Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site

One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 microM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites. The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane.     

Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet

The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.     

Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs

The P-glycoprotein efflux pump, an ABC superfamily member, can export a wide variety of hydrophobic drugs, natural products, and peptides from cells, powered by the energy of ATP hydrolysis. Transport substrates appear to first partition into the membrane and then interact with the protein within the cytoplasmic leaflet. Two drug binding sites within P-glycoprotein have been described which interact allosterically, the H-site (binds Hoechst 33342) and the R-site (binds rhodamine 123); however, the structural and functional relationship between the various binding sites appears complex. In this work, we have used fluorescence spectroscopic approaches to characterize the interaction of the transporter with LDS-751 and rhodamine 123, both of which are believed to bind to the putative R-site based on functional transport studies. By carrying out single and sequential dual fluorescence titrations of purified P-glycoprotein with the two substrates, we observed that bound LDS-751 interacted with bound rhodamine 123. Rhodamine 123 and LDS-751 showed a reciprocal negative interaction, each reducing the binding affinity of the other by 5-fold, indicating that the two compounds were simultaneously bound to the protein to form a ternary complex. Fitting of the dependence of the apparent Kd for LDS-751 binding on rhodamine 123 concentration suggested that the two compounds interacted noncompetitively. We conclude that the two-site drug binding model for P-glycoprotein requires modification. The putative R-site appears large enough to accommodate two compounds simultaneously. The locations where LDS-751 and rhodamine 123 bind are likely adjacent to each other, possibly overlapping, and may be within a hydrophobic pocket.     

Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.     

The secondary multidrug transporter LmrP contains multiple drug interaction sites.

The secondary multidrug transporter LmrP of Lactococcus lactis mediates the efflux of Hoechst 33342 from the cytoplasmic leaflet of the membrane. Kinetic analysis of Hoechst 33342 transport in inside-out membrane vesicles of L. lactis showed that the LmrP-mediated H(+)/Hoechst 33342 antiport reaction obeyed Michaelis-Menten kinetics, with a low apparent affinity constant of 0.63 microM Hoechst 33342 (= 0.5 mmol Hoechst 33342/mol phospholipid). Several drugs significantly inhibited LmrP-mediated Hoechst 33342 transport through a direct interaction with the protein rather than through dissipation of the proton motive force or reduction of the membrane partitioning of Hoechst 33342. The characterization of the mechanism of inhibition of LmrP-mediated Hoechst 33342 transport indicated competitive inhibition by quinine and verapamil, noncompetitive inhibition by nicardipin and vinblastin, and uncompetitive inhibition by TPP(+). The three types of inhibition of LmrP-mediated Hoechst 33342 transport in inside-out membrane vesicles indicate for the first time the presence of multiple drug interaction sites in a secondary multidrug transporter.     

Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence

The ATP-dependent transport enzyme known as P-glycoprotein (P-gp) confers multidrug resistance (MDR) against many unrelated drugs and xenobiotics. To understand better the broad substrate specificity of the enzyme as well as the mechanism of substrate transport out of the cell, it is critical to characterize the substrate binding sites. Since approximately 1 ATP is hydrolyzed per transport event, phosphate release rate provides a steady-state kinetics assay. Notably, the substrate H33342 causes a decrease in the baseline hydrolysis of ATP (probably due to competition for transport with an endogenous membrane lipid substrate) providing an excellent tool for a comprehensive graphical kinetic analysis of the interaction of substrate pairs at the transport site(s) allowing the determination of inhibition type and hence characterization of transport binding sites. The substrate H33342 interacted with quinidine, progesterone, and propranolol in a non-competitive manner, indicating that binding of H33342 precludes active transport of these other substrates at a distinct site. Compounds such as TPP+ and verapamil, and perhaps also nicardipine, interacted with H33342 as mixed-type inhibitors. This type of interaction results from a reduced affinity at the opposing active site by a factor of alpha and sometimes a partial activity of a fraction beta. Indeed, H33342 binding caused a roughly four-fold reduced affinity for TPP+. Using this definitive approach to inhibition kinetics, we were able to establish traits of a second transport site in P-gp. Therefore, the sites are unequal; however, the performance at one site is contingent on the other being unoccupied, and transport is also sometimes mitigated when the other site is occupied.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism

The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.     

