Allosteric Modulation of Human P-glycoprotein

INHIBITION OF TRANSPORT BY PREVENTING SUBSTRATE TRANSLOCATION AND DISSOCIATION*

Nazli Maki, Peter HafkemeyerDagger , and Saibal Dey§

From the Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, Maryland 20814-4799 and the Dagger  Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany

Received for publication, October 10, 2002, and in revised form, February 28, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z)-flupentixol, a thioxanthene derivative, occurs through an allosteric mechanism. Unlike competitive inhibitors, such as cyclosporin A and verapamil, cis-(Z)-flupentixol does not interfere with substrate ([125I]iodoarylazidoprazosin) recognition by Pgp, instead it prevents substrate translocation and dissociation, resulting in a stable but reversible Pgp-substrate complex. cis-(Z)-Flupentixol-induced complex formation requires involvement of the Pgp substrate site, because agents that either physically compete (cyclosporin A) for or indirectly occlude (vanadate) the substrate-binding site prevent formation of the complex. Allosteric modulation by cis-(Z)-flupentixol involves a conformational change in Pgp detectable by monoclonal antibody UIC2 binding to a conformation-sensitive external epitope of Pgp. The conformational change observed is distinct from that induced by Pgp substrates or competitive inhibitors. A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Taken together, our results describe the molecular mechanism by which the Pgp modulator cis-(Z)-flupentixol allosterically inhibits drug transport.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cellular expression of human P-glycoprotein (Pgp),1 the product of the MDR1 gene, confers resistance to a broad variety of structurally unrelated chemotherapeutic agents and restricts bioavailability of many therapeutic drugs in experimental models (1, 2). Pgp is a 1280-amino acid plasma membrane protein that has two homologous halves separated by a linker region of about 80 amino acids (3). Each half of the protein contains a hydrophobic region with six putative transmembrane (TM) helices, followed by a cytoplasmic consensus ATP-binding/hydrolysis site (3). The TM regions are presumed to form the drug-translocating pathway (4), whereas the ATP sites, through ATP hydrolysis, provide the necessary driving force for transport (5, 6).

Pgp-mediated drug transport is inhibited by a number of structurally unrelated compounds known as reversing agents or modulators (for review see Ref. 2). Although some of the modulators are currently being tested for their clinical effectiveness, there remains a growing need for molecules with higher efficacy (7-9). To develop such compounds, a clear knowledge of the mechanisms of action of the existing repertoire is essential. Some of the Pgp modulators themselves, such as verapamil (10) and cyclosporin A (11), are substrates of the pump and inhibit drug transport in a competitive manner without interrupting the catalytic turnover (catalytic cycle) of Pgp (12-15). However, for many others, the inhibitory mechanisms are yet to be fully understood.

Recent studies on the mechanism of action of Pgp modulators indicated an allosteric mode of action for several compounds. Martin et al. (16) demonstrated that inhibition of vinblastine transport by the anthranilic acid derivative XR9576 is not through direct physical competition for the drug translocating pathway, indicating an allosteric effect on substrate recognition or ATP hydrolysis (17). A similar study suggested that the indolizin sulfone SR33557 affected vinblastine binding to Pgp through interaction with a site distinct from the site of substrate recognition (17). Boer et al. (18) and Ferry et al. (19) have shown that modulators like dexniguldipine and prenylamine inhibited vinblastine interaction with Pgp by a non-competitive mechanism. Based on these and other similar studies, drug interaction sites of Pgp have been categorized into the following two discrete types: 1) transport sites, where translocation of drug across the lipid bilayer can occur, and 2) regulatory sites, which modulate Pgp function (20, 21). Most of these studies investigated the effect of the modulators on substrate binding in isolated membranes, and relied exclusively on the kinetic parameters of substrate interaction with Pgp. However, little is known about the site(s) of modulator interaction with Pgp and the molecular mechanism by which the allosteric site(s) communicate with the other domains to inhibit drug transport.

Based on our prior studies (21, 22) and studies of other groups (23), we have proposed an allosteric mode of action for Pgp inhibitors with a thioxanthene backbone. In this study using a photoaffinity compound [125I]iodoarylazidoprazosin ([125I]IAAP) as a transport substrate, we provide experimental evidence for an allosteric modulator site in Pgp through which drug transport is inhibited by preventing substrate translocation and dissociation, without interfering with the initial step of substrate recognition.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- cis-(Z)-Flupentixol was from Research Biochemicals International. Sodium orthovanadate, vincristine, sodium azide, and 2-deoxyglucose were purchased from Sigma. Cyclosporin A and rapamycin were supplied by Calbiochem. [125I]IAAP (2200 Ci/mmol) was supplied by PerkinElmer Life Sciences. cis-(Z)-[3H]Flupentixol was custom-labeled by American Radiochemical Inc., and [3H]cyclosporin A was purchased from Amersham Biosciences. Monoclonal antibody UIC2 was obtained from Immunotech (Westbrook, ME), and FITC-labeled anti-mouse IgG2a secondary antibody was purchased from Pharmingen. The human Pgp-specific polyclonal antibody PEPG13 was a generous gift from the laboratory of Dr. Michael M. Gottesman, NCI, National Institutes of Health. Goat anti-rabbit IgG conjugated with horseradish peroxidase was obtained from Invitrogen.

Cell Lines and Plasmid Construction-- Previously characterized mouse cell line NIH3T3 fibroblasts (drug-sensitive cell line) and the drug-resistant wild-type human Pgp-expressing NIHMDR1 cells (14) were used for this study. In addition, two cell lines NIHMDR1-WT and NIHMDR1-F983A were generated. NIHMDR1-WT and NIHMDR1-F983A were created by stepwise selection of NIH3T3 cells transfected with pHaMDR1 and pHaMDR1-F983A plasmids, respectively. The plasmid pHa-MDR1, which contains the human MDR1 cDNA in its entirety, was kindly provided by S. Kane (City of Hope, Duarte, CA) (24). To construct the vector pHaMDR1-F983A, the NsiI-XhoI fragment of the previously described plasmid pTM1MDR1 containing the F983A mutation (25) was cloned into the corresponding sites of pHaMDR1. The region corresponding to the NdeI-PstI fragment within pHaMDR1-F983A including the insertion points and the flanking regions was sequenced in its entirety in both directions by automated sequencing (PRISM Ready Reaction DyeDeoxy Terminator Sequencing Kit, PerkinElmer Life Sciences). The plasmids pHaMDR1 and pHaMDR1-F983A were calcium phosphate-transfected into NIH3T3 cells and selected with vincristine. Clones were then picked and cultured to confluency. A stepwise selection was carried out with increasing concentrations of vincristine to generate NIHMDR1-WT and NIHMDR1-F983A cell lines that were able to grow in the presence of 1 µM vincristine. For maintenance of the cultures, cells were grown in monolayers at 37 °C in the presence of 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as described earlier (15). Pgp-expressing cells were maintained in the presence of respective drugs that were used for their selection.

