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J Biol Chem, Vol. 274, Issue 35, 24759-24765, August 27, 1999


The Transmembrane Domains of the Human Multidrug Resistance P-glycoprotein Are Sufficient to Mediate Drug Binding and Trafficking to the Cell Surface*

Tip W. Loo and David M. ClarkeDagger

From the Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human multidrug resistance P-glycoprotein (P-gp)1 is an ATP-dependent drug pump that extrudes a broad range of hydrophobic substrates from the cell (reviewed in Refs. 1 and 2). Its likely physiological role is to protect the vital organs of the body from the cytotoxic effects of exogenous and endogenous compounds (3-5). The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during cancer (6, 7) and AIDS chemotherapy (8, 9).

P-gp, encoded by the MDR1 gene, has 1280 amino acids organized in two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids (10). Each repeat has an N-terminal hydrophobic domain containing six transmembrane sequences followed by a hydrophilic domain containing a nucleotide binding site (11, 12). The protein is a member of the ABC (ATP-binding cassette) superfamily of transporters (13).

There has been considerable effort in understanding the role of various domains in the mechanism of transport by P-gp. Both halves of P-gp have been expressed as separate polypeptides, but substrate-stimulated ATPase activity was detected only when the two halves were expressed simultaneously (14). The nucleotide-binding domains have been expressed in bacteria, and can bind ATP and its analogues (15-18). Both ATP-binding sites are essential because inactivation of either site by mutagenesis or chemical modification inhibits substrate-stimulated ATPase activity (19-22).

There is, however, some controversy concerning the interaction of substrates with the various domains. The transmembrane domains of P-gp are thought to form the translocation pathway for the drug substrates as they exit the membrane. Labeling of P-gp with photoactive analogues of drug substrates (23-26), cysteine-scanning mutagenesis studies using a thiol-reactive substrate (27), and mutational analysis studies (28-31) suggest that TM6 and TM12 are particularly important for drug-protein interactions. These two segments are close to each other in the tertiary structure of P-gp (32, 33). There is evidence, however, that suggests that the nucleotide binding domains also contribute to drug binding. For example, mutations in the nucleotide-binding domains of mouse mdr3 P-gp have also been reported to cause large alterations in the drug resistance phenotypes in transfected cells (34). Recently, it was reported that the nucleotide-binding domains could also interact with hydrophobic molecules such as steroids and flavonoids (35).

The aim of our study was to determine if P-gp deletion mutants missing one or both nucleotide-binding domains could still interact with drug substrates. An in vivo assay was used to test for drug binding by the deletion mutants. The rationale for this approach was that if the transmembrane domains alone could form the drug-binding site, then expression in the presence of drug substrates should result in a tightly and correctly folded protein that would be trafficked to the cell surface.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Deletion Mutants-- The cDNAs coding for full-length MDR1 (36), mutant P709G (37), or the N-half and C-half P-gp molecules (14) and containing the epitope for monoclonal antibody A52 at the C-terminal ends were subcloned into the mammalian expression vector pMT21.

The cDNAs coding for TMD1 (residues 1-379) and TMD2 (residues 681-1025) tagged at the C-end with A52 (38) were joined together to give TMD1+2 (residues 1-379 and 681-1025; transmembrane segments 1-6 and 7-12 only) and inserted into pMT21. P-gp cDNA coding for residues 1-1023 (i.e. deletion of the second nucleotide-binding domain) and tagged with A52 at the C-end was inserted into vector pMT21 to give the Delta NBF2 construct.

For expression studies involving drug resistance assays, the cDNAs of the A52-tagged wild-type, mutant P709G, N-half, C-half, and TMD1+2 were also inserted into the Epstein-Barr virus-based vector, pREP4 (Invitrogen Inc.).

Expression of P-gp Mutants-- For simple expression studies, HEK 293 cells were transfected with the cDNAs coding for the mutant P-gps. After 48 h, the cells were lysed with SDS sample buffer (63 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 2% (v/v) beta -mercaptoethanol) containing 50 mM EDTA and protease inhibitors (10 µM E-64, 12 µg/ml leupeptin, 100 units/ml aprotinin, 50 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 25 µg/ml benzamidine). The samples were then subjected to SDS-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose, and developed with monoclonal antibody A52 (36) or a rabbit anti-P-gp polyclonal antibody that is specific for the N-terminal nucleotide-binding domain (38), followed by enhanced chemiluminescence (Life Technologies, Inc.).

For drug resistance assays, HEK 293-EBNA cells (Invitrogen) constitutively expressing the Epstein-Barr virus nuclear antigen 1 (EBNA-1) gene and resistant to neomycin (G418) were used. The HEK 293(EBNA-1) cells were transfected with the mutant cDNA constructs in vector pREP4 (Invitrogen). After 24 h, the medium was replaced with fresh medium containing 10 µM cyclosporin A. Forty-eight hours after transfection, the medium was again replaced with fresh medium containing various concentrations of vinblastine. Then 72 h after transfection, the medium was again replaced with fresh medium containing no drug substrates. After another 4 days, the concentration of vinblastine that caused inhibition of cell growth by 50% was determined as described previously (36).

