(Received for publication, September 2, 1994; and in revised form, December 15, 1994)
From the
Human MDR1 encodes an ATP-binding cassette transporter,
P-glycoprotein, that mediates multiple drug resistance (MDR) to
antitumor agents. It has been previously shown that photoaffinity
drug-labeling sites reside within, or near, the last transmembrane loop
of each cassette within P-glycoprotein (transmembrane domains (TM)
5-6 and 11-12). A genetic approach was used to determine if
the drug-labeling site in the second cassette contains functionally
important amino acids. Since human MDR3 is 77% identical to MDR1 but does not mediate MDR, the region from TM10 to the C
terminus of MDR1 was replaced with the corresponding sequences
from MDR3. The resultant chimeric protein was expressed but
not functional. By using progressively smaller replacements, we show
that replacements limited to TM12 markedly impaired resistance to
actinomycin D, vincristine, and doxorubicin, but not to colchicine. The
phenotype was associated with an impaired ability to photoaffinity
label the chimeric P-glycoprotein with
[I]iodoaryl azidoprazosin. In contrast,
replacement of the loop between TM11 and 12 appears to create a more
efficient drug pump for actinomycin D, colchicine, and doxorubicin, but
not vincristine. These results suggest that, similar to voltage-gated
ion channels, amino acids within and immediately N-terminal to the last
transmembrane domain of P-glycoprotein compose part of the drug-binding
pocket and are in close proximity to photoaffinity drug-labeling
domains.
P-glycoprotein is a plasma membrane protein which is believed to
utilize ATP to export, and thereby mediate multiple drug resistance
(MDR) ()to, widely used anticancer drugs including
anthracyclines, taxanes, Vinca alkaloids, mitoxantrone, and
etoposide(1, 2, 3) . It is one of many
ATP-binding cassette (ABC) proteins which contain six putative
transmembrane (TM)
-helices and an ATP-binding motif(4) .
As with many ABC transporters, P-glycoprotein contains and requires two
cassettes to form a functional unit(1, 4) . Other ABC
transporters permit the movement of a variety of small molecules
through the membrane including Cl
(the cystic
fibrosis transmembrane conductance regulator), peptides associated with
antigen processing (TAP proteins), and multiple hydrophobic anticancer
drugs (the multidrug resistance-associated
protein)(4, 5) . Indeed, P-glycoprotein itself may
also be a volume-regulated Cl
channel(6) , an
efflux pump for ATP(7) , and a pH regulator(8) . On a
broader level, many membrane proteins contain one or more cassettes,
which are composed of six putative transmembrane
-helices, and
participate in the transport of a variety of small molecules including
ions (e.g. voltage-gated ion channels), sugars, peptides, and
neurotransmitters(9, 10) .
P-glycoprotein overexpression has been associated with a poor outcome in the treatment of some cancers(11) . In addition, a variety of small molecules, such as verapamil, dihydropyridines, forskolin, and cyclosporine A, bind to P-glycoprotein and inhibit its function(12) . Therefore, the clinical utility of non-toxic derivatives of these and other molecules as chemosensitizing agents has been studied intensively (12) . However, the molecular basis of interaction of anticancer drugs and inhibitors with P-glycoprotein are poorly understood.
We have attempted to define drug-binding domains
in P-glycoprotein, and thereby to understand how multiple agents are
exported by, or inhibit, P-glycoprotein. Based on photoaffinity
labeling studies, probes derived from anticancer drugs or
chemosensitizing agents are preferentially competed by vinblastine >
doxorubicin > colchicine(13, 14) . Azidopine (a
photoactive Ca channel blocker), and photoaffinity
analogs of forskolin and prazosin bind to small common domains (15, 16) that are present in each cassette of
P-glycoprotein(17, 18) . The photoaffinity labeling
domains map to TM5-6 (forskolin analog;(15) ) and within,
or immediately C-terminal to, TM6 and TM12 (prazosin
analog;(19) ). Similarly, TM6 and the loop between TM5-6
(known as the H5, P, or SS1-SS2 region) of cassettes which compose
voltage-gated Ca
, Na
, and
K
channels form part of the ion pore and contain
receptor sites for inhibitors and photoaffinity labeled
drugs(9, 20, 21, 22) . This may help
explain why some agents, such as verapamil and azidopine, inhibit both
P-glycoprotein and the L-type Ca
channel.