[125I]IAAP Accumulation in Intact Cells-- 0.5 × 106 cells/well were grown in monolayers in a 24-well tissue culture plate at 37 °C in the presence of 5% CO2 in DMEM supplemented with 10% FBS (DMEM + 10% FBS). Cells were washed once with 1 ml/well of DMEM + 10% FBS 15 min prior to the initiation of the assay. The assay was initiated by incubating cells with 1.5 nM [125I]IAAP in 0.3 ml of DMEM + 10% FBS under subdued light (to avoid photocross-linking) at 37 °C in the presence of 5% CO2. After incubation for varying times, cells were washed twice with 1 ml/well ice-cold PBS. Washed cells were harvested by treatment with 0.5 ml/well trypsin/EDTA at 37 °C for 15 min. Harvested cells were diluted in 5 ml of Biosafe II scintillation fluid and mixed well by vortexing. Radioactivity associated with the cells was measured in a scintillation counter. Cells washed with ice-cold PBS immediately after addition of the assay mix was used as the "0"-min time point, and the value for accumulated [125I]IAAP was subtracted from each data point as nonspecific binding of [125I]IAAP to the cells. The counts/min values were converted to pmol/million cells. Accumulation of [3H]cyclosporin A and cis-(Z)-[3H]flupentixol in intact cells was measured following the same procedure as for [125I]IAAP, except the concentrations of [3H]cyclosporin A and cis-(Z)-[3H]flupentixol in the assay mix were 0.1 and 1 µM, respectively. For metabolic starvation, cells were incubated in a glucose-free DMEM containing 10 mM sodium azide and 10 mM 2-deoxyglucose for 30 min at room temperature.

[125]IAAP Efflux from Intact Mammalian Cells-- Similar to the drug accumulation assay, 0.5 × 106 cells/well were incubated for a period of 60 min in an assay mix containing 1.5 nM [125I]IAAP supplemented with either 5 µM cyclosporin A or 5 µM cis-(Z)-flupentixol. Following incubation cells were washed twice with 1 ml/well ice-cold PBS and incubated for varying times at 37 °C (and 5% CO2) in 0.6 ml of DMEM + 10% FBS either in the presence or in the absence of modulators. The incubations were stopped by washing the cells twice in 1 ml/well ice-cold PBS. The radioactivity associated with the cells was determined as described. The rate constants for [125I]IAAP release from cells were determined by non-linear regression (in GraphPad PRISM program) using a first order rate equation [I]t = [I]0·e-kt, where [I]t and [I]0 denote the amounts of intracellular [125I]IAAP at times t and 0 min, respectively, and k represents the rate constant of [125I]IAAP release from the cells. The values for t1/2 (time for half-maximal release) were calculated from the relationship t1/2 = 0.69/k.

Isolation of Crude Membranes Form Mammalian Cells-- Crude membranes were prepared according to Dey et al. (22) except that membranes were collected by centrifugation at 300,000 × g for 25 min instead of 100,000 × g for 1 h.

[125I]IAAP Photocross-linking of Pgp in Intact Cells-- For [125I]IAAP photocross-linking of Pgp in intact cells, 0.5 × 106 cells/well were grown in monolayers as for the transport assay. Cells were washed once with 1 ml/well DMEM + 10% FBS and incubated at 37 °C for 60 min with 0.3 ml of IMEM + 10% FBS containing 1.5 nM [125I]IAAP either in the presence or in the absence of 5 µM cyclosporin A, 5 µM cis-(Z)-flupentixol, or 1 mM sodium orthovanadate. Cells were exposed to UV light (SPECTROLINE, model XX-15A, 365 nm) for 5 min at room temperature. After photocross-linking, cells were resuspended in 100 µl/well (0.5 × 106 cells/100 µl) of cell lysis buffer containing 10 mM Tris, pH 8.0, 0.1% (v/v) Triton X-100, 10 mM MgSO4, 2 mM CaCl2, 1 mM dithiothreitol, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 50 units/ml micrococcal nuclease (Staphylococcus aureus). Resuspended cells were lysed by three cycles of freezing (on dry ice) and thawing (at 37 °C), and resolved by SDS-PAGE. The gels were dried and exposed to an x-ray film or to a PhosphorImager screen. For photocross-linking during [125I]IAAP efflux, cells at varying times were washed twice with 1 ml/well ice-cold PBS and photocross-linked for 5 min in the absence of any medium to minimize dissociation of Pgp-[125I]IAAP during photocross-linking. Photocross-linking was carried out at room temperature.

[125]IAAP Photoaffinity Labeling of Pgp in Isolated Membranes-- Photo-affinity labeling of crude membranes was carried out according to Dey et al. (22) in the presence of 3 nM [125I]IAAP.

SDS-PAGE and Immunoblot Analysis-- Electrophoresis and immunoblot analyses were performed as described previously (22). Human Pgp-specific polyclonal antibody PEPG13 was used at a dilution of 1:4000 for detection of the wild-type and the mutant Pgps. A goat anti-rabbit IgG conjugated with horseradish peroxidase, at a dilution of 1:10000, was used as secondary antibody. Horseradish peroxidase-conjugated secondary antibody bound to the nitrocellulose was detected by using the horseradish peroxidase-catalyzed luminal-based chemiluminescence reaction (ECL Western blotting system) kit from Amersham Biosciences. The light emission signal was captured on a Kodak Bio-Max MR film.

Quantification of Radioactivity in Protein Bands-- To determine the amount of [125I]IAAP photocross-linked to Pgp, the radioactivity associated with each band was quantified from the dried gels by exposing to a PhosphorImager screen and analyzed using a STORM 860 PhosphorImaging system (Amersham Biosciences). Values were expressed either in arbitrary units or as percentage of a control experiment.

UIC2 Reactivity Shift Assay-- Cells grown in monolayers were harvested by trypsinization, washed, and resuspended in IMDM supplemented with 5% FBS. 0.5 × 106 cells were incubated at 37 °C for 30 min with 5 µg of the monoclonal antibody UIC2 in 0.4 ml of IMDM + 5% FBS. Following incubation, cells were diluted with IMDM to 4.5 ml and centrifuged at 200 × g for 7 min. Washed cell pellets were resuspended in 0.4 ml of IMDM + 10% FBS containing 1 µg of the FITC-conjugated anti-mouse IgG and incubated at 37 °C for 30 min under subdued light. Cells were washed two times with IMDM + 5% FBS and resuspended in 0.4 ml of cold PBS and analyzed in a fluorescence-assisted cell sorter (FACS). Wherever mentioned, cells were preincubated at 37 °C for 3 min with 5 µM cyclosporin A, 5 µM cis-(Z)-flupentixol, or 1 mM sodium orthovanadate prior to addition of UIC2. The fluorescence intensity associated with cells was expressed on a log scale.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cis-(Z)-Flupentixol Induces an Elevated Level of Substrate ([125]IAAP) Association with Pgp-expressing Cells-- cis-(Z)-Flupentixol and certain structurally related analogs are potent inhibitors of Pgp-mediated drug transport (26-28). Characterization of its effect on substrate binding (23) suggested an allosteric mode of action for the modulator (21, 22); however, the exact mechanism of action remained unresolved. To understand the mechanism better, we developed a cell-based assay suitable for studying both Pgp-mediated transport and Pgp-substrate interaction, simultaneously, without altering experimental conditions.

