Evidence for the vectorial nature of drug (substrate)-stimulated ATP hydrolysis by human P-glycoprotein.

P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp.ADP.Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp in the absence of substrate occurs both in the presence of 8-azido-[alpha-(32)P]ATP (following 8-azido-ATP hydrolysis) or 8-azido-[alpha-(32)P]ADP (without hydrolysis) and, the transition state intermediates generated under either condition are functionally indistinguishable. In this study, we compare the effect of substrates on Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of 8-azido-[alpha-(32)P]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi, indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp x ADP x P(i) (Vi), can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.     

ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein

P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.     

Evidence for assembly of small multidrug resistance proteins by a "two-faced" transmembrane helix

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism.     

The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis

Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments.     

Characterization of drug transport,ATP hydrolysis,and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation

The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.     

Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural and functional characteristics, this bacterial protein is classified as a member of the P-glycoprotein cluster of the ABC transporter superfamily. LmrA can even substitute for P-glycoprotein in human lung fibroblast cells, suggesting that this type of transporter is conserved from bacteria to man. The functional similarity between bacterial LmrA and human P-glycoprotein is further exemplified by their currently known spectrum of substrates, consisting mainly of hydrophobic cationic compounds. In addition, LmrA was found to confer resistance to eight classes of broad-spectrum antibiotics, and homologs of LmrA have been found in pathogenic bacteria, supporting the clinical and academic value of studying this bacterial protein. Current studies are focused on unraveling the mechanism by which ABC multidrug transporters, such as LmrA, couple the hydrolysis of ATP to the translocation of drugs across the membrane. Recent evidence indicates that LmrA mediates drug transport by an alternating two-site transport mechanism.     

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.     

Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2)

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.     

The fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug resistance-related protein (MRP)-1.

Several fluorescent probes have been used in functional studies to analyze drug transport in multidrug-resistant cells by fluorescent microscopy. Because many of these molecules have some drawbacks, such as toxicity, nonspecific background, or accumulation in mitochondria, new fluorescent compounds have been proposed as more useful tools. Among these substances, Bodipy-FL-Verapamil, a fluorescent conjugate of the drug efflux blocker verapamil, has been used to study P-glycoprotein activity in different cell types. In this study we tested by fluorescent microscopy the accumulation of Bodipy-FL-Verapamil in cell lines that overexpress either P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1). Expression of P-gp and MRP1 was evaluated at the mRNA level by RT-PCR technique and at the protein level by flow cytometric analysis using C219 and MRP-m6 monoclonal antibodies. Results indicate that Bodipy-FL-Verapamil is actually a substrate for both proteins. As a consequence, any conclusion about P-gp activity obtained by the use of Bodipy-FL-Verapamil as fluorescent tracer should be interpreted with caution.     

The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy.

The human multidrug resistance P-glycoprotein (P-gp) contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. A potential novel strategy to circumvent the effects of P-gp during chemotherapy is to prevent maturation of P-gp during biosynthesis so that the transporter does not reach the cell surface. Here we report that immature, core-glycosylated P-gp that is prevented from reaching the cell surface by processing mutations or by proteasome inhibitors such as lactacystin or MG-132 exhibited no detectable drug-stimulated ATPase activity. Disulfide cross-linking analysis also showed that the immature P-gp did not exhibit ATP-induced conformational changes as found in the mature enzyme. In addition, the immature P-gp was more sensitive to trypsin than the mature enzyme. These results suggest that P-gp is unlikely to be functional immediately after synthesis. These differences in the structural and enzymatic properties of the mature and core-glycosylated, immature P-gp could potentially be used during chemotherapy, and should result in the search for compounds that can specifically inhibit the maturation of P-gp.     

Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments

P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala₁₂₂₇ to Ser₁₂₅₁), which contains a single Trp residue (W₁₂₄₆) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala₁₂₂₇ was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring (∼50% α-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp₁₂₄₆ to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including λ_max, lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala(1227) to Ser(1251)), which contains a single Trp residue (W(1246)) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala(1227) was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring ( approximately 50% alpha-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp(1246) to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including lambda(max), lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.     

Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA

The development of multiple drug resistance in tumor cells is a significant problem in cancer therapy. In human, one of the reasons causing the resistance is due to the overexpression of the mdr1 gene product, P-glycoprotein. In our study, we had developed multiple drug resistant HepG2 cell line (HepG2/DR). To reverse the resistance, HepG2-DR cells were treated with antisense RNA against mdr1 gene. Total RNA and protein were extracted from the transfected cells. Northern analysis showed that mRNA level of mdr1 was decreased whereas a reduction in P-glycoprotein was detected by Western blot. By using flow cytometry, the ability of intracellular doxorubicin retention increased and drug efflux decreased in the treated cells. The result also showed that the cellular sensitivity to doxorubicin, vincristine and methotrexate measured in IC50 increased 83.3% 84.6% and 50% respectively. All these findings suggested that the expression of p-glycoprotein was successfully inhibited by antisense RNA and the drug resistance was reduced.     

