(Received for publication, February 12, 1996; and in revised form, March 28, 1996)
From the
The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.
Overexpression of the multidrug transporter proteins,
P-glycoprotein (MDR1) ()or the multidrug
resistance-associated protein (MRP) provides the molecular basis of the
multidrug resistance phenotype in tumor cells. The possible clinical
importance fuels an intensive research activity toward a better
understanding of the molecular structure and mechanism of action of
these membrane transporters(1, 2, 3) .
Both P-glycoprotein and MRP, together with several other bacterial
and eukaryotic transporters, are members of the ABC-transporter
(ATP-binding cassette) protein family. These proteins share a common
molecular architecture, i.e. they contain two large
transmembrane domains and two cytoplasmic ATP utilization (ABC) units (4) . Due to the difficulty of crystallizing large membrane
proteins, no detailed three-dimensional structure of any members of
these transporters is currently available, and empirical prediction
methods are used to obtain molecular models of their structure,
especially to predict the locations and numbers of the
membrane-spanning helices. In most cases, these methods identify six
short transmembrane segments in each of the two transmembrane
domains(1, 2, 3, 4) . The relevance
of the prediction for the membrane topology of CFTR has been confirmed
experimentally by insertional mutagenesis(5) , thus proving the
2 6 transmembrane helix model. The same arrangement of
transmembrane helices has been suggested in the case of
P-glycoprotein(6, 7) , and a large body of
experimental data strongly favors this model(8, 9) .
On the other hand, Ling and co-workers(10, 11) , by
suggesting an alternative 6- and 4-helix conformation, raised the
possibility that P-glycoprotein may exist in two different topological
forms in the cell membrane.
When the multidrug resistance-associated protein (MRP) was cloned and sequenced, analysis of its primary amino acid sequence revealed that MRP is more closely related to CFTR than to P-glycoprotein(3) . Cole et al.(3) predicted a unique transmembrane topology for MRP, with eight N-terminal and four C-terminal transmembrane segments. In the present experiments, we have examined the membrane topology of MRP by immunodetection with flow cytometry in intact and permeabilized cells, by limited proteolysis of isolated membranes with trypsin and chymotrypsin, and by immunoblotting of the proteolytic fragments with antipeptide antibodies, reacting either with the N-terminal or the C-terminal half of the protein. By using glycosylated and underglycosylated forms of MRP, its major sites of glycosylation could be determined. We have compared the experimental findings with a newly developed membrane topology model of MRP, based on the experimentally confirmed transmembrane topology of CFTR. According to our results, the transmembrane topology of MRP does not follow the model predicted by Cole et al.(3) , but this protein has a characteristic, triple membrane-bound domain structure.
Sf9 (Spodoptera frugiperda) cells were
cultured and infected with a baculovirus as described in (15) .
Recombinant baculoviruses, carrying the human MRP cDNA, were generated
by using the BaculoGold Transfection Kit (PharMingen), according to the
manufacturer's suggestions. Baculovirus transfer vector
containing the human MRP cDNA was constructed as follows: a SalI-BamHI fragment was removed from pJ3-MRP
vector(13) ; this segment contains 115 base pairs of the
5`-nontranslated region and the 1-840 region of the human MRP
cDNA. It was subcloned into M13mp18, and a new XbaI site was
introduced by site-directed mutagenesis at position -5 of MRP
cDNA(16) , and the entire insert was sequenced. The fragment
containing the mutation was inserted back into its original position in
pJ3
-MRP. The MRP cDNA was isolated from this modified plasmid by
digestion with XbaI and NotI and subcloned into
pVL1393 baculovirus transfer vector (Invitrogen).
In these experiments, we have used two drug-resistant human cell lines, the adriamycin-selected HL60 leukemia cells (14) and the MRP-transfected S1 lung tumor cells(13) , both expressing MRP but not MDR1(22, 23, 24, 25) . We have also generated an Sf9 cell-baculovirus system producing human MRP (see ``Materials and Methods''). In human tumor cells, MRP is known to be extensively glycosylated, with an apparent molecular mass of about 180-190 kDa(3, 13, 14, 18) . As shown in Fig. 1A, in an immunoblot MRP is well recognized at the expected molecular mass both in the HL60 ADR and the S1MRP cells by the two monoclonal MRP-specific antibodies, R1 and M6, generated against peptide segments 192-360 (located at the N-terminal half), and two fused sequences from the C-terminal half (1294-1430 plus 1497-1531), respectively (18) (these peptide segments are darkened on the model in Fig. 4B). As also shown in Fig. 1A, when the HL60 ADR or S1MRP cells are pretreated with tunicamycin (which prevents post-translational N-glycosylation) for 72 h, they express an underglycosylated form of MRP with an apparent molecular mass of 150 kDa, in only a slightly decreased quantity. In the baculovirus-infected Sf9 cells, in which no complete glycosylation of large membrane proteins can be observed(15) , the underglycosylated form of MRP, with an apparent molecular mass of 150 kDa was found to be expressed (Fig. 1A).