TPCK-treated Trypsin Digestion-- HEK 293(EBNA-1) cells were transfected with the cDNA coding for mutant TMD1+2 in pREP4 and grown for 24 h in the presence or absence of 10 µM cyclosporin A as described above. Membranes were prepared from the transfected cells (22) and suspended in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl). The membranes (5 mg/ml protein) were treated for 5 min at 22 °C with various amounts of TPCK-treated trypsin (Sigma; 12,000 BAEE units/mg), and the reaction was stopped by the addition of lima bean trypsin inhibitor (Worthington).

Endoglycosidase Digestion-- Digestion with endoglycosidase Hf (New England Biolabs) or with endoglycosidase F (PNGase F; New England Biolabs) was carried out as described previously (39).

Cell Surface Labeling-- HEK 293 cells were transfected with mutant TMD1+2 cDNA and then treated for 24 h with or without 10 µM cyclosporin A or 25 µM verapamil. The cells were chilled, washed twice with ice-cold PBS (phosphate-buffered saline, pH 7.4), and then incubated in the dark with phosphate-buffered saline containing 10 mM NaIO4 for 10 min. The cells were washed with phosphate-buffered saline and then treated for 10 min with 2 mM biotin-LC-hydrazide (Pierce) in 100 mM sodium acetate buffer, pH 5.5. The cells were washed once with 100 mM sodium acetate buffer, pH 5.5, and then solubilized with Tris-buffered saline, pH 7.4 containing 1% (w/v) Triton X-100. Biotinylated TMD1+2 was immunoprecipitated with monoclonal antibody A52. The immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis, transferred onto a sheet of nitrocellulose, and probed with streptavidin-conjugated horseradish peroxidase.

Expression in Escherichia coli-- The cDNAs coding for the A52-tagged TMD1+2 P-gp mutant or the A52-tagged TMD1+2 in which residues 1-27 were deleted were inserted into the NheI to XhoI sites of vector pET-21a(+) (Novagen Inc.), transformed into E. coli BL21(DE3) (Novagen), and selected in the presence of 100 µg/ml ampicillin. The clones were grown in LB medium containing 100 µg/ml ampicillin and then grown for 2 h at 37 °C in the presence of various concentrations (0-1 mM) of isopropyl-beta -D-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation at 12,000 × g for 5 min and then lysed with SDS sample buffer as described above. Equivalent volumes of samples were subjected to Western blot analysis, and the mutant P-gps were detected with monoclonal antibody A52 followed by enhanced chemiluminescence.

Resistance to tetraphenylarsonium chloride (TPA+) (Sigma) was done as described by Bibi et al. (40). Briefly, the same number of E. coli BL21(DE3) cells expressing the mutant TMD1+2 or TMD1+2 missing residues 1-27 were plated on LB agar plates containing 100 µg/ml ampicillin, 100 µM IPTG, and various concentrations (0-5 mM) of TPA+. The number of colonies in each plate was determined after several (1-7) days at 30 °C and compared with the plates with the control cells (vector only). For measuring resistance to TPA+ in liquid culture, the same number of E. coli BL21(DE3) cells expressing the mutant TMD1+2 or TMD1+2 missing residues 1-27 were grown at 37 °C in LB medium containing 100 µg/ml ampicillin, 100 µM IPTG, and various concentrations (0-5 mM) of TPA+. At hourly intervals, the absorbance at 600 nm was measured and compared with cells containing only the vector.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction of P-gp Deletion Mutants with Drug Substrates-- Each of the nucleotide-binding domains of P-gp (Fig. 1A) is predicted to contain 275-300 residues. To examine the role of the various domains of P-gp in drug-protein interactions, we first constructed a mutant that lacked the C-terminal nucleotide-binding domain (Delta NBF2; Fig. 1B). The Delta NBF2 mutant consisted of residues 1-1023 followed by the A52 antibody epitope tag. The presence of the tag facilitated detection of the protein after expression in HEK 293 cells and allowed us to distinguish it from any endogenous expression of P-gp. As shown in Fig. 2A (lane 1), immunoblot analysis of whole cell extracts of HEK 293 cells transfected with Delta NBF2 mutant cDNA showed the presence of a protein of 110 kDa as the major product. The 110-kDa Delta NBF2 protein was sensitive to digestion by endoglycosidase H (data not shown), suggesting that it was present mainly as a core-glycosylated immature protein.


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  		Fig. 1.   Schematic representation of P-gp and the deletion mutants. Full-length human P-gp (A) and deletion mutants missing the C-terminal nucleotide binding domain (Delta NBF2) (B) or missing both nucleotide-binding domains (TMD1+2) (C) are shown. The four domains of P-gp (TMD1, NBF1, TMD2, and NBF2) are shown in A. The location of the epitope tag for monoclonal antibody A52 is also shown. The arrows in A show the position of the deletions. The 12 TMs are represented by the numbered rectangles; the zigzag lines represent the linker region; and branched lines represent the consensus glycosylation sites.


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  		Fig. 2.   Effect of drug substrates on expression of P-gp deletion mutants. HEK 293 cells transfected with the cDNAs of mutant Delta NBF2 (A) or mutant TMD1+2 (B) were treated for 24 h with (+) or without (-) 3 µM cyclosporin A (Cyclo), 25 µM verapamil (Ver), 6 µM vinblastine (Vin), or 100 µM capsaicin (Caps). The cells were then solubilized with SDS sample buffer and subjected to immunoblot analysis with monoclonal antibody A52 followed by enhanced chemiluminescence. The locations of the 130- and 110-kDa (A) and the 100- and 80-kDa (B) proteins are indicated.