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.     

Induction of P-glycoprotein functional activity and multidrug resistance by a soluble factor(s) produced by some mammalian cells.

In the previous study we have found that Djungarian hamster fibroblasts with high levels of multidrug resistance (MDR) (colchicine-resistance index RI of 1000 to 42000) produce soluble factor(s) communicating MDR to the drug-sensitive cells of the same species by elevating the functional activity of P-glycoprotein (Pgp). Here we have shown that these cells can influence human tumor cells in the same fashion. Rat hepatoma McA RH7777 cells and their colchicine-resistant derivatives are shown to produce a factor with similar effects (induction of MDR and Pgp functional activity in the drug-sensitive cells). These effects seem to depend on the drug resistance level of the donor cells. Our results show that induction of the Pgp-mediated MDR is not species-specific and the tumor cells with intrinsic MDR (arising from the tissue with a high level of Pgp expression) can produce a factor(s) communicating this type of drug resistance to the sensitive cells.     

Developmental expression of P-glycoprotein (multidrug resistance gene product) in the rat brain.

P-Glycoprotein (PGP), a product of the multidrug resistance gene (mdr), acts as an adenosine triphosphate-dependent drug efflux system in cells. Initially, PGP was found in cancer cells, but it is now known that PGP is richly distributed in the adult brain. Passage to the central nervous system is limited by the blood-brain barrier (BBB), and mdr1 gene-deficient mice showed up-regulation of BBB permeability. In this study, we examined the expression and localization of PGP in the rat brain during development. PGP protein was predominantly detected in the membrane fraction of the adult rat brain, although it was also faintly detected in the cytosolic fraction. PGP protein in the membrane fraction was undetectable in the embryo and early stages of postnatal development by immunoblotting studies, was first detected on postnatal day (P) 7, and then gradually increased to reach a plateau. Such changes were observed commonly in the cerebral cortex, hippocampus, and cerebellum. Immunohistochemical studies showed that PGP immunoreactivity was first detected on P7, and intense PGP immunoreactivity was observed in the adult rat brain. Double-immunolabeling studies revealed that PGP was colocalized with von Willebrand factor-immunoreactive capillaries. We further examined the colocalization of PGP and astrocytes using glial fibrillary acidic protein (GFAP) as a marker. Three-dimensional analysis showed that the GFAP-immunoreactive astrocytes possessed fine processes which ensheathed capillaries, but the PGP immunoreactivity did not colocalize with the GFAP immunoreactivity. These results indicate that PGP expression increased with postnatal development and is localized in the brain capillaries.     

Modulation of the expression of a multidrug resistance gene (mdr-1/P-glycoprotein) by differentiating agents

Acquired resistance to multiple natural products in vitro is mediated by P-glycoprotein (Pgp). Expression of this protein has been demonstrated in some normal tissues and in tumor samples obtained from both untreated and treated patients. In situ hybridizations with RNA probes have demonstrated higher levels of expression of mdr-1/Pgp in well-differentiated tumors and in well-differentiated areas in tumors with mixed histologies. Expression of mdr-1/Pgp in human colon carcinoma cell lines was increased by the differentiating agents sodium butyrate, dimethyl sulfoxide, and dimethylformamide. In the SW-620 cell line addition of sodium butyrate resulted in a rapid induction of mdr-1/Pgp mRNA that was sustained for the duration of the exposure. The levels of P-glycoprotein were measured by immunoblotting and were also increased. Similar results were obtained in three other cell lines including the HCT-15 line. This induction occurred without alterations in nuclease sensitivity. Discontinuation of sodium butyrate was followed by a rapid fall in the levels of mRNA. The levels of P-glycoprotein returned to normal with a half-life of about 24 h. In spite of a 20-25-fold increase in the level of mdr-1/Pgp mRNA and P-glycoprotein, the SW-620 cell line did not demonstrate increased resistance to doxorubicin and vinblastine or decreased accumulation of vinblastine. In contrast, in the HCT-15 cell line, a 5-fold increase of mdr-1/Pgp was accompanied by a comparable fall in vinblastine accumulation which was reversed by verapamil. In the SW-620 cell line, the induced protein could be photolabeled using [3H]azidopine. Expression of mdr-1/Pgp appears to correlate with the degree of differentiation. However, its induction is not always accompanied by expression of the multidrug-resistance phenotype.     

The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface.

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.     

Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-gamma-S, a nonhydrolyzable analogue of ATP

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.