Figure 1: Estimation of MRP expression by immunoblotting and flow cytometry. A, immunoblot detection of human MRP by the R1 (I, lanes 1-6) and the M6 (II, lanes 1-6) anti-MRP monoclonal antibodies. Lane 1, HL60 control cells; lane 2, HL60 ADR cells; lane 3, HL60 ADR cells pretreated
with tunicamycin (+T); lane 4, Sf9 cells expressing human MRP; lane 5, S1MRP cells; lane 6, S1MRP cells pretreated with tunicamycin (+T). On the 7.5% running gel, each lane contained 20 µg of cellular protein. B, detection of MRP with the R1 antibody (filled histograms) in intact (graphs 1, 3, and 5) or permeabilized (2, 4, 6) HL60 cells. The corresponding isotype controls are indicated as empty histograms. Graphs 1 and 2, HL60 control cells; 3 and 4, HL60 ADR cells; 5 and 6, HL60 ADR cells pretreated with tunicamycin (+Tun).
Figure 4: Membrane topology model for the human MRP. A, hydrophobicity plots of the aligned sequences of CFTR and MRP. Transmembrane regions common in CFTR and MRP are shown as shaded areas. B, model for the membrane topology of MRP. I and II represent alternative possibilities for the arrangement of transmembrane helices in the N-terminal membrane-bound domain. The predicted transmembrane segments of the protein include the following amino acid residues: 33-53, 74-94 (lacking in version II), 99-119, 134-154, 171-191, 320-340, 360-380, 438-458, 464-484, 551-571, 574-594, 970-990, 1018-1038, 1091-1111, 1114-1134, 1203-1223, and 1230-1250. Predicted and experimentally supported N-linked glycosylation sites are shown by solid lines, a possible extra glycosylation site is shown by a dotted line. The two predicted ATP-binding sites are circled. The predominant trypsin (Try) and chymotrypsin (Chy) cleavage areas are indicated by the lower arrows. The peptide sequences used to generate the applied R1 and M6 monoclonal antibodies are darkened.
According to the membrane topology model of MRP by Cole et al.(3) , the 192-360 peptide segment, the binding site of the R1 mAb, is localized extracellularly. However, as noted by Flens et al.(18) , this mAb recognizes MRP only in permeabilized cells. Flens et al.(18) suggested that the lack of reaction of R1 with MRP in intact cells might be due to an epitope-shielding effect of glycosylation or to a complex conformation of this epitope in the matured MRP protein. As shown in Fig. 1B, in flow cytometry experiments with HL60 ADR cells, both the fully glycosylated and the underglycosylated (tunicamycin-treated) MRP was recognized by R1 mAb when the cells were permeabilized by Triton X-100, while there was no mAb recognition in the intact cells, even when MRP was underglycosylated. These experiments exclude the possibility that a large carbohydrate side-chain shields the epitope of R1 in intact cells and suggest that the peptide region recognized by this mAb is located intracellularly.
