(Received for publication, December 7, 1995; and in revised form, January 26, 1996)
From the
P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P.(1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.
Overexpression of P-glycoprotein (P-gp) ()is
associated with the onset of multidrug resistance in cancer cells in vivo and in cultured cells in vitro (Gottesman and
Pastan, 1993; Shustik et al., 1995). P-gps are integral
membrane phosphoglycoproteins that can directly bind photoactivatable
analogs of ATP (Cornwell et al., 1987; Schurr et al.,
1989) and drug molecules (Cornwell et al., 1986; Safa et
al., 1986; Safa, 1993) and use the energy of ATP hydrolysis to
reduce intracellular drug accumulation in resistant cells through an
active efflux mechanism (Gottesman and Pastan, 1993; Ruetz and Gros,
1994). P-gps are encoded by a small gene family that comprises three
members in rodents (mdr1, mdr2, mdr3) (Gros et al., 1986, 1988; Devault and Gros, 1990) and two members in
humans (MDR1, MDR2) (Chen et al., 1986; Van
der Bliek et al., 1986). Primary amino acid sequence analysis
from cloned cDNAs (Gros et al., 1986) indicates that P-gps are
formed by two sequence homologous halves, each encoding six putative
transmembrane (TM) domains and one nucleotide binding domain (Walker et al., 1982). Furthermore, P-gps contain a putative
glycosylated loop between predicted TM1 and TM2, with a cluster of N-linked glycosylation signals (Schinkel et al.,
1993). The P-gp family is also a member of a much larger family of
structurally and functionally related membrane proteins that together
form the ATP-binding cassette (ABC) superfamily of transport proteins
(for review, see Higgins(1992)). The majority of the members of this
ATP-binding cassette superfamily share a similar structure consisting
of 12 TM domains and two nucleotide binding domains, either linearly
joined in the same molecule (P-gp, CFTR, STE-6; Gros et
al.(1986), Riordan et al.(1989), McGrath and
Varshavsky(1989)) or expressed as half-molecules in the form of
heterodimers (TAP1/TAP2, Trowsdale et al.(1990), Spies et
al.(1990); ALDP/PMP70, Kamijo et al.(1990), Mosser et
al.(1993)).
Some of the major topological features of P-gp, including the cytoplasmic localization of the two ATP binding sites, the extracellular location of the TM1-TM2 glycosylated loop, and the intracellular position of the carboxyl terminus, have been verified by biochemical and immunological methods (Kartner et al., 1985; Yoshimura et al., 1989; Bruggemann et al., 1989; Schinkel et al., 1993). In addition, localization studies of the epitope for monoclonal anti-Pgp antibody MRK-16 in intact cells revealed that the short protein segments located between predicted TM1 and TM2 and between TM7 and TM8 are indeed extracellular and in close proximity (discontinuous epitope), in agreement with the topological model proposed above by hydropathy analysis (Georges et al., 1993). The exact number and proposed topology of individual TM domains of P-gp have been analyzed by several groups, but a clear consensus has yet to emerge. Two strategies have been used to map the topology of individual TM domains: (a) in vitro methods in which truncated P-gps are fused to an indicator molecule, and polarity is deduced biochemically after insertion into microsomes or heterologous expression systems; and (b) in vivo methods in which discrete tags are inserted into key locations. In this case, the full-length mutant cDNAs are expressed in mammalian cells, and the tags are mapped using epitope-specific antibodies or labeling reagents. These approaches have resulted in contradictory topological models for P-gp. Methods using truncated proteins fused to indicator molecules suggested topological models different from hydropathy prediction, whereas methods using the full-length functional P-gp suggested a topological model in agreement with hydropathy prediction.