To test whether the Delta NBF2 mutant protein retained the ability to interact with drug substrates, we used an in vivo drug-binding assay. The rationale was that the Delta NBF2 mutant protein was not completely folded, since it was still sensitive to digestion by endoglycosidase H, and it most likely failed to be transported to the plasma membrane. We had previously shown that some point mutations in P-gp can cause malfolding of the protein such that it remains as a core-glycosylated protein that is not present at the cell surface. By expressing these mutant proteins in the presence of drug substrates, it was possible to induce the mutant proteins to fold in an active conformation, become resistant to endoglycosidase H, and be transported to the plasma membrane (41). Accordingly, we expressed the 110-kDa Delta NBF2 protein in the presence of various substrates. Fig. 2B shows that expression in the presence of 3 µM cyclosporin A (lane 2), 25 µM verapamil (lane 4), 6 µM vinblastine (lane 6), or 100 µM capsaicin (lane 8) all resulted in the appearance of another polypeptide of apparent mass 130 kDa. The 130-kDa Delta NBF2 P-gp protein was resistant to digestion by endoglycosidase H but not to endoglycosidase F (data not shown). The appearance of the 130-kDa protein in the presence of drug substrates suggests that the 110-kDa Delta NBF2 protein retained the ability to bind drug substrates, resulting in maturation of the protein.

We then tested whether a deletion mutant that was missing both nucleotide-binding domains, and containing only the transmembrane segments 1-6 and 7-12 (TMD1+2; Fig. 1C) could also interact with drug substrates. The cDNA for mutant TMD1+2 was expressed in HEK 293 cells in the presence or absence of drug substrates. In the absence of drug substrates, the major product for TMD1+2 was an 80-kDa protein (Fig. 2B, lanes 1, 3, 5, and 7). Expression of mutant TMD1+2 in the presence of 3 µM cyclosporin A (lane 2); 25 µM verapamil (lane 4), 6 µM vinblastine (lane 6), or 100 µM capsaicin (lane 8), however, resulted in the appearance of two products of 80 and 100 kDa. These results suggest that the mutant TMD1+2 protein could interact with drug substrates and undergo maturation.

Effect of Drug Substrates on Glycosylation of Mutant TMD1+2 and Trafficking to the Cell Surface-- The mutant TMD1+2 protein was treated with endoglycosidases to test whether expression in the presence of drug substrates had indeed induced maturation of the protein. Fig. 3 shows that the 80-kDa TMD1+2 protein was sensitive to digestion by endoglycosidase H when expressed in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of drug substrate (cyclosporin A). The 80-kDa protein decreased in apparent mass to 72 kDa following digestion by endoglycosidase H. Similar results were obtained when the 80-kDa protein was digested with endoglycosidase F (Fig. 3, lanes 5-8). These results suggest that the 80-kDa protein is the core-glycosylated immature form. In contrast, synthesis in the presence of drug substrate resulted in the appearance of the 100-kDa protein that was insensitive to digestion by endoglycosidase H (Fig. 3, lanes 1 and 2) but not to endoglycosidase F (Fig. 3, lanes 5 and 6). Apparently, the expression of mutant TMD1+2 in the presence of substrate allowed the mutant protein to leave the endoplasmic reticulum and pass through the Golgi apparatus for extensive modification of the carbohydrate groups.


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  		Fig. 3.   Digestion by endoglycosidases. HEK 293 cells expressing mutant TMD1+2 were treated with (+Drug) or without (-Drug) 10 µM cyclosporin A for 24 h. The cells were solubilized with SDS sample buffer, and the cell extracts were treated with (+) or without (-) endoglycosidase Hf (Endo H) or PNGase F (Endo F). The samples were subjected to immunoblot analysis with monoclonal antibody A52, followed by enhanced chemiluminescence. The positions of the 100-, 80-, and 72-kDa proteins are indicated.

Cells expressing TMD1+2 were then subjected to cell surface labeling to determine if the mutant protein was correctly targeted to the cell surface. Cells expressing the A52-tagged TMD1+2 protein were grown for 24 h in the presence or absence of cyclosporin A, treated with sodium periodate to convert the carbohydrate moieties to aldehydes, and then reacted with biotin-LC-hydrazide (Pierce). Biotin-LC-hydrazide is a cell-impermeant compound that covalently attaches biotin groups to glycoproteins after periodate oxidation of the carbohydrate moieties. The biotinylated TMD1+2 proteins were immunoprecipitated with monoclonal antibody A52 and subjected to Western blot analysis with streptavidin-conjugated horseradish peroxidase, followed by enhanced chemiluminescence. Fig. 4 shows that the mutant TMD1+2 protein (100 kDa) was detected at the cell surface after expression in the presence of cyclosporin A (lane 2) or verapamil (lane 4). There was no labeling of TMD1+2 (80 kDa) when expressed in the absence or presence of drug substrate (lanes 1 and 3). These results showed that TMD1+2 is capable of interacting with drug substrates, resulting in maturation of the carbohydrate residues and trafficking to the cell surface. These results also suggested that the presence of drug substrates during synthesis induces structural changes in the protein.