Here we examine if the photoaffinity drug-labeling domains of P-glycoprotein contain amino acid residues that mediate resistance to anticancer drugs. To do this, we have made and evaluated the function of chimeric P-glycoproteins encoded by human MDR1 and MDR3. This method is logical since, in man, P-glycoprotein is encoded by two highly related genes, MDR1 and MDR3, which share 77% amino acid identity and similar putative topology(23, 24) . (MDR3 is also known as MDR2(25) ). However, only MDR1, or its mouse equivalent, mdr1 and mdr3 (also known as mdr1a and mdr1b(26) ), mediates MDR in transfected cells(27, 28, 29, 30, 31) . The human MDR3 homologue in the mouse, mdr2, appears to be a translocase for phospholipids (32, 33) . Functional differences between MDR1 and MDR3 may be mediated by regions of greatest amino acid diversity between these genes. Such regions are located in the transmembrane domains as well as the so-called linker region connecting each cassette. Consistent with this, chimeric P-glycoproteins composed of human MDR1 and MDR3 (34) or mouse mdr1 and mdr2(35) , were defective or had altered ability to mediate MDR if a large span of transmembrane domains or the first intracytoplasmic loop (between TM2 and 3) from MDR3 replaced the corresponding region in MDR1, respectively. However, the nucleotide binding regions in mdr2 (which is highly conserved between all MDR genes) were functional in the mdr1 environment(35) .
In this report, we focused on the second cassette in P-glycoprotein. In particular, sequences from TM10 to the C terminus, or subdivisions within this region, from human MDR3 replaced the corresponding region in human MDR1. We find that TM12 and the loop between TM11 and TM12 of P-glycoprotein is intimately involved in substrate specificity and alters the ability to photoaffinity label P-glycoprotein.
To
facilitate the construction of chimeric MDR 1/3 genes,
pCMVHMDR1 underwent several rounds of site-directed mutagenesis to
introduce and eliminate several restriction enzyme sites (Fig. 1). This resulted in pCMVHMDR1C, D, E, and F. In all
cases, no changes in amino acid identity occurred. Compared to
pCMVHMDR1, pCMVHMDR1C had 1) a HindII site introduced at the
bases encoding amino acid residues 856 and 857 of MDR1, and 2) two PstI sites eliminated; one at the multiple cloning site and
the other in the -lactamase gene. Compared to pCMVHMDR1C,
pCMVHMDR1D had a KpnI site eliminated at the multiple cloning
site. Compared to pCMVHMDR1D, pCMVHMDR1E had a PstI site
introduced at bases that correspond to amino acid residue 954 of MDR1.
Compared to pCMVHMDR1E, pCMVHMDR1F had a PstI site eliminated
at bases encoding amino acid residue 1125 in MDR1.
Figure 1: Amino acid sequence alignment from residue 858 to the C terminus of human P-glycoprotein encoded by MDR1 with the homologous region (from residue 857 to the C terminus) of human MDR3 gene product. Dashes represent amino acid identity between the gene products. Brackets above sequence indicates predicted TM domains or the Walker motifs A and B of the nucleotide binding fold. Vertical lines inserted in the sequence indicate the positions of restriction enzymes sites used to construct chimeric P-glycoprotein genes. Chimeric construct K was made by replacing MDR1 with MDR3 in the region spanning the KpnI to the second PstI site. Hatched bars indicate the position and sequence of mutagenesis primers used with the K construct to create chimeric construct R, Q, and P. These primers were also used in combination to create chimeric constructs M, N, and O.