We have used an alternative strategy for topology mapping of the membrane-associated portion of P-gp (Kast et al., 1995). A nine-amino acid hemagglutinin peptide derived from influenza virus and recognized by the monoclonal antibody 12CA5 was inserted within individual predicted intracellular and extracellular loops of P-gp. The mutant proteins were expressed in mammalian cells to test their capacity to confer multidrug resistance. Immunofluorescence with 12CA5 using light microscopy in normal or permeabilized cells was then used to deduce the polarity of the tag with respect to the plasma membrane (intracellular versus extracellular). In addition, confocal microscopy was used to monitor proper targeting to the plasma membrane of mutants showing intracellular localization of the tag. Using this approach, we previously mapped the topology of loops connecting TM1-TM2 and TM5-TM6 as extracellular and that connecting TM2-TM3 and the segment downstream of TM6 as intracellular (Kast et al., 1995). Here we report a more complete topographical map of Mdr3 localizing three additional intracellular and three additional extracellular loops of P-gp. Our results are in agreement with the topology predicted by hydropathy analysis and recent results obtained with the cysteineless MDR1 mutant (Loo and Clarke, 1995a). In contrast, our findings contradict topological models deduced from the study of truncated P-gps fused to reporter genes and expressed in heterologous in vitro or in vivo systems (Zhang and Ling, 1991, 1993; Zhang et al., 1993; Bibi and Béjà, 1994; Béjà and Bibi, 1995).
Figure 1: Construction of fusion Mdr3 proteins containing hemagglutinin epitope tags. Panel A, schematic representation of the mdr3 cDNA, including predicted structural domains of P-gp. Panel B, hydropathy analysis of P-gp encoded by mouse mdr3 (amino acids 1-400 and 650-1050, corresponding to the membrane-associated regions). The average local hydrophobicity at each residue was calculated by the algorithm of Kyte and Doolittle(1982), and the predicted TM domains are identified as dark boxes. Panel C, location of hemagglutinin A epitope tag onto the secondary structure of P-gp proposed by hydropathy analysis. The fusion proteins are identified as 1-21, with the amino acid residue targeted for hemagglutinin tag insertion identified in parentheses (panel D). Filled triangles indicate insertion sites that produced functional proteins in which the tag could be mapped by immunofluorescence. Dotted triangles indicate insertion sites that produced functional proteins in which the tag could not be mapped by immunofluorescence. Empty triangles identify insertions that inactivated the protein. Single triangles represent single epitope tags; double or triple triangles represent two or three epitope tags side by side.
The expression of mutant P-gps in
drug-resistant cell clones was analyzed by immunoblotting with an
isoform-specific anti-mouse Mdr3 antibody (Devault and Gros, 1990) or
the mouse anti-hemagglutinin epitope monoclonal antibody 12CA5 (Niman et al., 1983). As detailed below, mutants 11 (predicted EC4),
15 (predicted EC5), 16 (predicted EC5), and 19 (predicted EC6) were
nondetectable by immunofluorescence analysis. Therefore, immunoblotting
results are shown only for informative mutants 8 (predicted IC2), 10
(predicted EC4), 17 (predicted EC5), 18 (predicted IC4), 20 (predicted
EC6), and 21 (second ATP binding fold). Crude membranes prepared from
mass populations of drug-resistant colonies transfected with mutant or
wild type cDNA showed a specific immunoreactive band of molecular mass
160 kDa, which was absent in untransfected LR73 cells (Fig. 2A). The same band of molecular mass
160 kDa
was also recognized with the hemagglutinin epitope-specific antibody
12CA5 in all mutants analyzed but was not present in cells transfected
with wild type mdr3 or in nontransfected LR73 cells (Fig. 2B). Taken together, these results confirm that
mutants 8, 10, 17, 18, 20, and 21 are functional and present within the
membrane-enriched fraction. Cells transfected with mutants 10
(predicted EC4), 17 (predicted EC5), 20 (predicted EC6), and 21 (second
ATP binding fold) expressed levels of Mdr3 similar to those measured in
vinblastine-resistant cells expressing wild type Mdr3, whereas cells
transfected with mutants 8 (predicted IC2) and 18 (predicted IC4)
expressed significantly higher amounts of protein. The high level
expression detected in mutants 8 and 18 suggests a decreased activity
of these two mutants, necessitating higher level of expression than
wild type Mdr3 to survive the same level of drug selection.
Figure 2: Expression of the mouse Mdr3 fusion proteins 8, 10, 17, 18, 20, and 21 in LR73 cells. LR73 Chinese hamster ovary cells were transfected with either wild type (WT) or mutant mdr3 cDNAs containing inserted hemagglutinin epitopes and selected in drug-containing medium. Crude membrane fractions were isolated from mass populations of transfected cells. Proteins (6 µg/lane) were resolved on 7.5% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with the isoform specific anti-Mdr3 polyclonal antibody B2037 (panel A) or the mouse monoclonal anti-hemagglutinin epitope antibody 12CA5 (panel B). The positions of the molecular mass markers (in kDa) are shown to the left.