Drug-sensitive NIH3T3 cells and drug-resistant NIHMDR1 (NIH3T3 cells transfected with human MDR1 cDNA) cells were incubated with the Pgp substrate analog [125I]IAAP. The steady-state level of [125I]IAAP accumulation in NIHMDR1 cells (0.15 pmol/million cells), expressing human Pgp, was 2-fold less than that of the NIH3T3 cells (0.3 pmol/million cells) lacking any detectable Pgp expression, indicating a Pgp-mediated efflux of [125I]IAAP from the cells (Fig. 1A). Inclusion of 5 µM cyclosporin A, a competitive inhibitor of Pgp-mediated drug transport (29), increased the steady-state level of [125I]IAAP accumulation in NIHMDR1 cells (0.3 pmol/million cells) to that of the control NIH3T3 cells (Fig. 1A), substantiating the role of Pgp in [125I]IAAP efflux from cells. Vanadate, a phosphate analog, inhibits Pgp-ATPase activity by stabilizing a catalytic transition state conformation of the protein with the substrate site poorly accessible (21, 30). Addition of 1 mM sodium orthovanadate induced a similar increase in [125I]IAAP accumulation in NIHMDR1 cells, further supporting the role of Pgp in [125I]IAAP transport. Neither cyclosporin A nor vanadate had any effect on the [125I]IAAP accumulation in NIH3T3 cells indicating Pgp as the only transporter responsible for [125I]IAAP efflux from NIHMDR1 cells.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of modulators on [125I]IAAP transport by Pgp (A) and Pgp-[125I]IAAP interaction (B). A, drug-sensitive NIH3T3 (open symbols) and drug-resistant NIHMDR1 (closed symbols) cells were incubated at 37 °C for indicated times with 1.5 nM [125I]IAAP in the absence ( and black-square) or presence of either 5 µM cyclosporin A (open circle  and ), 5 µM cis-(Z)-flupentixol (triangle  and black-triangle), or 1 mM sodium orthovanadate (black-diamond ). Intracellular accumulation of [125I]IAAP was measured and expressed as pmol per million cells. Data presented are average of three independent experiments. B, effect of modulators on Pgp-[125I]IAAP interaction in NIHMDR1 cells. Monolayers of NIHMDR1 cells were incubated at 37 °C with 1.5 nM [125I]IAAP for 60 min either in the presence of 5 µM cyclosporin A (CsA) (2nd lane), 5 µM cis-(Z)-flupentixol (Cis(Z)) (3rd lane), 1 mM sodium orthovanadate (Vi) (4th lane), or in the absence of any modulator (None) (1st lane). Following incubation, cells were exposed to UV irradiation for 5 min, lysed, and resolved by SDS-PAGE (80,000 cells per well) as indicated under "Experimental Procedures." Radioactivity associated with Pgp was detected in an autoradiogram (lower panel) and quantified in a PhosphorImager (upper panel). Data are expressed as fold stimulation of the basal [125I]IAAP binding to Pgp in the absence of any modulator.

Interestingly, in the presence of 5 µM cis-(Z)-flupentixol, cellular association of [125I]IAAP with NIHMDR1 cells (0.8 pmol/million cells) increased to a level 2.5-fold higher than that of cyclosporin A- or vanadate-treated NIHMDR1 cells (0.3 pmol/million cells) (Fig. 1A). No such increase was observed in NIH3T3 cells, indicting that the phenomenon of elevated association was Pgp-dependent. Therefore, the results indicated that in the presence of cis-(Z)-flupentixol the amount of [125I]IAAP molecules associated with Pgp-expressing cells exceeded (2.5-fold) the level usually reached in the absence of Pgp-mediated outward transport (Fig. 1A). The increased amount of this excess of [125I]IAAP molecules associated with NIHMDR1 cells varied with increasing concentrations of cis-(Z)-flupentixol and reached a maximum at around 30 µM (data not shown). Under optimal conditions, an excess of about 7 pmol of [125I]IAAP (relative to that achieved by simple inhibition of Pgp-mediated transport by cyclosporin A) was associated per million NIHMDR1 cells, which was equivalent to 0.4 × 106 molecules per cell. Interestingly, this value approximates the number of Pgp molecules (1.5 × 106 molecules per cell) present on the cell surface of each NIHMDR1 cell (31), indicating a possible complex formation of [125I]IAAP with Pgp in NIHMDR1 cells, in the presence of cis-(Z)-flupentixol. Preincubation for 60 min with 5 µM cyclosporin A (which competes for the substrate-binding site) or 1 mM sodium orthovanadate (which lowers the substrate binding affinity), prior to the addition of cis-(Z)-flupentixol in the assay, effectively inhibited (by 80 and 100%, respectively) the elevated association of [125I]IAAP with NIHMDR1 cells induced by cis-(Z)-flupentixol without altering the level of intracellular [125I]IAAP accumulation resulting from inhibition of transport (data not shown). These results indicated that cis-(Z)-flupentixol-induced elevated association of [125I]IAAP with NIHMDR1 cells can be prevented by blocking access of the substrate molecule to the substrate-binding site of Pgp.

cis-(Z)-Flupentixol-induced Elevated Association of [125I]IAAPIs Due to Formation of a Stable Complex between Pgp and [125I]IAAP-- To determine the possibility of a complex formation between the excess [125I]IAAP molecules associated with the NIHMDR1 cells and Pgp expressed in the cells, in the presence of cis-(Z)-flupentixol, cells were briefly exposed to UV irradiation at the end of the accumulation assay, and photocross-linking of [125I]IAAP to cellular proteins was determined. In the absence of a modulator, where [125I]IAAP was efficiently transported out of the cells, a detectable amount of [125I]IAAP was photocross-linked to Pgp, indicating a momentary association of the substrate molecule with Pgp during transport (Fig. 1B, 1st lane). In the presence of 5 µM cyclosporin A or 1 mM sodium orthovanadate, which inhibited [125I]IAAP transport by Pgp, no measurable photocross-linking was observed (Fig. 1B, 2nd and 4th lanes). On the other hand, in the presence of 5 µM cis-(Z)-flupentixol, which induced an elevated level of [125I]IAAP association with NIHMDR1 cells, an 80-fold increase in the amount of [125I]IAAP photocross-linked was observed (Fig. 1B, 3rd lane). This result indicated that in the presence of cis-(Z)-flupentixol, a large fraction of the accumulated [125I]IAAP in NIHMDR1 cells remained physically associated with Pgp. Consistent with that, preincubation of the cells with either 5 µM cyclosporin A or 1 mM sodium orthovanadate prior to addition of cis-(Z)-flupentixol reduced the stimulatory effect on photocross-linking by 80 and 98%, respectively (data not shown). Therefore, the excess [125I]IAAP associated with NIHMDR1 cells apparently remained in a stable Pgp-[125I]IAAP complex, presumably in the plasma membrane.