Plasmid
pHepG2-16 contains a human MDR3 cDNA fragment (43) and was obtained from the American Type Culture Collection
(Rockville, MD). A 21-base pair insert in the MDR3 cDNA
fragment in pHepG2-16 was eliminated by site-directed mutagenesis
before being used for construction of chimeric genes. Chimeric
expression vectors pMDR1/3 I, H, G, J, K, and L were constructed by
ligation of inserts from pHepG2-16 and corresponding backbones
from pCMVHMDR1C, D, or E. Prior to ligation, construction of pMDR1/3 G
and I required the introduction of an XhoI site, by the
polymerase chain reaction, at the 3` non-translated region of the MDR3 cDNA fragment in pHepG2-16. Construction of pMDR1/3
K, required the introduction of a KpnI site in MDR3
cDNA fragment in pHepG2-16 prior to ligation. This necessitated
the conversion of two consecutive codons at residues 924 and 925 from MDR3 identity to MDR1 identity (see Fig. 1).
As a result, codon 926 in pMDR1/3 K and its derivatives, pMDR1/3 R, Q,
P, M, O, and N, was valine instead of glycine. Chimeric expression
vectors pMDR1/3 R, Q, P, M, O, and N were constructed from pMDR1/3 K by
site-directed mutagenesis with the mutagenesis primers shown in Fig. 1. Under this condition, preservation of Leu from MDR3 was allowed in constructs pMDR1/3 R, Q, P, M, N, and O (Fig. 1). No attempt was made to return this base pair change to MDR1 identity, since point mutation of MDR1 at this residue
(I1003L) did not alter the drug resistance phenotype compared to
wild-type MDR1 (data not shown). All constructs were verified by DNA
sequencing.
The level of P-glycoprotein was also estimated by
immunoblot analysis using crude membranes. To prepare crude membranes
from cell lines, 10 cells were washed in phosphate-buffered
saline and then lysed in 1 ml of 10 mM Tris, pH 8.0, 10 mM NaCl, 1 mM MgCl
in the presence of a protease
inhibitor mixture (100 units/ml aprotinin, 30 µM leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride) for 20 min at 4 °C. All
subsequent steps were conducted at 4 °C. After homogenization with
a Tenbrock mortar and pestle (30 strokes), the suspension was
centrifuged for 10 min at 450
g. The supernatant was
centrifuged at 100,000
g for 1 h. Subsequently, the
pellet was resuspended in 10 mM Tris, pH 7.4, in the presence
of 100 units/ml aprotinin. Protein was determined by the Lowry
method(46) . Proteins within crude membranes were resolved on
gels and transferred to nitrocellulose. Blots were probed by a
peptide-directed antibody (1:500) that specifically recognized the
sequence after the Walker B-motif of the second cassette in
P-glycoprotein (obtained from Oncogene Sciences Inc, Uniondale, NY).
Bound antibody was detected by using 1:2000 donkey anti rabbit Ig whole
antibody linked to horseradish peroxidase (Amersham Corp.), followed by
ECL detection (Amersham) using XAR-5 film (less than 1 min).
P-glycoprotein levels were quantitated using an image scanner
(Molecular Devices, Menlo Park, CA).
The experimental strategy initially required expression of wild-type MDR1 in drug-sensitive cells. Therefore, a clonal cell line derived from human melanoma cells was transfected with MDR1. Multiple cell colonies were established and evaluated. One clone, SK2-1F8, was established as a standard with which future constructs were compared. Relative to SK2, 1F8 cells were resistant to colchicine, vincristine, actinomycin D, and doxorubicin, but not to the non-MDR drug, cisplatin (Table 1).