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  		Fig. 4.   Cell surface labeling of cells expressing mutant TMD1+2. HEK 293 cells expressing mutant TMD1+2 in the presence (+) or absence (-) of 10 µM cyclosporin A or 25 µM verapamil for 24 h were treated with sodium periodate and then incubated with biotin-LC-hydrazide. The cells were lysed with TBS containing 1% (w/v) Triton X-100, and the biotinylated protein was immunoprecipitated with monoclonal antibody A52. The immunoprecipitates were subjected to Western blot analysis with horseradish peroxidase conjugated to streptavidin. The locations of the 100- and 80-kDa proteins are indicated.

Detection of Structural Changes in TMD1+2 Using Protease Digestion-- We previously showed that immature and mature forms of full-length wild-type P-gp were different in their sensitivity to digestion by trypsin (42). Core-glycosylated P-gp was about 100-fold more sensitive to digestion by trypsin compared with the mature enzyme. This enhanced sensitivity to trypsin of the core-glycosylated form of P-gp suggested that it existed in a more "relaxed" conformation than the mature enzyme. Accordingly, the TMD1+2 protein was subjected to trypsin digestion to test for potential structural differences between the 80- and 100-kDa forms of the protein. Membranes prepared from cells expressing the A-52-tagged TMD1+2 protein were treated with trypsin and then subjected to Western blot analysis with monoclonal antibody A52. Fig. 5 (lane 1) shows that the 80-kDa form of TMD1+2 is the major product in the membranes of cells grown in the absence of drug substrate, while the 100-kDa form is the major product when grown in the presence of substrate. In addition, the 80-kDa form of the protein showed relatively higher sensitivity to trypsin (10 µg/ml trypsin; lane 3) than the 100-kDa form of the protein (1000 µg/ml trypsin; lane 5). These results suggested that the trypsin-sensitive sites are more readily accessible in the 80-kDa protein but are probably hidden in the 100-kDa protein as a result of tighter or proper folding when synthesized in the presence of drug substrate.


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  		Fig. 5.   Protease digestion of TMD1+2. Membranes were prepared from HEK 293 cells expressing TMD1+2 and which were grown for 24 h with (+drug) or without (no drug) 10 µM cyclosporin A. The membranes (5 mg/ml protein) were treated with various concentrations (0-1000 µg/ml) of TPCK-treated trypsin, and the reactions were stopped by the addition of trypsin inhibitor. Equivalent amounts of protein were subjected to immunoblot analysis with monoclonal antibody A52. The positions of the 100- and 80-kDa proteins are indicated.

Drug Resistance Assays-- We then tested whether TMD1+2 could confer resistance to cytotoxic drugs when expressed in mammalian cells. The usual approach for determining if a mutant P-gp can confer resistance to different drugs is to generate stable cell lines. The cells are first transfected with the mutant P-gp cDNA followed by selection of drug-resistant colonies after incubation in different cytotoxic drug substrates. A technical problem in using this method for studying mutant TMD1+2 is that the protein does not reach the cell surface in the absence of substrate. Compounding this problem is the observation that the concentrations of drug substrate required for trafficking of TMD1+2 to the cell surface would initially cause extensive cell death, thus complicating the results. Another problem with generating stable lines is that other mechanisms of drug resistance (endogenous) could develop during the relatively long selection period (months) needed to generate highly drug-resistant colonies.

To circumvent these potential problems, we developed a faster assay for measuring P-gp-mediated drug resistance that does not require the generation of drug-resistant stable cell lines. The assay relies on the use of an Epstein-Barr virus vector, pREP4, that can be maintained extrachromosomally in HEK 293 cells that constitutively express EBNA-1. The cDNAs for the A52-tagged wild-type, and P-gp mutants P709G, N-half, C-half, and TMD1+2 were inserted into the pREP4 vector and transfected into HEK 293(EBNA-1) cells. Mutant P709G was included because it shows similar processing defects as mutant TMD1+2. When mutant P709G is expressed in HEK 293 cells in the absence of drug substrate, the major product is a protein of 150 kDa that is retained in the endoplasmic reticulum as a core-glycosylated intermediate (37). Expression in the presence of drug substrate, however, corrects this processing defect (Fig. 6A). The P-gp half-molecules were also included because the processing of the N-half is also sensitive to the presence of substrate. When N-half P-gp is expressed alone or co-expressed with the C-half P-gp in the absence of drug substrate, it is retained in the endoplasmic reticulum as a core-glycosylated intermediate. Maturation of the N-half P-gp and trafficking to the cell surface is restored when it is expressed in the presence of the C-half P-gp and drug substrate (42). Fig. 6A shows expression of the P-gp mutants using the pREP4 vector and HEK 293(EBNA-1) cells. The major product for wild-type P-gp was the 170-kDa mature protein in the presence or absence of cyclosporin A. For mutant P709G, the major product in the absence of drug substrate was the 150-kDa (core-glycosylated) protein, while the 170-kDa protein was the major product in the presence of substrate. When N-half (90-kDa) P-gp was co-expressed with C-half P-gp in the presence of substrate, another N-half protein of 115 kDa was present. The 115-kDa protein is the fully mature form of the core-glycosylated N-half (90 kDa) protein. This was confirmed in two ways. When A52-tagged N-half protein and histidine-tagged C-half protein were co-expressed in the presence of drug substrate, the 90- and 115-kDa proteins, but not the histidine-tagged C-half, were detected with monoclonal antibody A52 (42). Only the 115-kDa (N-half) protein was sensitive to digestion with endoglycosidase F (42). Also, when the blot (Fig. 6A, Halves panel) was probed with a rabbit anti-Pgp polyclonal antibody that was specific for the N-terminal nucleotide-binding domain (38), the 90- and 115-kDa proteins, but not the C-half protein, were detected (data not shown). Similarly, the expression in the presence of substrate resulted in the appearance of the 100-kDa protein of mutant TMD1+2.