cis-(Z)-Flupentixol-induced Pgp-[125I]IAAP Complex Is Formed Prior to the Translocation Step-- Stabilization of Pgp-[125I]IAAP complex by cis-(Z)-flupentixol could occur either prior to or following the ATP-dependent translocation step. To distinguish between the two possibilities, we investigated the effect of cis-(Z)-flupentixol on [125I]IAAP accumulation in NIHMDR1 cells that were metabolically starved using NaN3 and 2-deoxyglucose. Energy depletion of NIHMDR1 cells resulted in a 2-fold increase in the intracellular accumulation of [125I]IAAP (Fig. 2A), indicating a requirement for cellular ATP in Pgp-mediated [125I]IAAP extrusion. However, cis-(Z)-flupentixol-mediated elevated association of [125I]IAAP with NIHMDR1 cells remained unaffected by metabolic starvation (Fig. 2A). Because substrate recognition by Pgp does not require ATP, whereas the translocation step does, the results suggested that the cis-(Z)-flupentixol-induced Pgp-[125I]IAAP complex formation occurs prior to the translocation step. The elevated [125I]IAAP association with NIHMDR1 cells induced by cis-(Z)-flupentixol was efficiently reversed by 1 mM vanadate but not in the energy-depleted cells (Fig. 2A). Because vanadate trapping requires ATP hydrolysis, the lack of reversal in starved cells suggested that the cells were effectively depleted of ATP.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of deenergization on cis-(Z)-flupentixol-mediated Pgp-[125I]IAAP complex formation. A, NIHMDR1 cells were incubated with ( and open circle ) or without (black-square and ) sodium azide and 2-deoxyglucose at room temperature for 30 min (for deenergization), prior to incubation with 1.5 nM [125I]IAAP in the presence (open circle  and ) or absence ( and black-square) of 5 µM cis-(Z)-flupentixol. Intracellular accumulation of [125I]IAAP was measured at indicated time intervals and expressed as pmol/million cells. As indicated by the arrow, 1 mM sodium orthovanadate (Vi) was added to the assay at the 30-min time point, and intracellular accumulation of [125I]IAAP was measured for another 30 min at different intervals. Data presented are average of three independent experiments. B, in a similar experiment as described above, after 30 (1st and 2nd lanes) and 90 min (3rd lane) of incubation, normal (upper panel) and energy-depleted (lower panel) cells were exposed to UV irradiation for 5 min. Cells were lysed and resolved in an SDS-PAGE, and [125I]IAAP cross-linked to Pgp was detected in an autoradiogram.

To investigate directly the Pgp-[125I]IAAP interaction (under the condition of elevated [125I]IAAP association), cells were subjected to photocross-linking immediately prior to and 60 min after addition of vanadate. Energy depletion that inhibited [125I]IAAP extrusion (Fig. 2A) had no effect on Pgp-[125I]IAAP interaction and its stimulation by 5 µM cis-(Z)-flupentixol (Fig. 2B), further suggesting that stabilization of the Pgp-[125I]IAAP complex by cis-(Z)-flupentixol occurred prior to the translocation step. On the other hand, addition of vanadate effectively destabilized the Pgp-[125I]IAAP complex induced by cis-(Z)-flupentixol (Fig. 2B), presumably by stabilizing the nucleotide-trapped transition state of Pgp. Consistent with the requirement of ATP hydrolysis for vanadate trapping, no such destabilization of the complex was observed in the energy-depleted cells.

Stabilization of the Pgp-[125I]IAAP Complex Blocks Pgp-mediated [125I]IAAP Transport from the Cells-- It is conceivable that stabilization of the Pgp-[125I]IAAP complex prior to the translocation step would result in inhibition of transport and accumulation of [125I]IAAP molecules inside the cells. This would lead to two distinct [125I]IAAP pools in the cell, one bound to Pgp in a complex and the other a free [125I]IAAP pool. To experimentally distinguish between the two populations, NIHMDR1 cells were loaded with [125I]IAAP in the presence of modulators, and the kinetics of [125I]IAAP release from the cells in a drug-free medium was determined. Cells loaded in presence of cyclosporin A showed a one-phase exponential release of [125I]IAAP with a rate constant (k) of 0.21 ± 0.0027/min (t1/2 = 3.2 min) (R2 = 0.986) (Fig. 3A). Inclusion of 5 µM cyclosporin A in the efflux medium had no effect on the rate of [125I]IAAP release (0.189 ± 0.028/min) (t1/2 = 3.6 min) (R2 = 0.981), indicating no involvement of Pgp in the process. Under the same experimental conditions, a similar one-phase kinetics of [125I]IAAP release was also observed in NIH3T3 cells (data not shown), which has no detectable level of Pgp expression, further suggesting that the single phase kinetics represented passive diffusion of the accumulated drug analog across the lipid bilayer. In contrast, [125I]IAAP release from cells loaded in the presence of 5 µM cis-(Z)-flupentixol was clearly biphasic (Fig. 3B). The initial phase was more rapid with a rate constant (k) of 0.18 ± 0.028/min (t1/2 = 3.8 min) (R2 = 0.99) comparable with [125I]IAAP release from NIH3T3 cells or from the cyclosporin A-treated NIHMDR1 cells, and likely represented transmembrane diffusion of [125I]IAAP molecules accumulated inside the cells. In contrast, the second phase of [125I]IAAP release was considerably (10-fold) slower with a rate constant k of 0.0158 ± 0.0011/min (t1/2 = 43.6 min) (R2 = 0.99). Inclusion of 5 µM cis-(Z)-flupentixol in the efflux medium selectively reduced the rate of release during the second phase to a considerable extent (k = 0.0058 ± 0.0003/min, t1/2 = 118 min) (R2 = 0.989) with a negligible effect on the initial phase (k = 0.131 ± 0.021/min, t1/2 = 5.2 min) (R2 = 0.989). When similar experiments were carried out in NIH3T3 cells, [125I]IAAP release from the cells followed a single exponential kinetics, irrespective of inclusion of cis-(Z)-flupentixol in the efflux medium (data not shown). This suggested that the second phase of [125I]IAAP release from cis-(Z)-flupentixol-treated NIHMDR1 cells likely reflected dissociation of [125I]IAAP molecules from a Pgp-[125I]IAAP complex.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   A and B, extrusion of [125I]IAAP from NIHMDR1 cells. NIHMDR1 cells were incubated with 1.5 nM [125I]IAAP in DMEM either in the presence of 5 µM cyclosporin A ( and black-square) (A) or 5 µM cis-(Z)-flupentixol (open circle  and ) (B) for 60 min. Following incubation, cells were transferred to DMEM without [125I]IAAP either in the absence ( and open circle ) or in the presence of 5 µM cyclosporin A (black-square) or 5 µM cis-(Z)-flupentixol (). At the indicated time points the amount of [125I]IAAP associated with the cells was measured and expressed as pmol of [125I]IAAP retained per million cells. Values are average of three independent experiments. C, status of Pgp-[125I]IAAP complex during [125I]IAAP efflux from cells. NIHMDR1 cells were preincubated with 1.5 nM [125I]IAAP and 5 µM cis-(Z)-flupentixol for 60 min prior to transferring into DMEM containing 5 µM cis-(Z)-flupentixol and no [125I]IAAP. After 0, 5, and 90 min of incubation, cells were exposed to UV light, lysed, resolved by SDS-PAGE, and exposed to an x-ray film (upper panel). The amount of [125I]IAAP photocross-linked to Pgp was quantified using a PhosphorImager and expressed as percentage of the amount incorporated at 0 min (lower panel). Values represent an average of three independent experiments. The presence of an equal amount of Pgp in all three samples was confirmed by immunoblot analysis using polyclonal antibody PEPG13 (middle panel).