P-glycoprotein expressed in SK2-1F8 cells was detected by fluorescent staining using an antibody, 4E3.16, specific for a cell surface epitope in P-glycoprotein (Fig. 2A) as well as by immunoblot analysis (see below). No P-glycoprotein was found in SK2 cells by either method. Since the human MDR1 gene was used to transfect human cells, we were concerned that the selection procedures used to clone such transfected cells might permit the elevation of endogenous MDR1. While this seemed unlikely since no colonies arose in mock transfected cells, RT-PCR analysis was used to test this possibility (Fig. 2B). In this case, RT-PCR exploited the differences in the 3`untranslated region between the endogenous and exogenous MDR mRNAs. The primer pair specific for endogenous MDR1 was able to detect a strong signal in KB-85 cells. These cells are known to have low level expression of P-glycoprotein and low level resistance(38) . No signal was detected in drug-sensitive KB cells or SK2-1F8 cells. A very weak signal was detected with SK2 cells. In contrast, the primer pair specific for exogenous MDR1 was able to detect exogenous MDR mRNA only in SK2-1F8 cells. Sequence analysis of the resultant PCR products verified that the products came from the expected genes (data not shown).
Figure 2: P-glycoprotein expression in transfected cells. Panel A, expression of P-glycoprotein on the surface of non-transfected cells (SK2) and cells transfected with MDR1 (SK2-1F8). Cells were incubated in 4E3.16 as described and analyzed by fluorescence analysis. Histogram displays the number of cells that bound the 4E3.16 antibody as indirectly monitored by fluorescent intensity. Panel B, RT-PCR analysis of endogenous and exogenous MDR gene expression. Primer pairs specific for the 3`-untranslated region of MDR1 (endogenous gene; primer pair 1A/1AP) or the expression vector (exogenous gene; primer pair 1A/135B) were used to amplify reversed-transcribed mRNA from the indicated cell line. Control cell lines were KB and its drug-selected counterpart, KB-85. PCR product was resolved on a 1% agarose gel. Standard markers are from top to bottom: 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234-220, and 154 base pairs.
A series of MDR3
replacements within the MDR1 environment was done (Fig. 3). After transfection of SK2 cells and subsequent
selection, individual colonies were expanded and then evaluated for
resistance to actinomycin D, colchicine, vincristine, and doxorubicin
using 3-day survival assays. P-glycoprotein levels varied from 0.4 to
1.5 times the amount found in cells transfected with MDR1.
Therefore, the amount of resistance was normalized based on
P-glycoprotein levels. The data were most striking for actinomycin D
resistance. The largest replacement, which spanned Thr to
the C terminus (Leu
), contained 72 amino acid
differences ( Fig. 1and 3) and is designated construct
pMDR1/3 I (or I). In this case, resistance to actinomycin D (as well as
the other three drugs) was markedly impaired. The I region was
subdivided into the H and G regions which contained 59 and 13 amino
acid differences compared to MDR1, respectively. The H construct
encoded a P-glycoprotein that was defective for actinomycin D
resistance. By sequential subdivision of the H and K regions, the
defect in actinomycin D resistance was localized to TM12 which
contained 6 different amino acids compared to MDR1 (construct P; see Fig. 1and Fig. 3). In contrast, replacement of the
TM 11 region from MDR3 (construct R), which has only six
differences compared to MDR1, had no effect on actinomycin D
resistance. Surprisingly, the chimera that contained the loop between
TM11 and 12 from MDR3 (construct Q) was associated with a
2.7-fold elevation in actinomycin D resistance above that found in
cells transfected with MDR1. As shown below in immunoblot
analysis, in cells transfected with the Q construct, P-glycoprotein was
expressed at approximately 0.36-fold of the wild-type levels. Since the
amount of actinomycin D required to kill 50% of 1F8 and Q3 cells was
similar (21 ± 2.9 versus 22.6 ± 3.3; mean
± S.E.; n = 5 or 7, respectively),
P-glycoprotein encoded by construct Q behaved as a more efficient drug
efflux pump compared to wild-type MDR1. Similar results were
obtained with another P and Q clonal cell line (data not shown).