Transition state P-glycoprotein binds drugs and modulators with unchanged affinity,suggesting a concerted transport mechanism

The P-glycoprotein multidrug transporter is a plasma membrane efflux pump for hydrophobic natural products, drugs, and peptides, driven by ATP hydrolysis. Determination of the details of the catalytic cycle of P-glycoprotein is critical if we are to understand the mechanism of drug transport and design ways to inhibit it. It has been proposed that the vanadate-trapped transition state of P-glycoprotein (Pgp x ADP x V(i) x M(2+), where M(2+) is a divalent metal ion) has a very low affinity for drugs compared to resting state protein, thus leading to binding of substrate on the cytoplasmic side of the membrane and release of substrate to the extracellular medium (or the extracellular membrane leaflet). We have used several different fluorescence spectroscopic approaches to show that isolated purified P-glycoprotein, when trapped in a stable transition state with vanadate and either Co(2+)or Mg(2+), binds drugs with high affinity. For vinblastine, colchicine, rhodamine 123, and doxorubicin, the affinity of the vanadate-trapped transition state for drugs was only very slightly (less than 2-fold) lower than the binding affinity of resting state Pgp, whereas for the modulators cyclosporin A and verapamil and the substrate Hoechst 33342, the binding affinity was very similar for the two states. The drug binding affinity of the ADP-bound form of the transporter was also comparable to that of the unoccupied transporter. These results suggest that release of drug from the transporter during the catalytic cycle precedes formation of the transition state.     

Functional expression of a multidrug P-glycoprotein transporter of Leishmania

P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Baculovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-beta-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line overexpressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents.     

Interaction of transported drugs with the lipid bilayer and P-glycoprotein through a solvation exchange mechanism

Broad substrate specificity of human P-glycoprotein (ABCB1) is an essential feature of multidrug resistance. Transport substrates of P-glycoprotein are mostly hydrophobic and many of them have net positive charge. These compounds partition into the membrane. Utilizing the energy of ATP hydrolysis, P-glycoprotein is thought to take up substrates from the cytoplasmic leaflet of the plasma membrane and to transport them to the outside of the cell. We examined this model by molecular dynamics simulation of the lipid bilayer, in the presence of transport substrates together with an atomic resolution structural model of P-glycoprotein. Taken together with previous electron paramagnetic resonance studies, the results suggest that most transported drugs are concentrated near the surface zone of the inner leaflet of the plasma membrane. Here the drugs can easily diffuse laterally into the drug-binding site of P-glycoprotein through an open cleft. It was concluded that the initial high-affinity drug-binding site was located in the interfacial surface area of P-glycoprotein in contact with the membrane interface. Based on these results and our recent kinetic studies, a “solvation exchange” drug transport mechanism of P-glycoprotein is discussed. A molecular basis for the improved colchicine transport efficiency by the much-studied colchicine-resistance G185V mutant human P-glycoprotein is also provided.     

New Insights into the Drug Binding,Transport and Lipid Flippase Activities of the P-Glycoprotein Multidrug Transporter