Figure 3:
Drug survival characteristics of control
LR73 cells and drug-resistant mdr3 transfectants expressing
wild type (WT) or fusion proteins 8, 10, 17, 18, 20, and 21.
Control drug-sensitive LR73 cells () and mass populations of
cell clones expressing either wild type mdr3 (
) or mdr3 constructs 8 (
), 10 (
), 17 (
), 18
(
), 20 (
), and 21 (
) were plated in increasing
concentrations of either vinblastine, colchicine, adriamycin, or
actinomycin-D and further incubated for 72 h. Drug cytotoxicity was
measured using a sulforhodamine B staining procedure (see
``Experimental Procedures''). The relative plating efficiency
of each cell population was calculated by dividing the absorbance
measured at a given drug concentration by the value obtained for the
same clone in the absence of drug and was expressed as a percentage
(%). Each point represents the average of at least two independent
experiments performed in duplicate dishes.
Figure 4: Detection of epitope-tagged Mdr3 fusion proteins by immunofluorescence. Control LR73 cells and transfectants stably expressing mutant P-gps 8, 10, 17, 18, 20, and 21 were exposed to the mouse monoclonal anti-hemagglutinin epitope antibody 12CA5 with (permeabilized cells, right column) or without pretreatment (intact cells, left column) with 0.05% Nonidet P-40. Cells were then incubated with a second goat anti-mouse antibody conjugated to rhodamine, and the cells were photographed using a fluorescence microscope. The hemagglutinin epitopes in constructs 8 (panels C and D), 18 (panels I and J), and 21 (panels M and N) were only detected in permeabilized cells, whereas the hemagglutinin epitopes in constructs 10 (panels E and F), 17 (panels G and H), and 20 (panels K and L) were detected in both permeabilized and nonpermeabilized cells.
Figure 6: Proposed membrane topology of P-gp as determined by epitope insertion. Immunofluorescence with hemagglutinin epitopes confirmed the extracellular localization of the third, fourth, and fifth predicted EC loops of the protein, and the cytoplasmic localization of the second and fourth predicted IC loops of P-gp and the second ATP binding site. Using the same method, we had previously established the topology of the first and third predicted EC loop as well as the first predicted IC loop and the first ATP binding domain (Kast et al., 1995) (italicized and underlined positions). Loops for which an unambiguous intra- or extracellular localization has been established are shown in filled circles; loops that could not be mapped are identified as empty circles. Filled arrowheads indicate the position of hemagglutinin epitope insertions that resulted in functional P-gps; empty arrowheads indicate the position of hemagglutinin epitopes that abrogate P-gp function. Dotted arrowheads indicate the position of hemagglutinin epitope insertions that resulted in functional P-gps but in which the epitope tag could not be localized by immunofluorescence. Single triangles represent a single epitope; double or triple triangles represent two or three contiguous epitopes. The position of the nucleotide binding site is identified as the ATP site. This drawing is a modification of that published by Gottesman and Pastan(1988).
Figure 5:
Subcellular localization of the
epitope-tagged Mdr3 fusion proteins by confocal laser microscopy.
Transfected cells stably expressing Mdr3 fusion protein 8 (panel
A), 18 (panel B), and 21 (panel C) were
permeabilized, and localization of the tagged proteins was revealed by
immunofluorescence with anti-hemagglutinin antibody 12CA5. Laser
confocal microscopy was done on optical sections scanned through cells
at 3-5 µm above the surface of the
coverslip.