Figure 3: Effects of chimeric P-glycoproteins on actinomycin D resistance in transfected cells. Bottom left, proposed orientation of human MDR1 based on hydropathy plot (see Refs. 63, 64 for modifications of this model). Upper left, map of replacement from TM10 to the C terminus of MDR1 with the corresponding region from MDR3 is shown. Top line shows the C-terminal one-third of P-glycoprotein encoded by MDR1. Three filled rectangles represent TM10, 11, and 12. Two ovals indicate the Walker A and B consensus sites within the nucleotide binding fold (NBF). Hatched bars indicate replacements from MDR3. Selected restriction sites (dashed lines) in the nucleotide sequence were either present in MDR1 or created by site-directed mutagenesis and were used to allow replacement with the corresponding region from MDR3. Replacement regions from MDR3 are coded by letters indicated to the right. The number after letter indicates clone number. Checkered bar at the bottom of the replacement map indicates the proposed photoaffinity drug-binding site for iodoaryl azidoprazosin identified by immunological mapping(19) . Upper right, drug resistance profile of cells transfected with MDR1/3 constructs compared to MDR1. The amount of actinomycin D resistance was determined as described in Table 1. This value was adjusted to account for difference in P-glycoprotein expression and then compared with the resistance found in cells transfected with wild-type MDR1 (SK2-1F8 cells, abbreviated as 1F8). Resistance in non-transfected cells (SK2) is shown for comparison. Values are mean ± S.E., n = 3-6 independent experiments except for construct I where n = 2. Significant differences compared to 1F8 cells were determined by two-tailed Student's t test (* p < 0.05,** p < 0.01,*** p < 0.005).
A detailed drug resistance profile of chimeric proteins with mutations in the TM11-TM 12 region was compiled (Fig. 4). The most striking observation was that replacement of TM 12 from MDR3 (construct P) impaired resistance to vincristine and doxorubicin. However, colchicine resistance was normal. Therefore, the TM12 region of MDR3 is capable of mediating resistance to at least one P-glycoprotein substrate. The loop between TM11 and 12 mediated supernormal levels of resistance for colchicine and doxorubicin, but no enhancement of vincristine resistance was observed.
Figure 4: Resistance profile of P-glycoproteins with replacement in the TM11-12 region. Resistance to colchicine, doxorubicin, and vincristine were evaluated in chimeric P-glycoproteins that contain a replacement of TM11, the loop between TM11-12, and TM12 from MDR3. Methodology is described in the legend to Fig. 3. Comparisons were made with cells transfected with full-length MDR1 (WT). The resistance profile to actinomycin D is repeated from Fig. 3for comparison.
If the loop between TM11-12 and TM12 alone has a positive and negative effect on actinomycin D resistance, respectively, these regions may interact to form a drug binding domain in P-glycoprotein. Therefore, we determined if the effects of TM12 and the loop between TM11 and TM12 were additive. The TM11 region from MDR3, since it had no effect on resistance, was used a control. To do this, constructs M, N, and O were transfected into SK2 cells (Fig. 3, lower portion of the graph). Expression of construct M, which encodes TM11 and TM12 from MDR3, resulted in a defect for actinomycin D resistance. Construct O, which encodes TM11 plus the loop between TM11 and TM12 from MDR3, mediated a 2.9-fold increase in resistance to actinomycin D compared to 1F8 cells. However, construct N, which encodes TM12 plus the loop between TM11 and TM12 from MDR3, mediated normal levels of resistance to actinomycin D compared to 1F8 cells. Therefore, the neutral, positive and negative effects on drug resistance from TM11, the loop between TM11 and TM12, and TM12 regions, respectively, were additive. Less prominent, but consistent effects were seen with doxorubicin, colchicine, or vincristine (data not shown).
Since a major photoaffinity drug
labeling site in P-glycoprotein has been localized to the TM11-12
region (see Fig. 3), the photoaffinity drug binding capacity of
chimeric P-glycoproteins was investigated (Fig. 5).