To determine definitely the nature of the two [125I]IAAP pools, the status of the Pgp-[125I]IAAP complex during [125I]IAAP release was studied by briefly exposing the cells to UV irradiation at three different time points. A reduction of only 5% was observed in Pgp-bound [125I]IAAP at the end of the initial phase (5 min) (Fig. 3C, autoradiogram), during which almost 20% of the total [125I]IAAP molecules associated with NIHMDR1 cells was released to the extracellular medium. This suggested that most of the [125I]IAAP released during the initial rapid phase was not associated with Pgp but instead remained free inside the cell. These molecules, not in complex with Pgp, most likely accumulated because of inhibition of outward transport. On the other hand, at 90 min, the amount of photocross-linked [125I]IAAP was reduced to 58% compared the initial level (at 0 min) (Fig. 3C, autoradiogram). This closely matched the percentage of [125I]IAAP molecules released from the cells (58%) during the same period. Immunoblotting with Pgp-specific polyclonal antibody PEPG13 of the same samples showed equal amounts of Pgp present in each lane (Fig. 3C, immunoblot). This result strongly suggested that the initial phase of [125I]IAAP release from the cis-(Z)-flupentixol-treated NIHMDR1 cells represented release of free [125I]IAAP accumulated inside the cells due to inhibition of outward transport, whereas the latter phase reflected dissociation of [125I]IAAP from its complex with Pgp.

A Single Amino Acid Substitution That Alters the Inhibitory Potential of cis-(Z)-Flupentixol Also Affects cis-(Z)-Flupentixol-induced Formation of Pgp-[125I]IAAP Complex-- To determine the functional significance of the Pgp-[125I]IAAP complex formation and the elevated level of [125I]IAAP associated with Pgp-expressing cells in the presence of cis-(Z)-flupentixol, both phenomena were tested in the Pgp mutant F983A, which is largely insensitive to modulation by cis-(Z)-flupentixol (22). The intracellular accumulation of [125I]IAAP in cells expressing either the wild-type Pgp (NIHMDR1-WT) or the mutant F983A Pgp (NIHMDR1-F983A) was considerably lower than that of the control NIH3T3 cells, suggesting efficient transport of [125I]IAAP by both the wild-type and F983A mutant Pgp. However, the elevated level of cellular [125I]IAAP association, in the presence of 5 µM cis-(Z)-flupentixol, was selectively abrogated in NIHMDR1-F983A (Fig. 4A). Photocross-linking for 5 min at the end of the uptake assay showed no increase in interaction (photocross-linking) between [125I]IAAP and F983A by cis-(Z)-flupentixol, although under the same conditions increased complex formation was observed between wild-type Pgp and [125I]IAAP in NIHMDR1-WT cells (Fig. 4B, upper panel). In the absence of cis-(Z)-flupentixol, there was no significant difference in the interaction with [125I]IAAP between the wild-type and the F983A mutant. Cyclosporin A (5 µM), a competitive inhibitor of Pgp, inhibited both transport and [125I]IAAP binding by wild-type and F983A with similar efficiency (Fig. 4B, upper panel). Similar results were obtained from experiments with membranes isolated from NIHMDR1-WT and NIHMDR1-F983A cells (Fig. 4B, lower panel).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   A, [125I]IAAP association with NIHMDR1WT and NIHMDR1F983A cells. NIH3T3, NIHMDR1-WT (wild-type Pgp), and NIHMDR1-F983A (mutant F983A) cells were incubated with 1.5 nM [125I]IAAP in the presence and absence of 5 µM cis-(Z)-flupentixol for 60 min at 37 °C. Cells were washed, and intracellular accumulation of [125I]IAAP was measured in a scintillation counter. Data were expressed as picomoles of [125I]IAAP accumulated per million cells. Data points represent average value of three identical experiments. B, interaction of [125I]IAAP with wild-type (WT) Pgp and F983A, in intact cells (upper panel) and in isolated membranes (lower panel). For the experiment with intact cells (upper panel), NIHMDR1WT and NIHMDR1F983A cells in monolayer were incubated with IMEM containing 1.5 nM [125I]IAAP in the presence or absence (None) of 5 µM cyclosporin A (CsA) and 5 µM cis-(Z)-flupentixol (Cis(Z)) for 60 min at 37 °C. After incubation, cells were exposed to UV irradiation, lysed, and subjected to SDS-PAGE. Alternatively, membranes (lower panel) were isolated from NIHMDR1WT and NIHMDR1F983A cells and incubated with 1.5 nM [125I]IAAP for 10 min at room temperature with or without (None) preincubation for 3 min in the presence of 5 µM cyclosporin A (CsA) or 5 µM cis-(Z)-flupentixol (Cis(Z)). Photocross-linked samples were resolved by SDS-PAGE. [125I]IAAP photocross-linked to Pgp was identified by exposing the gels to an x-ray film.

cis-(Z)-Flupentixol Induces a Conformational Change in Pgp That Is Distinct from the Change Induced by Substrates and Competitive Inhibitors-- Substrates and competitive inhibitors induce a conformational change in Pgp, which causes an increased reactivity to UIC2, a monoclonal antibody specific for a conformation-sensitive Pgp external epitope (32). A similar increase in reactivity was observed with Pgp-expressing NIHMDR1 cells in the presence of the competitive inhibitor cyclosporin A (32) (Fig. 5A) or the Pgp substrate vinblastine (data not shown), suggesting a conformational change in the protein-enhancing accessibility of the UIC2 epitope. Interestingly, cis-(Z)-flupentixol induced a clear decrease in the level of UIC2 binding to NIHMDR1 cells (Fig. 5A) indicating a conformational change distinct from that induced by the competitive inhibitor cyclosporin A. 1 mM sodium orthovanadate induced a more profound reduction in UIC2 binding (Fig. 5A). Because vanadate inhibits Pgp-mediated drug transport by trapping Pgp in a transition-state conformation without physically competing for the substrate site, the result confirmed that reduced binding of UIC2 represented a mechanism of Pgp modulation that was distinct from competitive inhibition. To investigate the mechanistic significance of this conformational change, UIC2 reactivity was studied in cells expressing the Pgp mutant F983A, which has impaired sensitivity to inhibition by cis-(Z)-flupentixol (Fig. 5B). cis-(Z)-Flupentixol was unable to induce any negative effect on UIC2 binding to NIHMDR1-F983A cells (Fig. 5B), suggesting that inhibition of transport and the conformational change induced by cis-(Z)-flupentixol could be mechanistically related. Because increased or decreased UIC2 reactivity induced by cyclosporin A or vanadate, respectively, was unaffected in NIHMDR1-F983A (Fig. 5B), compared with that observed in NIHMDR1-WT cells, the possibility of a nonspecific effect could be ruled out.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5.   A, monoclonal antibody UIC2 reactivity of Pgp in NIHMDR1 cells. NIHMDR1 cells expressing Pgp were incubated with monoclonal antibody UIC2 in the presence and absence (filled histogram) of 5 µM cyclosporin A (thin line), 5 µM cis-(Z)-flupentixol (thick line), or 1 mM sodium orthovanadate (dotted line) as described under "Experimental Procedures." Cells were then stained with a FITC-conjugated secondary antibody and subjected to FACS analysis. The fluorescence intensity (x axis) is plotted against cell counts (y axis). B, UIC2 reactivity of Pgp mutant F983A. UIC2 binding to NIHMDR1-WT and NIHMDR1-F983A cells was carried out as mentioned above either in the presence of 1 mM sodium orthovanadate (Vi), 5 µM cis-(Z)-flupentixol (Cis(Z)), 5 µM cyclosporin A (CsA), or in the absence of any modulators. Values were expressed as mean fluorescence and represent average of two independent experiments.