In order to examine the possible effect of glycosylation on the functional structure of MRP, we have studied the extrusion of calcein AM from S1MRP and HL60 ADR cells. As demonstrated earlier, calcein AM is actively extruded both from MDR1-expressing (12, 19, 20) and MRP-expressing (25, 26) tumor cells, and this extrusion is blocked by cytostatic drugs or by drug-resistance reversing agents. As shown in Fig. 2, the increase in cellular calcein fluorescence is much slower in the HL60 ADR (A) or in the S1MRP (B) cells than in the drug-sensitive parent cell lines, and in the MRP-expressing cells this rate is greatly increased by the addition of vinblastine. As shown previously(25, 26) , the decreased formation of intracellular free calcein (restored to the control rate, for example, by vinblastine) in the MRP-expressing cells closely correlates with the drug extrusion function of MRP. Fig. 2demonstrates that this calcein AM extrusion (that is the lower rate of free calcein production, restorable to the control value by vinblastine) is not significantly different in the HL60 ADR or S1MRP cells cultured for 72 h without or with tunicamycin, respectively (tunicamycin, which slightly interferes with calcein AM extrusion, was removed by washing before the experiments). It has been demonstrated earlier that neither the HL60 ADR nor the S1MRP cells contain any measurable amount of MDR1 (13, 14, 18) . Thus, the function of MRP, at least according to this fluorescent dye extrusion assay, is unimpaired in the tumor cells expressing an underglycosylated protein, as compared to its fully glycosylated form (see Fig. 1A). This is in line with previous findings for the human MDR1 protein, which is also fully functional in an underglycosylated form(1, 2, 27) . The flow cytometry experiments with R1 mAb and the above functional assay altogether strongly suggest that the N-linked glycosylation of MRP does not result in a major conformational alteration in its molecular structure. It is worth noting that tunicamycin, when present in the assay, inhibits calcein AM extrusion and increases the cytotoxic effect of adriamycin, thus probably directly inhibits MRP function.
Figure 2: Functional assay (calcein AM extrusion) in human MRP-expressing cells; effect of tunicamycin pretreatment. A, HL60 and HL60 ADR cells; B, S1 and S1MRP cells. Drug-sensitive (HL60 and S1) and drug-resistant (HL60 ADR and S1MRP) cells were incubated in the presence of 0.25 µM calcein AM (CaAM), and, at the times indicated by the arrows, 10 µM vinblastine was added to the media. The plots show the increase in calcein fluorescence (in arbitrary units) against time.
When the cultured HL60 ADR or S1MRP cells were treated with up to 2 mg/ml trypsin or chymotrypsin for 10 min at room temperature, there was no visible change in the immunoblot pattern of MRP. The transport function of the protein, as measured by calcein AM extrusion, remained intact as well under these conditions (data not shown). Thus, MRP has no easily accessible proteolytic cleavage sites on its extracellular surface. In the following experiments, we have performed limited proteolysis experiments in the isolated membranes of three different MRP-expressing cells, i.e. in human tumor cells expressing the native, glycosylated MRP, the same cells grown in the presence of tunicamycin, and in insect cells (Sf9), the latter both expressing an underglycosylated MRP. By using low concentrations of trypsin and chymotrypsin at 4 °C, we obtained relatively large proteolytic fragments of MRP which could be distinguished clearly by immunoblotting with the two monoclonal anti-peptide antibodies, R1 and M6.
As shown in Fig. 3, trypsin digestion of the glycosylated MRP, with an original molecular mass of about 180 kDa (lane 1), resulted in the predominant formation of a 110-kDa band, as detected by the N-terminal R1 mAb (A, lane 2), and in the formation of a wide 70-kDa band, as detected by the C-terminal M6 mAb (B, lane 2). Chymotrypsin digestion (lanes 3 on both panels) produced a fuzzy, 35-45-kDa band, as seen by the N-terminal mAb (A), and a predominant 140-150-kDa band, as seen by the C-terminal mAb (B, with further chymotrypsin digestion the bands corresponding to the trypsin digestion also appeared).
Figure 3: Limited proteolysis of isolated membranes containing human MRP by trypsin and chymotrypsin. Immunoblot detection by the R1 (A) and the M6 (B) anti-MRP monoclonal antibodies. Lanes 1-3, membranes of HL60 ADR cells; lanes 4-6, membranes from HL60 ADR cells pretreated with tunicamycin (+Tun); lanes 7-9, membranes from Sf9 cells expressing human MRP. Lanes 1, 4, and 7, nondigested membranes; lanes 2, 5, and 8, membranes digested with trypsin; lanes 3, 6, and 9, membranes digested with
chymotrypsin. On 10% running gels, each lane contained 10 µg of original membrane protein.
In the case of the underglycosylated MRP, as expressed in the membranes of HL60 ADR + tunicamycin cells, and of Sf9 cells, the original 150-kDa MRP band (lanes 4 and 7 on both panels), after trypsin digestion yielded a 90-kDa band with the N-terminal R1 mAb (A, lanes 5 and 8), while a 50-55-kDa band (in some cases a doublet in this region) with the C-terminal M6 mAb (B, lanes 5 and 8). Chymotrypsin digestion in these cases produced a sharp 30-kDa band as seen by the N-terminally located mAb (A, lanes 6 and 9), and a predominant 120-kDa band, as seen by the C-terminally located mAb (B, lanes 6 and 9, again, with further chymotrypsin digestion the bands corresponding to the trypsin digestion also appeared).