Characterization was limited to the TM11-12 region. To properly
interpret the results, both the photoaffinity drug binding capacity and
the amount of P-glycoprotein need to be compared. Therefore, after
photoaffinity labeling with [I]iodoaryl
azidoprazosin, material was resolved on gels and transferred to
nitrocellulose. Blots were quantitated for both immunoreactivity and
photolabeling of P-glycoprotein. S1 cells and their drug-resistant
counterpart designated S1-B1-20, which highly overexpress MDR1
P-glycoprotein, were used as controls(37) . It was found that
all cell lines that expressed chimeric genes also expressed
P-glycoprotein which could be photoaffinity labeled (Fig. 5A). The binding was specific since labeling of
the species that migrated at the position of P-glycoprotein was
competed by 20 µM vinblastine (Fig. 5B). In the chimera that contained TM11
from MDR3, which was expressed in the R1 cell line, both the amount of
photoaffinity labeling and immunoreactive P-glycoprotein were increased
80% above the level found in wild-type MDR1 (Fig. 5C). However, replacement of TM12 from MDR3 markedly inhibited
photolabeling since the level of P-glycoprotein in the P2 cell line was
15% higher than wild-type P-glycoprotein, yet photoaffinity labeling
was actually reduced 75% compared to wild-type P-glycoprotein. In
chimeras that contained the TM11-12 loop from MDR3, expressed in
the Q3 cell line, the P-glycoprotein level was reduced, but
photoaffinity labeling of P-glycoprotein was reduced even more than
expected. Similar results occurred with the Q2 cell line (data not
shown).
Figure 5:
Photoaffinity labeling of P-glycoprotein. Panel A, crude membranes from the indicated cell lines were
photoaffinity labeled with [I]iodoaryl
azidoprazosin. After gel electrophoresis and transfer to
nitrocellulose, the resultant labeled proteins were detected by
autoradiography (6 h of exposure). The blot was subsequently reacted
with antibody for the detection of P-glycoprotein and detected by the
ECL method using a 15-s exposure. Panel B, photoaffinity
labeling as in panel A in the presence (+) or
absence(-) of 20 µM vinblastine. Panel C,
quantitative analysis of photoaffinity labeled P-glycoprotein encoded
by MDR1/3 chimeras. The relative amount of P-glycoprotein (as
determined by densitometic scans of immunoblots) or photoaffinity
labeled P-glycoprotein was determined and compared. Values (mean
± S.E.; n = 3 independent experiments) were
standardized to cells that expressed wild-type MDR1
(1F8).
Previously, we and other others have shown that TM11-12 of P-glycoprotein, and the corresponding region of the first cassette, TM5-6, contain photoaffinity labeling sites which are likely to form part of the drug binding domain in P-glycoproteins that mediate MDR (human MDR1, and mouse mdr1 and mdr3)(15, 17, 19) . Because of the technical limitations with the immunological mapping studies of drug binding sites, and in particular, the multiple conformations that photoaffinity labeled molecules can assume(49) , the drug labeling site may or may not contain residues that form the actual drug binding domain in P-glycoprotein. Therefore, a genetic approach was used to further this analysis. The genetic approach used here demonstrates that mutations in TM12 and the loop between TM11 and TM12 of MDR1 can alter the specificity of P-glycoprotein for anticancer drugs. These data verify and refine the current model. However, we still cannot rule out the possibility that these regions indirectly participate in drug efflux (e.g. by inducing conformational changes at a distant site within P-glycoprotein).