cis-(Z)-Flupentixol Is Not a Transport Substrate for Pgp-- Competitive inhibitors such as cyclosporin A or verapamil are high affinity substrates of Pgp. When NIH3T3 and NIHMDR1 cells were incubated with cis-(Z)-[3H]flupentixol, no difference in intracellular accumulation of the radioactive compound was observed between the two cell types (Fig. 6A), suggesting no Pgp-mediated outward transport of the modulator from the cells. Inclusion of the competitive inhibitor cyclosporin A did not have any effect (Fig. 6A), further suggesting lack of cis-(Z)-[3H]flupentixol transport by Pgp. Under similar experimental conditions, the steady-state level of [3H]cyclosporin A accumulation in NIHMDR1 cells was 6-fold lower than that of the NIH3T3 cells (Fig. 6B). This low level of [3H]cyclosporin accumulation in NIHMDR1 cells was effectively increased to that of the control NIH3T3 cells by inclusion of 5 µM rapamycin, a Pgp modulator, in the assay (Fig. 6B). No comparable increase in [3H]cyclosporin accumulation was observed in NIH3T3 cells (Fig. 6B), demonstrating that cyclosporin A extrusion from NIHMDR1 cells was Pgp-dependent.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of Pgp on intracellular accumulation of cis-(Z)-[3H]flupentixol (A) and [3H]cyclosporin A (B). NIH3T3 ( and black-square) and NIHMDR1 (open circle  and ) cells, in monolayers, were incubated either with 0.5 µM cis-(Z)-[3H]flupentixol (A) or with 0.1 µM [3H]cyclosporin A (B) for indicated times. Cells were washed, and radioactivity associated with the cells was determined in a scintillation counter. Values are expressed as nanomoles of radioactive drug accumulated per million cells. A, cells were incubated either in the presence () or in the absence (open circle ) of 5 µM cyclosporin A; B cells were incubated in the presence (black-square) and absence () of 5 µM rapamycin. Values are average of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An allosteric mode of action has been proposed for a number of Pgp modulators (16-20). However, the exact mechanism by which these compounds inhibit Pgp-mediated drug transport is not yet fully understood. Knowledge about the mode of allosteric modulation will benefit rational design of Pgp inhibitors with higher efficacy. In the absence of high resolution structural information about Pgp, such studies rely heavily on biochemical and molecular biology approaches. In this study, we demonstrate that cis-(Z)-flupentixol, a thioxanthene derivative, allosterically inhibits Pgp-mediated drug transport by blocking translocation of substrate molecules and their subsequent dissociation from the transporter, without affecting the initial recognition of the substrate.

cis-(Z)-Flupentixol and its structurally related analogs are potent inhibitors of Pgp-mediated drug transport (26-28). A number of these thioxanthene derivatives do not physically compete for the substrate-binding site of Pgp (21, 23), but instead they seem to interact at a distinct (allosteric) site within the protein (22), indicating an allosteric mode of action. However, the molecular events leading to inactivation of the pump remained unresolved. To determine experimentally the mechanism of action of these modulators, we developed a cell-based assay that allows independent but simultaneous monitoring of substrate binding and substrate translocation by Pgp, without the need for changing experimental conditions. Because Pgp recruits substrate molecules directly from the lipid bilayer (33, 34), the integrity and polarity of the plasma membrane were kept unperturbed using intact cells instead of inside-out membrane vesicles. The drug-sensitive NIH3T3 cells and the drug-resistant NIHMDR1 cells (NIH3T3 cells transfected with human MDR1 cDNA) provided a good signal to noise ratio in both drug extrusion and drug binding assays. The use of the photoaffinity analog of prazosin ([125I]IAAP) as a substrate in the assay provided two distinct advantages. The high specific activity of the 125I group in [125I]IAAP allowed convenient detection of the molecule, whereas the photoactivable-N3 group provided an efficient means to covalently immobilize the drug analog to its site of interaction, whenever required. Therefore, assessing both substrate recognition and substrate transport by Pgp could be monitored using the same experimental set up.

Although the photoaffinity analog [125I]IAAP has been extensively used for studying substrate-binding properties of Pgp (35, 36), no prior report has demonstrated Pgp-mediated transport of the molecule. In this study, we demonstrate that [125I]IAAP is transported by Pgp out of the cells affecting a 2-fold reduction in its intracellular steady-state level (Fig. 1A). Photocross-linking of [125I]IAAP with Pgp demonstrated a transient association between the two during transport (Fig. 1B). Cyclosporin A, which competes for the Pgp substrate-binding site (21), and vanadate, which traps Pgp in a conformation with reduced affinity for substrates (21, 30), independently blocked both recognition as well as transport of 125I]IAAP by Pgp (Fig. 1). On the other hand, metabolic starvation that depletes cells of intracellular ATP inhibited transport (Fig. 2A) but not [125I]IAAP recognition (Fig. 2B) by Pgp. Because ATP (hydrolysis) is specifically required for the translocation step but not for substrate recognition, the results are in clear agreement with the proposed mechanism of action of Pgp. Therefore, the assay proves to be a reliable tool for studying the mode of action of Pgp modulators with unknown mechanisms of action.

By using this assay, we observed that the thioxanthene derivative cis-(Z)-flupentixol inhibits Pgp-mediated drug transport by a distinct mechanism. Unlike most other Pgp modulators, cis-(Z)-flupentixol did not compete for substrate binding or translocation, but instead induced formation of a stable but reversible complex between Pgp and its substrate, [125I]IAAP. Because Pgp is present in the plasma membrane of the cells, formation of this complex resulted in an additional build up of [125I]IAAP molecules in Pgp-expressing cells (Fig. 1A). Sequestration of these [125I]IAAP molecules by Pgp led to a 2-3-fold higher cellular association than the steady-state level achieved by simple inhibition of Pgp-mediated outward transport. Consistent with this, photocross-linking of Pgp-bound [125I]IAAP revealed an 80-fold increase in direct interaction between cellular Pgp and [125I]IAAP in the presence of cis-(Z)-flupentixol (Fig. 1B). Although the association between Pgp and [125I]IAAP was stabilized by cis-(Z)-flupentixol, the phenomenon was reversible, because removal of the cells to a drug-free medium resulted in dissociation of the complex (Fig. 3, B and C). The fact that cis-(Z)-flupentixol neither stimulated Pgp-[125I]IAAP photocross-linking nor induced any build up of excess [125I]IAAP in the cells, when the substrate-site of Pgp was blocked by cyclosporin A or made poorly accessible by vanadate trapping (data not shown), suggested a direct involvement of the Pgp substrate site in the complex formation.