Since the membrane-associated regions of P-gp are responsible for recognition and interaction with its various substrates and inhibitors (Greenberger et al., 1991; Zhang et al., 1995), much effort has been directed toward the elucidation of the number, location, and polarity of individual TM domains. Hydropathy profiling of the primary amino acid sequences of the murine and human P-gp families (Devault and Gros, 1990; Chen et al., 1986) has consistently led to a proposed structure of 12 TM domains grouped into two symmetrical and sequence homologous halves, with the amino and carboxyl termini intracellular (Fig. 7A). Such a model would position the two predicted nucleotide binding sites intracellularly and the predicted glycosylated loop located between predicted TM1 and TM2 extracellularly, two predictions verified experimentally by epitope mapping (Kartner et al., 1985; Georges et al., 1993). However, despite several attempts by different groups using unrelated experimental strategies, a clear topological picture for this region of the protein has yet to emerge. A summary of the current topological models for the membrane associated portion of P-gp is shown in Fig. 7. Studies of in vitro translated truncated P-gp molecules containing an epitope tag whose polarity with respect to the membrane was monitored after translocation into pancreatic microsomes suggested that P-gp may also exist in a different conformation with each hydrophobic domain spanning the membrane four rather than six times. In this model, predicted TM5 and TM8 were extracellular, and predicted TM3 and TM10 were located intracellularly (Fig. 7B; Zhang and Ling, 1991, 1993; Zhang et al., 1993). Independent studies of truncated P-gp molecules fused to a prolactin reporter gene and expressed in Xenopus laevis oocytes suggested six TM domains in the amino-terminal half and four TM domains for the carboxyl-terminal half of the protein (Fig. 7C; Skach et al., 1993; Skach and Lingappa, 1993, 1994). Finally, topological studies of truncated P-gps fused to alkaline phosphatase and expressed in Escherichia coli proposed a six-TM domain topology for the amino-terminal half of the protein, but with a different arrangement than predicted by hydropathy: TM4 was extracellularly located and replaced by a novel TM4 mapping in a further carboxyl-terminal segment (Fig. 7D; Bibi and Béjà, 1994). The reasons for discrepancies between topological models derived from the study of truncated proteins and that predicted by hydropathy are not clear, but they have prompted the search for methods that would allow topology mapping in intact and active proteins. Recently, Loo and Clarke (1995a) inserted single cysteine residues in the backbone of a cysteineless but biologically active human P-gp. Cysteines inserted in the third, fourth, or sixth predicted EC loop were biotinylated in intact cells by low concentrations of biotin maleimide, suggesting an extracellular location of these residues; cysteines inserted in the first, second, third, or fourth predicted IC loop were biotinylated only at higher concentrations of the same reagent, supporting an intracellular location (Fig. 7E; Loo and Clarke, 1995a), in agreement with the structural organization predicted by hydropathy.
Figure 7: Proposed models of P-gp membrane topology. Panel A, hydropathy-based model (Gros et al., 1986; Chen et al., 1986). Panel B, Zhang and Ling (1991, 1993), Zhang et al.(1993). Panel C, Skach and Lingappa(1993, 1994), Skach et al.(1993). Panel D, Bibi and Béjà(1994), Béjà and Bibi (1995). Arrowheads indicate position of insertion of alkaline phosphatase into P-gp. Panel E, single cysteine insertions (arrowheads) in a cysteineless MDR1 mutant (Loo and Clarke, 1995a). Panel F, hemagglutinin epitope insertions (Kast et al., 1995; this study).
To obtain topological information on the membrane-associated portion of P-gp, we have inserted discrete epitope tags in the full-length polypeptide followed by expression of the modified protein in mammalian cells where (a) the effect of epitope insertion on P-gp function can be monitored, and (b) the polarity of the epitope with respect to the plasma membrane can be established unambiguously by direct visualization with immunofluorescence in intact and permeabilized cells using an antibody directed against the tag. In addition, confocal microscopy ensures that the mutant proteins are expressed exclusively at the plasma membrane and that their intracellular localization does not reflect trapping in the endoplasmic reticulum or Golgi apparatus. Recently, we have used this method to establish that the segments separating TM1-TM2 and TM5-TM6 are extracellular, whereas segments linking TM2-TM3 and the one located downstream of TM6 are intracellular (Fig. 6, epitope locations indicated with underlined numbers and filled arrowheads) (Kast et al., 1995). In the present report, we have mapped the membrane polarity of a series of epitopes inserted at positions 235, 887, and 1024 (found to be intracellular) and 736, 849, and 961 (found to be extracellular). These results allowed us to establish unambiguously that the segments separating TM4-TM5, TM10-TM11, and located downstream of TM12 correspond to intracellular regions, whereas the segments linking TM7-TM8, TM9-TM10, and TM11-TM12 correspond to extracellular regions (Fig. 6, epitope locations indicated by filled arrowheads), in complete agreement with the topological model obtained by hydropathy profiling (Devault and Gros, 1990).
