possible higher order structures of members of this most recently identified branch of the ABC superfamily.

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Acknowledgements We gratefully acknowledge Dr James Gerlach for figures describing the structural and evolutionary relationships between MRP-related proteins, and the members of our laboratories, past and present, who contributed to some of the studies described. We also acknowledge support for our research from The Medical Research Council of Canada, the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Ontario Cancer Treatment and Research Foundation.

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References 1. Juliano RL, Ling V Ž1976. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455:152]162 2. Gottesman MM, Pastan I Ž1993. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:385]427 3. Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, Deeley RG Ž1992. Overexpression of a transporter gene in a multidrugresistant human lung cancer cell line. Science 258:1650]1654 4. Mirski SEL, Gerlach JH, Cole SPC Ž1987. Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin. Cancer Res 47:2594]2598 5. McGrath T, Center MS Ž1987. Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein. Biochem Biophys Res Commun 145:1171]1176 6. Slovak ML, Hoeltge GA, Dalton WS, Trent JM Ž1988. Pharmacological and biological evidence for differing mechanisms of doxorubicin resistance in two human tumor cell lines. Cancer Res 48:2793]2797 7. de Jong S, Zijlstra JG, de Vries EGE, Mulder NH Ž1990.

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