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  		Fig. 6.   Expression and vinblastine resistance assay of P-gp mutants in HEK 293(EBNA-1) cells. A, HEK 293(EBNA-1) cells were transfected with pREP4 vector (B, Control) or pREP4 vectors containing cDNAs for full-length wild-type P-gp (Wild), mutant P709G, or TMD1+2, or they were cotransfected with the cDNAs for the N-half and C-half of P-gp (Halves). After 24 h, the transfected cells were incubated with (+) or without (-) 10 µM cyclosporin A, and then all of the cells were incubated in the presence of 100 nM vinblastine for 24 h. Seventy-two hours after transfection, the cells were lysed with SDS sample buffer and subjected to immunoblot analysis with monoclonal antibody A52 and enhanced chemiluminescence. The positions of the mature (170-kDa), core-glycosylated full-length (150-kDa), N-half (115- and 90-kDa), C-half, and 100- and 80-kDa proteins of TMD1+2 are indicated. B, HEK 293(EBNA-1) cells expressing the above P-gp constructs and pretreated with 10 µM cyclosporin A were incubated with fresh medium containing various concentrations (0-200 nM) of vinblastine for 24 h. The medium was then replaced with medium containing no drug substrates, and the cells were grown for another 4 days. The concentration of vinblastine that inhibited cell growth by 50% was determined.

We then tested whether the mutant P-gps could confer drug resistance on the HEK 293(EBNA-1) cells. The cells expressing these mutants were initially treated for 24 h with cyclosporin A to allow trafficking of the mutant proteins to the cell surface. The cyclosporin A was then removed, and the cells were incubated for 24 h with various concentrations of vinblastine. Fig. 6A shows that the mature forms of wild-type, mutant P709G, N-half, and TMD1+2 P-gps were still present in the cells after treatment with cyclosporin A followed by vinblastine. The cells were then incubated for 4 days with plain medium, and cell survival characteristics were compared with control cells (concentration of vinblastine that caused a 50% reduction in cell growth). Fig. 6B shows that cells expressing mutant TMD1+2 and pretreated with or without cyclosporin A did not show any increased resistance to vinblastine relative to control cells. (LD50 about 1 nM vinblastine). By contrast, wild-type P-gp showed about a 45-fold increase in resistance to vinblastine (pretreated with or without cyclosporin A) compared with control cells that were transfected with pREP4 vector only. In mutant P709G or in the half-molecules of P-gp, preincubation of the cells with cyclosporin A greatly increased the relative resistance to vinblastine. The cells expressing mutant P709G that were not pretreated with cyclosporin A showed about an 8-fold increase in resistance to vinblastine relative to the control cells. After pretreatment with cyclosporin A, however, the relative resistance was increased to about 32-fold. Similarly, the cells expressing the half-molecules of P-gp showed little resistance to vinblastine (about 1 nM) unless they were pretreated with cyclosporin A (23 nM vinblastine). These results are consistent with the observation that the processing mutants and the half-molecules of P-gp can be induced to fold properly and be transported to the cell surface in an active conformation by synthesis in the presence of drug substrate.

Expression in E. coli and Resistance to Tetraphenylarsonium-- Expression of hamster P-gp in E. coli has been reported to confer resistance on E. coli to lipophilic cations such as tetraphenylphosphonium and TPA+ (40). An interesting observation was that mutations in the nucleotide-binding domains of hamster P-gp that abolished its ability to confer drug resistance on mammalian cells had no effect on P-gp-mediated efflux of TPA+ in E. coli (40). These results suggested that in the absence of functioning nucleotide-binding domains, the membrane potential across the E. coli membrane may be used by P-gp for drug efflux.

We had reported previously that full-length wild-type human P-gp is extremely unstable when expressed in E. coli, such that we could detect the presence of only digestion products of P-gp (43). By removing the nucleotide-binding domains, we hoped that this would make the TMD1+2 protein more stable and that it too might use the energized membrane of E. coli to confer resistance to TPA+. Another potential advantage of expression in bacteria is that membrane proteins are inserted directly into the bacterial membrane such that the problems associated with trafficking to the plasma membrane seen in mammalian cells are avoided. Accordingly, the cDNA for the A52-tagged mutant TMD1+2 was cloned into the inducible bacterial expression vector, pET-21a(+). An A52-tagged mutant TMD1+2 cDNA that was missing the first 27 amino acids of P-gp was also inserted into the expression vector (TMD1+2 (-1-27)). In this mutant, an initiating methionine residue was placed in front of residue 28. E. coli BL21(DE3) cells were transformed with the mutant P-gps, and expression of the mutant protein was induced by the addition of IPTG. Fig. 7 shows that proteins corresponding to the predicted size of TMD1+2 (72 kDa) or TMD1+2 (-1-27) (69 kDa) were synthesized after induction with IPTG. In the absence of IPTG, these proteins were not synthesized. Although the same number of cells were induced with various concentrations of IPTG, the number of cells recovered at the end of the induction period (2 h) was considerably less in the tubes with the higher concentrations of IPTG. This is a common occurrence, since the T7 RNA polymerase of the pET-21a(+) vector is so active and its T7 transcription and translation signals are so strong that most of the cell's machinery is directed to the production of the target protein upon induction with IPTG (44). This makes direct comparison between the lanes (Fig. 7) difficult to interpret, since the amount of IPTG affects both cell growth and metabolism. Therefore, only low concentrations (0.1 mM) were used in the experiments to test if TMD1+2 could confer resistance to TPA+.