ATP-dependent transport by Pgp involves three major steps as follows: 1) substrate recognition, 2) substrate translocation (coupled to ATP hydrolysis), and 3) substrate dissociation. It is conceivable that interference with any of these three steps may lead to inhibition of transport. Depletion of cellular ATP specifically inhibited the translocation step without affecting substrate recognition by Pgp (Fig. 2), substantiating that ATP hydrolysis is required for substrate translocation and not for substrate binding. Interestingly, ATP depletion had no effect on stabilization of the Pgp-[125I]IAAP complex by cis-(Z)-flupentixol (Fig. 2). Because substrate translocation by Pgp has an absolute requirement for ATP (binding/hydrolysis), this result clearly indicates that cis-(Z)-flupentixol-induced stabilization of the Pgp-[125I]IAAP complex occurs prior to the translocation step. It is likely that stabilization of the Pgp-substrate association preceding the translocation step stalls further progression of the catalytic cycle and could be the key event in inhibition by cis-(Z)-flupentixol of Pgp-mediated transport. According to this model, there should be two distinct pools of [125I]IAAP molecules in the cis-(Z)-flupentixol-inhibited cells as follows: one bound to Pgp molecules in the plasma membrane as Pgp-[125I]IAAP complex, and the other as free intracellular [125I]IAAP accumulated due to inhibition of transport. When [125I]IAAP release from NIHMDR1 cells was determined, biphasic kinetics did in fact indicate the existence of two distinct [125I]IAAP pools in cis-(Z)-flupentixol-treated cells (Fig. 3B). Consistent with that, a rapid initial phase, which had a rate constant comparable with that of the single phase [125I]IAAP release from NIH3T3 cells (data not shown) or from the cyclosporin A-inhibited NIHMDR1 cells (Fig. 3A), represented transmembrane diffusion of the free [125I]IAAP molecules accumulated due to inhibition of transport. In contrast, the second slower phase reflected the dissociation of [125I]IAAP from the Pgp-[125I]IAAP complexes in cells, because inclusion of cis-(Z)-flupentixol in the medium reduced the rate of [125I]IAAP release selectively during the latter phase (Fig. 3B). A direct investigation of the status of the Pgp-[125I]IAAP interaction, by photocross-linking during the biphasic release of the [125I]IAAP molecules from cis-(Z)-flupentixol-treated cells, demonstrated that dissociation of the Pgp-[125I]IAAP complex mainly occurred during the latter phase (Fig. 3C). Interestingly, formation of the stable Pgp-[125I]IAAP complex induced by cis-(Z)-flupentixol was remarkably affected in the Pgp mutant F983A (Fig. 4B), drug transport function by which is not inhibited by cis-(Z)-flupentixol (Fig. 4A). This emphasizes the mechanistic significance of the stable complex formation in inhibition of transport by cis-(Z)-flupentixol. Because the mutation F983A did not affect [125I]IAAP transport or the ability of cyclosporin A to block the substrate site (Fig. 4B) and inhibit transport (Fig. 4A), any global effect of the mutation can be ruled out.

Recently, by using bivalent cross-linking agents and cysteine-scanning mutagenesis, Loo and Clarke (4, 37) have mapped the substrate-binding site of Pgp. The proposed model shows the relative disposition of the putative transmembrane helices of Pgp and their contribution in constituting the substrate-binding site. Interestingly, residue Phe-983, which is necessary for cis-(Z)-flupentixol interaction with Pgp, maps well outside the substrate interaction site (4, 37). This is in clear agreement with our experimental data showing no physical competition for the substrate site by cis-(Z)-flupentixol (Fig. 1B) and no transport of cis-(Z)-flupentixol by Pgp (Fig. 6A). However, the spatial distinctness of the putative interaction site for cis-(Z)-flupentixol indicated a requirement for communication between the allosteric modulator site and the substrate translocating pathway. Communication of this nature often requires conformational changes in the protein. By using the monoclonal antibody UIC2, which is specific to a conformation-sensitive extracellular epitope of Pgp, we demonstrate that cis-(Z)-flupentixol induces a remarkable change in Pgp conformation, which is distinct from the changes induced by Pgp substrates (data not shown) or competitive inhibitors (Fig. 5A). No such conformational change was induced by cis-(Z)-flupentixol in the Pgp mutant F983A, whereas the ability of the competitive inhibitor cyclosporin A to induce its characteristic change in conformation remained unaltered (Fig. 5B), underscoring the functional distinctness of the allosteric site.

Allosteric regulations of ion channels and carrier proteins often play important functional roles in regulating ion fluxes across cellular membranes, and these have important implications in cell signaling and neuronal transmission (38). Although allosteric modulation of ligand-gated channels is well documented, allosteric modulation of ABC transporters is a novel and emerging concept. The basic structural plan of ABC transporters includes two hydrophobic transmembrane domains and two cytosolic nucleotide-binding moieties per functional unit (39). According to the most widely accepted model, the transmembrane regions associate with each other to form a single drug translocating pathway across the lipid bilayer, whereas the two nucleotide-binding sites constitute the catalytic domains that hydrolyze ATP to provide the driving force for the translocation step (1). The coupling of ATP hydrolysis to vectorial translocation of the drug substrate requires precise communication between the two domains. Stimulation of ATP hydrolysis by substrate binding to Pgp (6) and occlusion of the drug-binding site upon ATP hydrolysis (21, 30) suggested direct communication between the two domains via conformational changes (40-42). In this study, we demonstrate the presence of an allosteric modulator site within the Pgp capable of communicating with the substrate-binding/translocating site evidently through conformational change(s). Studying the details of the conformational change will reveal important information on the molecular events responsible for this communication. On the other hand, mapping the amino acid residues constituting the modulator site and the structural moieties of the modulator in contact with the site are likely to provide valuable clues for designing more efficient Pgp modulators. These avenues are currently being explored in our laboratory.

    ACKNOWLEDGEMENTS

We thank Dr. Michael Gottesman for the encouragement and support. We also thank Dr. Barry Rosen, Dr. Teresa Dunn, and Dr. Todd Martinsen for their critical assessment of the manuscript and Dr. Wilfred Stein for helpful discussions.

    FOOTNOTES

* This work was supported by Uniformed Services University of the Health Sciences Intramural Grant C071FU.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.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. Tel.: 301-295-3449; Fax: 301-295-3512; E-mail: sdey@usuhs.mil.