Based on these limited proteolysis experiments we can conclude that there are two preferentially accessible proteolytic sites on the cytoplasmic surface of the MRP protein, and that all three large fragments obtained are membrane-inserted (they are not removed by washing of the membranes after proteolysis). Moreover, the C-terminal and the N-terminal fragments are both glycosylated in the human HL60 cells, while the central fragment, based on the additive molecular mass values, is probably not glycosylated (we have no direct antibody detection for this fragment). As shown earlier for MDR1 and CFTR, these proteins are also preferentially proteolyzed at the large cytoplasmic loop between the two major membrane-inserted portions(1, 2, 28) ; thus, limited proteolysis provides a useful tool for examining the membrane topology of these ABC transporters.
The above described experimental findings have been compared to a newly developed membrane topology model of MRP. The model described here is based on the comparison of the corrected amino acid sequence of MRP (3, 29) with two members of the ABC protein family: the human CFTR, which has a close position to MRP on the relative similarity dendogram (3, 30) and whose membrane topology is experimentally established(5) , and with a recently cloned yeast cadmium resistance protein, YCF1, which seems to be the closest relative of MRP(30) . We found that when the CFTR and MRP sequences were aligned, the hydrophobicity analysis of the aligned sequences yielded a close matching of the transmembrane segments, thus suggesting a 6 + 6 transmembrane helix topology for MRP as well (Fig. 4A). However, MRP contains an additional N-terminal segment of about 200 amino acids, which has no counterpart in CFTRs or MDRs, but closely resembles the N-terminal region of the YCF1(30) . We suggest that the mostly hydrophobic N-terminal segments of both YCF1 and MRP are membrane-embedded, and the Kyte-Doolittle analysis (21) of this region predicts several transmembrane helices for both proteins.
On the basis of these above described predictions, our membrane topology model for MRP, as shown in Fig. 4B, proposes three major membrane-inserted regions, separated by large cytoplasmic loops. Potential N-linked glycosylation sites in this model can be found on the extracellular portions of all three membrane-bound regions. In the N-terminal membrane-bound region, extracellular glycosylation sites could be obtained by two possible transmembrane helix predictions based on the hydrophobicity analysis (versions I and II in the model of Fig. 4B). Glycosylation of the second membrane-bound domain may not be probable, as a consensus site closer to the membrane than about 10 amino acids, is unlikely to be glycosylated in naturally expressed membrane proteins (see (31) ). This model is strongly supported by our limited proteolysis data, producing three large, membrane-bound fragments of MRP, and experimentally confirming the glycosylation of both the N-terminal and the C-terminal membrane-spanning regions.
The original prediction of Cole et al.(3) for the membrane topology of MRP identified two major membrane-bound segments, the N-terminal part with eight and the C-terminal region with four transmembrane helices. The first major trypsin cleavage site found in our experiments coincides with the predicted ``linker'' region of this model, C-terminally located from the first ABC domain. However, in this model(3) , the whole 192-360 peptide region faces the extracellular space, with two possible N-glycosylation sites, while there is no glycosylation site further N-terminal from this region. In contrast, the membrane topology model described here with three membrane-bound domains suggests that a large portion of the 192-360 peptide region is intracellularly located and predicts an extra glycosylation site at the first N-terminal extracellular loop. Our model is strongly supported by (i) the lack of reactivity of R1 mAb with MRP in intact, nonpermeabilized cells, independently of the glycosylation status of MRP, (ii) preferential, intracellularly located proteolytic cleavage sites in this region, (iii) glycosylation of the protein further N-terminal from this proteolytic cleavage site. Altogether, the experimental findings are fully compatible with the model presented here for the membrane topology for MRP.
Addendum-While this manuscript was under editorial review, Paulusma et al.(32) reported the discovery of an MRP homolog protein (cMOAT), expressed in the liver canalicular membranes, and probably involved in the excretion of organic anions. The authors, based on the homology with MRP(3) , predicted a membrane topology for this protein with 8 + 4 transmembrane helices. Alignment of the cMOAT and the MRP sequences (not shown here) indicates that the membrane topology model described above for MRP may be valid for cMOAT as well, thus suggesting a special structural feature for these proteins among ABC transporters.