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  		Fig. 7.   Expression of TMD1+2 in E. coli. TMD1+2 cDNA or TMD1+2 cDNA missing the first 27 amino acids (-1-27) were inserted into the bacterial expression vector pET-21a(+). E. coli BL21(DE3) were transformed with the mutant cDNAs. An equivalent number of transformed cells were incubated with various concentrations (0-1 mM) of IPTG for 2 h at 37 °C. After induction with IPTG, the cells from an equivalent volume of culture were harvested and lysed with SDS sample buffer. An equal volume of lysed cells was subjected to immunoblot analysis with monoclonal antibody A52 followed by enhanced chemiluminescence. The positions of the 72- and 69-kDa proteins are indicated.

E. coli cells transformed with vector alone (control) or with vector containing the cDNAs for TMD1+2 or TMD1+2 (-1-27) were then tested for resistance to TPA+. Initially, equivalent numbers of transformed cells were plated on LB agar plates containing 100 µg/ml ampicillin, 100 µM IPTG, and various concentrations (0-5 mM) of TPA+ and grown for up to 7 days at 30 °C. The number of colonies on each plate was counted. There was no difference in the number of colonies when the cells expressing the TMD1+2 or TMD1+2 (-1-27) mutants in the presence of TPA+ were compared with control cells containing the vector (pET-21a(+)) alone. There was also no difference when similar experiments were done using liquid LB media. The growth rates of the cells expressing the mutant P-gps were similar to control cells. These results suggest that TMD1+2 does not confer resistance to TPA+.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The family of ABC transporters, of which P-gp is a member, typically contains four distinct domains: two nucleotide-binding domains and two transmembrane domains. The N- and C-terminal ATP binding domains of P-gp have been overexpressed in bacteria, purified, and characterized. Each nucleotide-binding domain has been shown to efficiently bind ATP and its analogs and will hydrolyze ATP (15-18). The rates of ATP hydrolysis, however, are low and are not affected by the presence of drug substrates. These observations indicate that ATP binding and hydrolysis do not require participation of the transmembrane domains. Mutational studies on the nucleotide-binding domains showed that point mutations can alter the substrate specificity of P-gp (45, 46) and that the nucleotide-binding domains can bind flavonoids (35), thus suggesting that the cytoplasmic domains may also participate in drug-protein interactions.

The results in this study, however, show that the transmembrane domains alone retained the ability to interact with a variety of drug substrates in the absence of both ATP-binding domains. There is also a distinct difference in the interaction of the transmembrane domains with drug substrates from that of ATP binding to the nucleotide-binding domains. It appears that both transmembrane domains must be present for drug-induced maturation of the protein to occur. It was reported previously that the N- and C-terminal transmembrane domains (TMD1 and TMD2, respectively) could be expressed as separate polypeptides (38). When expressed alone, TMD1 or TMD2 showed no evidence of maturation in the presence of drug substrate. TMD1 alone, when expressed with or without drug substrate, remained as a core-glycosylated protein.2 Similarly, TMD2 alone, when expressed in the presence or absence of drug substrate, remained relatively sensitive to trypsin (42).

This paper provides direct evidence that drug-induced maturation of P-gp requires the presence of both TMD1 and TMD2. It seems that efficient drug-protein interactions in P-gp require interactions with residues from both transmembrane domains. There is increasing evidence to support the hypothesis that residues from both the N- and C-halves of P-gp may interact and cooperate to form the drug binding site(s). The N- and C-terminal half-molecules will noncovalently associate in a drug-dependent manner (42). These half-molecules will couple drug binding to stimulation of ATPase activity only if co-expressed in the same cell (Ref. 14 and this study). Photolabeling studies of human P-gp have shown that both halves of P-gp are about equally labeled (23-25).

The spacing between TMD1 and TMD2 appears to be important for drug-induced maturation. TMD1+2 proteins containing residues 1-379 and 681-1025 could be induced to mature by drug substrates (Fig. 1B). Other TMD1+2 constructs such as residues 1-379 and 632-1025, residues 1-379 and 700-1025, residues 1-358 and 632-1025, residues 1-358 and 681-1025, and residues 1-358 and 700-1025 showed little or no drug-induced maturation of the mutant protein. These proteins were found to be present as a major core-glycosylated protein when expressed in the presence or absence of drug substrates (data not shown). Although the various domains or half-molecule forms of P-gp can carry out partial reactions such as binding to drug substrates or ATP, none of these partial molecules could confer drug resistance. Therefore, coupling of drug binding to ATP hydrolysis must require coordinated interactions between all four domains. It appears that there is considerable flexibility in the native full-length P-gp molecule during the transport cycle. Indeed, cross-linking studies have shown that movement between the transmembrane segments of P-gp occurs during substrate-stimulated ATP hydrolysis (33). The region of P-gp that is responsible for imparting such flexibility may partly be due to the linker region (residues 635-694) connecting the two halves of P-gp. Hrycyna et al. (47) showed that deletion of this region or replacement of the linker region with an inflexible segment blocked drug transport by P-gp. Replacement of the linker region with a flexible segment, however, did not inhibit function.