Additional evidence argues that TM5-6 and TM11-12 form part of the drug-binding domain in P-glycoprotein (although TM4, TM10, and the cytoplasmic loop between TM2 and TM3 also play functional roles(34, 50) ). This includes point mutations in TM6 and TM12 of human MDR 1 (F335A and F978A)(48) , in TM11 of mouse mdr genes (S941F of mdr1 or S943F of mdr3)(51) , and two consecutive mutations (G338A and A339P) in TM6 of hamster pgp1(50) , which cause complex perturbations in the drug resistance phenotype mediated by P-glycoprotein. Beyond this, point mutations in the fifth and sixth transmembrane domains of cassettes within other ABC transporters (cystic fibrosis transmembrane conductance regulator, plasmodium falciparum MDR, and the Drosophilia melanogaster eye color genes) either alter specificity or are associated with a loss in function of these proteins (53, 54, 55) . Future analysis of double mutants in TM5-6 and TM11-12 of P-glycoprotein is likely to further define the interaction between these regions.
Since the ``mutations'' were created in a cassette-like fashion using replacement regions from MDR3, the data may also help explain why MDR3 does not mediate drug resistance(30) . Human MDR3 may not be able to transport certain antitumor drugs because of the 6 amino acid residues within TM12 that differ from those in MDR1. Consistent with this, the mouse homolog of MDR3, designated mdr2, participates in the translocation of phosphotidylcholine in the membrane while one of the mouse genes that mediates MDR (mdr3) is incapable of this function(31, 33) . The amino acid residues in mouse mdr2 and human MDR3 are perfectly conserved within TM12. Whether or not residues in this region are critical for lipid translocation needs to be investigated.
The mutations in TM12 and the loop between TM11-12 are associated with fundamentally different phenotypes. Compared to cells transfected with wild-type P-glycoprotein, replacement in TM12 was associated with markedly impaired resistance to actinomycin D, vincristine, and doxorubicin, but not colchicine as well as reduced photoaffinity labeling of P-glycoprotein (construct P). Similar to this, a point mutation within TM12 of MDR1 eliminated resistance to all of these drugs(48) , and mouse mdr2 can be expressed at high amounts but had considerably reduced binding to the same photoaffinity probe used here (31) . In contrast, alteration of the loop between TM11-12 was associated with approximately a 2.5-fold reduction in P-glycoprotein expression but a 2-3-fold elevation in resistance to actinomycin D, doxorubicin, and colchicine. Therefore, the latter mutant protein behaves as a more efficient pump for certain drugs, although the molecular basis for this observation is unknown. A similar observation has been made when the two mouse genes that mediate MDR were compared (56) . These two regions are likely to interact with each other, since positive and negative effects on resistance were additive. Additivity between point mutations within other proteins is common(57) .
The molecular alterations induced by these mutations in P-glycoprotein probably alter the contact points between amino acids and substrates (or inhibitors) for the protein. This could influence initial drug binding, translocation of bound drug through the plasma membrane, release of drug, as well as energy coupling between ATP hydrolysis and the site of energy utilization. Consistent with this, mutations in TM12 within MDR1 (shown here) or TM11 from mouse mdr1 or mdr3 impair the ability of P-glycoprotein to mediate MDR to some drugs and bind photoaffinity analogs(58) . In contrast, the G185V point mutation (59) and the TM11-12 replacement (shown here) are associated with an increase in drug resistance to certain drugs and a decrease in photoaffinity labeling of P-glycoprotein. The mechanistic implications of changes in photoaffinity labeling of P-glycoprotein are not clear and will need further kinetic analysis with reversible ligand-binding and/or transport methods. This is true since photoaffinity labeling is not reversible, the fate of photoactivated ligands can be altered by changes in the local lipid environment, and covalent linkage of the photolabel to P-glycoprotein is known to be an inefficient process.
The chimeric approach itself has certain unique
limitations. It would not indicate if conserved residues between MDR1
and MDR3, such as Phe (in TM12 of MDR1) or the homolog of
Ser
(in TM11 of mdr1), which have been previously
implicated, participate in drug resistance. Additionally, because of
additivity, it is possible to miss regions of importance. Therefore,
point mutation of divergent amino acids within TM12 or the loop between
TM11-12 of MDR1 and MDR3 may or may not result in the same
phenotype as the chimeric molecules.