Published, JBC Papers in Press, March 17, 2003, DOI 10.1074/jbc.M210413200

    ABBREVIATIONS

The abbreviations used are: Pgp, P-glycoprotein; IAAP, iodoarylazidoprazosin; TM, transmembrane; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; FACS, fluorescence-assisted cell sorter; IMDM, Iscove's modified Dulbecco's medium; FITC, fluorescein isothiocyanate; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427[CrossRef][Medline] [Order article via Infotrieve]
2. Ambudkar, S. V., Dey, S., Hrycyna, C. A., Ramachandra, M., Pastan, I., and Gottesman, M. M. (1999) Annu. Rev. Pharmacol. Toxicol. 39, 361-398[CrossRef][Medline] [Order article via Infotrieve]
3. 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[Medline] [Order article via Infotrieve]
4. Loo, T. W., and Clarke, D. M. (2001) J. Biol. Chem. 276, 14972-14979[Abstract/Free Full Text]
5. Azzaria, M., Schurr, E., and Gros, P. (1989) Mol. Cell. Biol. 9, 5289-5297[Medline] [Order article via Infotrieve]
6. Ambudkar, S. V., Lelong, I. H., Zhang, J. P., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8472-8476[Abstract]
7. Tan, B., Piwnica-Worms, D., and Ratner, L. (2000) Curr. Opin. Oncol. 12, 450-458[CrossRef][Medline] [Order article via Infotrieve]
8. Murren, J. R. (2002) Clin. Cancer Res. 8, 633-635[Free Full Text]
9. Sikic, B. I. (1999) Oncology (Huntingt.) 13, 183-187[Medline] [Order article via Infotrieve]
10. Tsuruo, T., Iida, H., Tsukagoshi, S., and Sakurai, Y. (1981) Cancer Res. 41, 1967-1972[Abstract]
11. Twentyman, P. R., Reeve, J. G., Koch, G., and Wright, K. A. (1990) Br. J. Cancer 62, 89-95[Medline] [Order article via Infotrieve]
12. Spoelstra, E. C., Westerhoff, H. V., Pinedo, H. M., Dekker, H., and Lankelma, J. (1994) Eur. J. Biochem. 221, 363-373[Abstract]
13. Saeki, T., Ueda, K., Tanigawara, Y., Hori, R., and Komano, T. (1993) J. Biol. Chem. 268, 6077-6080[Abstract/Free Full Text]
14. Tsuji, A., Tamai, I., Sakata, A., Tenda, Y., and Terasaki, T. (1993) Biochem. Pharmacol. 46, 1096-1099[CrossRef][Medline] [Order article via Infotrieve]
15. Didier, A., Wenger, J., and Loor, F. (1995) Anti-Cancer Drugs 6, 669-680[Medline] [Order article via Infotrieve]
16. Martin, C., Berridge, G., Mistry, P., Higgins, C., Charlton, P., and Callaghan, R. (1999) Br. J. Pharmacol. 128, 403-411[Abstract/Free Full Text]
17. Martin, C., Berridge, G., Higgins, C. F., and Callaghan, R. (1997) Br. J. Pharmacol. 122, 765-771[Abstract]
18. Boer, R., Dichtl, M., Borchers, C., Ulrich, W. R., Marecek, J. F., Prestwich, G. D., Glossmann, H., and Striessnig, J. (1996) Biochemistry 35, 1387-1396[CrossRef][Medline] [Order article via Infotrieve]
19. Ferry, D. R., Malkhandi, P. J., Russell, M. A., and Kerr, D. J. (1995) Biochem. Pharmacol. 49, 1851-1861[CrossRef][Medline] [Order article via Infotrieve]
20. Martin, C., Berridge, G., Higgins, C. F., Mistry, P., Charlton, P., and Callaghan, R. (2000) Mol. Pharmacol. 58, 624-632[Abstract/Free Full Text]
21. Dey, S., Ramachandra, M., Pastan, I., Gottesman, M. M., and Ambudkar, S. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10594-10599[Abstract/Free Full Text]
22. Dey, S., Hafkemeyer, P., Pastan, I., and Gottesman, M. M. (1999) Biochemistry 38, 6630-6639[CrossRef][Medline] [Order article via Infotrieve]
23. Safa, A. R., Agresti, M., Bryk, D., and Tamai, I. (1994) Biochemistry 33, 256-265[Medline] [Order article via Infotrieve]
24. Metz, M. Z., Best, D. M., and Kane, S. E. (1995) Virology 208, 634-643[CrossRef][Medline] [Order article via Infotrieve]
25. Hafkemeyer, P., Dey, S., Ambudkar, S. V., Hrycyna, C. A., Pastan, I., and Gottesman, M. M. (1998) Biochemistry 37, 16400-16409[CrossRef][Medline] [Order article via Infotrieve]
26. Ford, J. M., Prozialeck, W. C., and Hait, W. N. (1989) Mol. Pharmacol. 35, 105-115[Abstract]
27. Ford, J. M., Bruggemann, E. P., Pastan, I., Gottesman, M. M., and Hait, W. N. (1990) Cancer Res. 6, 1748-1756
28. Ford, J. M., and Hait, W. N. (1993) Cytotechnology 12, 171-212[Medline] [Order article via Infotrieve]
29. Ayesh, S., Shao, Y. M., and Stein, W. D. (1996) Biochim. Biophys. Acta 1316, 8-18[Medline] [Order article via Infotrieve]
30. 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]
31. Ambudkar, S. V., Cardarelli, C. O., Pashinsky, I., and Stein, W. D. (1997) J. Biol. Chem. 272, 21260-21266[Abstract/Free Full Text]
32. Mechetner, E. B., Schott, B., Morse, B. S., Stein, W. D., Druley, T., Davis, K. A., Tsuruo, T., and Roninson, I. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12908-12913[Abstract/Free Full Text]
33. Raviv, Y., Pollard, H. B., Bruggemann, E. P., Pastan, I., and Gottesman, M. M. (1990) J. Biol. Chem. 265, 3975-3980[Abstract/Free Full Text]
34. Stein, W. D., Cardarelli, C., Pastan, I., and Gottesman, M. M. (1994) Mol. Pharmacol. 45, 763-772[Abstract]
35. Greenberger, L. M., Yang, C. P., Gindin, E., and Horwitz, S. B. (1990) J. Biol. Chem. 265, 4394-4401[Abstract/Free Full Text]
36. Greenberger, L. M. (1993) J. Biol. Chem. 268, 11417-11425[Abstract/Free Full Text]
37. Loo, T. W., and Clarke, D. M. (2000) J. Biol. Chem. 275, 39272-39278[Abstract/Free Full Text]
38. Christopoulos, A. (2002) Nat. Rev. Drug Discov. 1, 198-210[CrossRef][Medline] [Order article via Infotrieve]
39. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113[CrossRef][Medline] [Order article via Infotrieve]
40. Liu, R., and Sharom, F. J. (1996) Biochemistry 35, 11865-11873[CrossRef][Medline] [Order article via Infotrieve]
41. Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989[Abstract/Free Full Text]
42. Sonveaux, N., Shapiro, A. B., Goormaghtigh, E., Ling, V., and Ruysschaert, J. M. (1996) J. Biol. Chem. 271, 24617-24624[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.