The inability of TMD1+2 to confer drug resistance may be due to its inability to undergo conformational changes after interacting with drug substrates. The conformational changes, such as those observed between TM6 and TM12 during ATP hydrolysis (33) are likely to be essential for transport of substrate after it interacts with P-gp. ATP hydrolysis is not required for P-gp interaction with drug substrates, but it appears to be critical for subsequent release of the substrate during the reaction cycle (48). Conformation changes that occur during ATP hydrolysis by the native full-length molecule may not be the same as that induced by the energized membrane of E. coli. This may explain why mutant TMD1+2 could be expressed in the bacterial membrane and still not confer resistance to TPA+ as seen with hamster P-gp.

Maturation and trafficking to the cell surface also seems to be sensitive to global interactions in P-gp. None of the deletion mutants such as TMD1, N-half, or Delta NBF2 efficiently matured or was transported to the cell surface in the absence of drug substrate. The nucleotide-binding domains may influence the packing of the transmembrane segments so that a deletion mutant is readily recognized as being misfolded by the cellular quality control mechanisms. The influence of the nucleotide-binding domains on the interactions between the transmembrane domains has been shown in other studies. Cross-linking studies show that there is "cross-talk" between the nucleotide-binding and transmembrane domains (32, 33). Cross-linking between cysteines introduced at positions 332 (TM6) and 975 (TM12) of P-gp occurs only during ATP hydrolysis. Drug substrates also appear to influence the packing of the transmembrane segments. Disulfide cross-linking between cysteines introduced at positions 343 (TM6) and 986 (TM12) is blocked by drug substrates such as verapamil, vinblastine, cyclosporin A, and colchicine (33). It is possible that drug substrates promote maturation of TMD1+2 by inducing the protein to adopt a more "native" conformation.

    ACKNOWLEDGEMENTS

We thank Dr. David H. MacLennan for the A52 epitope and antibody and Dr. Randal Kaufman (Boston, MA) for pMT21.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Grant CA80900 and grants from the Medical Research Council of Canada and the Canadian Cystic Fibrosis Foundation. A scientist of the Medical Research Council of Canada and the Canadian Cystic Fibrosis Foundation Zellers Senior Scientist. To whom all correspondence should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1105; Fax: 416-978-1105.