The
results of our study differ from those deduced from the in vitro analysis of truncated molecules (Zhang and Ling, 1991, 1993, Zhang et al., 1993) or from studies of fusion proteins in E.
coli (Bibi and Béjà,
1994; Béjà and Bibi, 1995).
Whereas the results of Zhang et al.(1993) predicted that the
intervening segment between TM4 and TM5 would be extracellular, our
results with mutant 8 (insertion at position 235) indicate that this
segment is in fact intracellular (Fig. 7B). Likewise,
these authors predicted that the intervening segment between predicted
TM9 and TM10 would be intracellular (Fig. 7B), whereas
the results obtained here with mutant 17 (insertion of triple tag at
position 849) clearly indicate that it would be positioned
extracellularly. On the other hand, Bibi and
Béjà proposed a new
location for TM4 being flanked by residues 245 (out) and 266 (in),
based on the analysis in E. coli of various P-gps fused to
alkaline phosphatase and on the extracellular localization of an
alkaline phosphatase (50 kDa) inserted between positions 226 and
227 in P-gp that retains activity in E. coli (Fig. 7D). Our intracellular localization of the
epitope inserted at position 235 (mutant 8) supports the initial
positioning of TM4 between positions 210 (in) and 229 (out) as
predicted by hydropathy analysis. The study of alkaline phosphatase
fusions in E. coli (intracellular at positions 683, and 736;
extracellular at position 720; Fig. 7D) also led to the
proposition that TM7 may span the membrane twice
(Béjà and Bibi, 1995).
However, our extracellular localization of a tandem epitope inserted at
position 736 strongly suggests that TM7 spans the membrane only once
and that the intervening segment between predicted TM7 and TM8 is
extracellular (Fig. 7F). This conclusion is also
supported by the independent observations that (a) a cysteine
inserted in a cysteineless P-gp mutant at position 745 maps
extracellularly (Loo and Clarke, 1995a) and (b) the protein
segment spanning positions 740-747 is recognized in intact cells
by the monoclonal antibodies MRK-16 (Georges et al., 1993) and
MM4.17 (Cianfriglia et al., 1994). There may be various
reasons for the observed discrepancies between topologies deduced from
truncated proteins in vitro or expressed in E. coli and our results. First, truncated proteins may lack
carboxyl-terminal sequence determinants important for correct protein
folding and membrane insertion of the full-length peptide (Traxler et al., 1993). Second, the insertion of a small epitope tag of
9-19 amino acids in P-gp (this study) may be less detrimental to
proper membrane insertion and function than the insertion of a
50-kDa alkaline phosphatase (Manoil and Beckwith, 1985). Third,
the expression systems selected in these studies (X. laevis oocytes, intact E. coli cells) may only incompletely
reveal P-gp function. On the other hand, the advantages of the method
we have used are 4-fold. The topology was always assessed in the
context of the full-length protein. Expression of the tagged proteins
was in an intact mammalian cell background where proper
post-translational modification possibly important for folding and
targeting was expected to be preserved. All proteins analyzed were
functional as they conferred drug resistance in transfected cells,
strongly suggesting proper folding and targeting to the membrane. The
use of normal optics and confocal microscopy to localize the tag
ensures maximal confidence for plasma membrane targeting of the
protein. This direct visualization of plasma membrane association
distinguishes our method from all previous topological studies on P-gp (Fig. 7, B-E). Finally it is interesting to note
that independent topology mapping studies performed in the intact
full-length proteins in human cysteineless P-gp mutant (Fig. 7E) and those reported by our group in the mouse
Mdr3 isoform (Kast et al., 1995; this study; summarized in Fig. 7F) are in agreement with each other and with the
membrane topology initially proposed by hydropathy analysis. Studies in
both systems were complementary: although EC5 could not be mapped in
the cysteineless P-gp (Loo and Clarke, 1995a), it could be localized in
our study (Fig. 7F); on the other hand, although IC3
could not be localized by epitope tagging, it was successfully assigned
in the cysteineless MDR1 mutant (Fig. 7E).