2 T. W. Loo and D. M. Clarke, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: P-gp, P-glycoprotein; N-half P-gp, P-gp residues 1-682, containing transmembrane segments 1-6 and the first nucleotide-binding domain; C-half P-gp, P-gp residues 681-1280, containing transmembrane segments 7-12 and the second nucleotide-binding domain; Delta NBF2, residues 1-1023 of P-gp; TMD1+2, residues 1-379 and 681-1025 of P-gp; TMD1, P-gp residues 1-379, containing transmembrane segments 1-6; TMD2, P-gp residues 681-1025, containing transmembrane segments 7-12; TM, transmembrane; TPCK, L-1-tosylamido-2-phenylethylchloromethyl ketone; TPA+, tetraphenylarsonium; EBNA, Epstein-Barr virus nuclear antigen 1; IPTG, isopropyl-beta -D-thiogalactopyranoside.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Sharom, F. J. (1997) J. Membr. Biol. 160, 161-175[CrossRef][Medline] [Order article via Infotrieve]
2. Germann, U. A., and Chambers, T. C. (1998) Cytotechnology 27, 31-60
3. Schinkel, A. H., Smit, J. J., van Tellingen, O., Beijnen, J. H., Wagenaar, E., van Deemter, L., Mol, C. A., van der Valk, M. A., Robanus-Maandag, E. C., te Riele, H. P. J., Berns, A. J. M., and Borst, P. (1994) Cell 77, 491-502[CrossRef][Medline] [Order article via Infotrieve]
4. Sparreboom, A., van Asperen, J., Mayer, U., Schinkel, A. H., Smit, J. W., Meijer, D. K., Borst, P., Nooijen, W. J., Beijnen, J. H., and van Tellingen, O. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2031-2035[Abstract/Free Full Text]
5. Charuk, J. H., Grey, A. A., and Reithmeier, R. A. (1998) Am. J. Physiol. 274, F1127-F1139[Abstract/Free Full Text]
6. Chan, H. S., Grogan, T. M., DeBoer, G., Haddad, G., Gallie, B. L., and Ling, V. (1996) Eur. J. Cancer 6, 1051-1061[CrossRef]
7. Fisher, G. A., Lum, B. L., Hausdorff, J., and Sikic, B. I. (1996) Eur. J. Cancer 6, 1082-1088[CrossRef]
8. Lee, C. G., Gottesman, M. M., Cardarelli, C. O., Ramachandra, M., Jeang, K. T., Ambudkar, S. V., Pastan, I., and Dey, S. (1998) Biochemistry 37, 3594-3601[CrossRef][Medline] [Order article via Infotrieve]
9. Kim, R. B., Fromm, M. F., Wandel, C., Leake, B., Wood, A. J., Roden, D. M., and Wilkinson, G. R. (1998) J. Clin. Invest. 101, 289-294[Medline] [Order article via Infotrieve]
10. Chen, C. J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986) Cell 47, 381-389[CrossRef][Medline] [Order article via Infotrieve]
11. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 843-848[Abstract/Free Full Text]
12. Kast, C., Canfield, V., Levenson, R., and Gros, P. (1996) J. Biol. Chem. 271, 9240-9248[Abstract/Free Full Text]
13. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113[CrossRef]
14. Loo, T. W., and Clarke, D. M. (1994) J. Biol. Chem. 269, 7750-7755[Abstract/Free Full Text]
15. Shimabuku, A. M., Nishimoto, T., Ueda, K., and Komano, T. (1992) J. Biol. Chem. 267, 4308-4311[Abstract/Free Full Text]
16. Baubichon-Cortay, H., Baggetto, L. G., Dayan, G., and Di Pietro, A. (1994) J. Biol. Chem. 269, 22983-22989[Abstract/Free Full Text]
17. Sharma, S., and Rose, D. R. (1995) J. Biol. Chem. 270, 14085-14093[Abstract/Free Full Text]
18. Dayan, G., Baubichon-Cortay, H., Jault, J. M., Cortay, J. C., Deleage, G., and Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658[Abstract/Free Full Text]
19. Azzaria, M., Schurr, E., and Gros, P. (1989) Mol. Cell. Biol. 9, 5289-5297[Abstract/Free Full Text]
20. Urbatsch, I. L., Sankaran, B., Weber, J., and Senior, A. E. (1995) J. Biol. Chem. 270, 19383-19390[Abstract/Free Full Text]
21. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 22957-22961[Abstract/Free Full Text]
22. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 21449-21452[Abstract/Free Full Text]
23. Bruggemann, E. P., Currier, S. J., Gottesman, M. M., and Pastan, I. (1992) J. Biol. Chem. 267, 21020-21026[Abstract/Free Full Text]
24. Greenberger, L. M. (1993) J. Biol. Chem. 268, 11417-11425[Abstract/Free Full Text]
25. Morris, D. I., Greenberger, L. M., Bruggemann, E. P., Cardarelli, C., Gottesman, M. M., Pastan, I., and Seamon, K. B. (1994) Mol. Pharmacol. 46, 329-337[Abstract]
26. Zhang, X., Collins, K. I., and Greenberger, L. M. (1995) J. Biol. Chem. 270, 5441-5448[Abstract/Free Full Text]
27. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948[Abstract/Free Full Text]
28. Gros, P., Dhir, R., Croop, J., and Talbot, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7289-7293[Abstract/Free Full Text]
29. Kajiji, S., Talbot, F., Grizzuti, K., Van Dyke-Phillips, V., Agresti, M., Safa, A. R., and Gros, P. (1993) Biochemistry 32, 4185-4194[CrossRef][Medline] [Order article via Infotrieve]
30. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 19965-19972[Abstract/Free Full Text]
31. Loo, T. W., and Clarke, D. M. (1994) Biochemistry 33, 14049-14057[CrossRef][Medline] [Order article via Infotrieve]
32. Loo, T. W., and Clarke, D. M. (1996) J. Biol. Chem. 271, 27482-27487[Abstract/Free Full Text]
33. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989[Abstract/Free Full Text]
34. Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170[Abstract/Free Full Text]
35. Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J. M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9831-9836[Abstract/Free Full Text]
36. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 3143-3149[Abstract/Free Full Text]
37. Loo, T. W., and Clarke, D. M. (1994) J. Biol. Chem. 269, 28683-28689[Abstract/Free Full Text]
38. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 21839-21844[Abstract/Free Full Text]
39. Loo, T. W., and Clarke, D. M. (1996) J. Biol. Chem. 271, 15414-15419[Abstract/Free Full Text]
40. Bibi, E., Gros, P., and Kaback, H. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9209-9213[Abstract/Free Full Text]
41. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709-712[Abstract/Free Full Text]
42. Loo, T. W., and Clarke, D. M. (1998) J. Biol. Chem. 273, 14671-14674[Abstract/Free Full Text]
43. Loo, T. W., and Clarke, D. M. (1998) Methods Enzymol. 292, 480-492[Medline] [Order article via Infotrieve]
44. Mierendorf, R., Yaeger, K., and Novy, R. (1994) inNovations 1, 1-11
45. Bakos, E., Klein, I., Welker, E., Szabo, K., Muller, M., Sarkadi, B., and Varadi, A. (1997) Biochem. J. 323, 777-783
46. Beaudet, L., Urbatsch, I. L., and Gros, P. (1998) Biochemistry 37, 9073-9082[CrossRef][Medline] [Order article via Infotrieve]
47. Hrycyna, C. A., Airan, L. E., Germann, U. A., Ambudkar, S. V., Pastan, I., and Gottesman, M. M. (1998) Biochemistry 37, 13660-13673[CrossRef][Medline] [Order article via Infotrieve]
48. Ramachandra, M., Ambudkar, S. V., Chen, D., Hrycyna, C. A., Dey, S., Gottesman, M. M., and Pastan, I. (1998) Biochemistry 37, 5010-5019[CrossRef][Medline] [Order article via Infotrieve]

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