We noted a non-random distribution for tag insertion sites that were either completely tolerated by the protein versus those that caused a complete loss of function. In general, insertion of epitopes in the predicted EC loops was much better tolerated than insertion in predicted IC loops. Although only two of the nine insertion sites selected for typing EC loops proved to yield nonfunctional proteins (both are clustered in the TM3-TM4 interval, see discussion below), half of the sites selected in predicted IC loops (5 out of 10) resulted in complete loss of function. Interestingly, insertion of single epitopes in all three predicted EC loops of the carboxyl-terminal half (positions 741, 849, and 961) produced active proteins, but the tag at these locations could not be identified by immunofluorescence in the corresponding transfectants (although the tag was detected by immunoblotting). Additional copies of the tag at these sites were well tolerated to retain function and were required to allow detection by immunofluorescence (Fig. 3). Hydropathy analysis predicts that these loops should be fairly short at 21, 2, and 16 residues in length, respectively, and therefore may be tightly packed with the TM domains in a membrane-embedded bundle. Insertion of multiple epitopes in this context, although not detrimental to helix packing and protein function, may be required to reveal possible flexible extracellular loops. We note that predicted EC2 loop defined by the TM3-TM4 segment did not tolerate epitope insertion (positions 206 and 207), and the resulting proteins were nonfunctional. On the other hand, insertion of single cysteine residues in this loop in the P-gp cysteineless backbone (positions 209, 211, and 215), although producing active P-gps, failed to yield an identifiable location, suggesting inaccessibility of the inserted cysteine to the maleimide reagent (Loo and Clarke, 1995a). Together these results suggest that (a) this short loop (four residues) may not be exposed to the surface of the cell and may be in a much more compact structure; and (b) as opposed to its carboxyl-terminal counterparts, this loop does not tolerate alterations to maintain proper folding, maturation, or activity of P-gp.
Predicted IC loops were often more sensitive to tag insertion than EC loops. This was most evident for IC loops delineated by TM4-TM5 and TM8-TM9, where two out of three insertions in the former and all three tested in the latter seemingly inactivated the protein (Fig. 6). The sequence of these segments is fairly well conserved among the different isoforms of P-gps and among the same isoform in different species. Therefore, the integrity of the segments may be crucial for the mechanism of action of P-gp, as they may be important for drug transport, in particular for signaling to and from the drug binding site(s) located within the membrane portion of the protein and the ATP binding sites. Alternatively, these regions may play an important role in protein folding or proper targeting to the plasma membrane. Indeed, it has been observed that mutations in these regions often resulted in partially glycosylated proteins often retained in the endoplasmic reticulum, probably because of misfolding of the protein (Loo and Clarke, 1994). Therefore, amino acids in these regions may be responsible for proper protein folding because of their interaction with molecular chaperons, such as shown recently for calnexin (Loo and Clarke, 1995b).
In our study, some of the epitope insertions resulted in apparent alterations in the substrate specificity of P-gp, with the more pronounced ones associated with insertions in predicted IC loops (mutants 2, 8, and 18). In general, mutations in P-gp affecting substrate specificity map in the TM portion of the protein (Choi et al., 1988; Gros et al., 1991; Loo and Clarke, 1993a, 1993b) where they have been found to affect drug binding or drug release from the protein (Safa et al., 1990). A few mutations in certain IC loops (Currier et al., 1992; Loo and Clarke, 1994) or near the ATP binding sites (Hoof et al., 1994; Beaudet and Gros, 1995) have also been found to alter the profile of drug resistance encoded by the mutant P-gps. Such mutations have been proposed to impair a signal from the TM regions of the protein to the ATP binding sites, which normally underlies the drug-stimulatable ATPase activity characteristic to P-gp. In the case of a Gly to Val substitution at position 185 within the predicted IC loop defined by the TM2-TM3 interval of the human MDR1 P-gp, it was observed that the alteration in substrate specificity is associated with a change in the drug-stimulatable ATPase activity of the protein (Rao, 1995). Therefore, it is possible that the altered drug specificity caused by insertions in IC loops observed in some of our mutants may be mediated by alterations in this aspect of P-gp ATPase activity.
In conclusion, our epitope mapping studies (Fig. 7F), together with the parallel analysis by Loo and Clarke of single Cys mutants in the human MDR1 protein (Fig. 7E), provide a complementary and unambiguous topology for all TM domains of P-gp. This topology is strikingly close to that predicted by hydropathy analysis of the primary amino acid sequence of the protein but is in disagreement with data obtained with truncated proteins, suggesting that these methods are not optimal to establish the topology of P-gp.