The MDR1 P-glycoprotein, an ATP-binding cassette (ABC) superfamily member that functions as an ATP-driven drug efflux pump, has been linked to resistance of human tumors to multiple chemotherapeutic agents. P-glycoprotein binds and actively transports a large variety of hydrophobic drugs and peptides. P-glycoprotein in reconstituted proteoliposomes is also an outwardly directed flippase for membrane phospholipids and simple glycosphinglipids. This review focuses on recent advances in our understanding of P-glycoprotein structure and function, particularly through the use of fluorescence spectroscopic approaches. Progress is being made towards understanding the structure of the transporter, especially the spatial relationship between the two nucleotide-binding domains. Exploration of the P-glycoprotein catalytic cycle using vanadate-trapped complexes has revealed that drug transport likely takes place by concerted conformational changes linked to relaxation of a high energy intermediate. Low resolution mapping of the protein using fluorescence resonance energy transfer showed that both the H and R drug-binding sites are located within the cytoplasmic leaflet. Two drugs can bind to the R-site simultaneously, suggesting that the protein contains a large flexible binding region.     

哺乳动物精子质量的评价方法

精子的各种功能状态反应了精子的受精能力。检测精子质膜完整性的荧光探针有SYBR－14,SYTO－17,CFDA、CDMFDA、CAM、PI、Hoechst33258、Hoechst33342,其中以SYBR－14结合PI使用效果最好,检测线粒体活性的荧光探针有JC－1、MITO、Rh123,JC-1比MITO和Rh123更适用于检测精子线粒体功能,检测顶体状态的荧光探针有PNA－FITC、PSA－FITC、LYSO－G及CTC等。检测获能状态的荧光探针有CTC。此外,还可以通过检测精子与透明带的结合能力、精子穿入去透明带卵子的能力以及使卵子受精的能力和其后胚胎的发育能力等方面来评价精子的功能状态。     

P-glycoprotein (ABCB1) interacts directly with lipid-based anti-cancer drugs and platelet-activating factors.

The P-glycoprotein multidrug transporter (Pgp; ABCB1) is an ATP-binding cassette (ABC) protein that has been implicated in the multidrug resistance of human cancers. Pgp couples ATP hydrolysis to active extrusion from the cell of a broad array of amphipathic compounds via an ill-defined mechanism. Substrates are believed to interact with Pgp within the membrane. Reconstituted Pgp functions as an ATP-dependent flippase for a variety of fluorescently labelled membrane lipids. The protein may also function as a drug 'flippase', moving its substrates from the inner to the outer leaflet of the bilayer. We show that lipid-based anti-cancer drugs, such as miltefosine, and signaling molecules, such as platelet-activating factors, bind saturably to Pgp with Kd values in the low micromolar range, and modulate its ATPase activity. These compounds also inhibit Pgp-mediated flipping of fluorescent lipids and transport of Hoechst 33342 and tetramethylrosamine, which occupy different subsites in the drug-binding pocket. Bacterial lipid A modulates Pgp ATPase activity, and glycolipid flipping is inhibited by unlabelled glucosylceramide, suggesting that these lipids also interact with the transporter. These results indicate that Pgp treats a variety of lipid-based molecules as substrates, and likely interacts with lipids and drugs in the same manner.     

Highly efficient over-production in E. coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis

ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.     

P-glycoprotein possesses a 1,4-dihydropyridine-selective drug acceptor site which is alloserically coupled to a vinca-alkaloid-selective binding site.

[3H]Vinblastine bound with high affinity to surface membranes prepared from H69/LX4 cells which express P-glycoprotein (P-gp) and as a consequence are multidrug resistant (MDR). The KD was 9.8 +/- 1.5 nM and density of sites 31.2 +/- 8.6 pmol/mg of protein. [3H]Vinblastine binding was inhibited by cytotoxics and agents known to reverse MDR. 1,4-Dihydropyridine MDR reversing agents including nicardipine and nifedipine accelerated the dissociation of [3H]vinblastine from P-gp indicating a negative heterotropic allosteric effect. Cyclosporin A, vincristine and actinomycin D did not alter [3H]vinblastine dissociation kinetics. It is concluded that P-gp possesses at least two allosterically coupled drug acceptor sites, receptor site-1 that is selective for vinca alkaloids and cyclosporin A, and receptor site-2 that is selective for 1,4-dihydropyridines.     

Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site

The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied.     

Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.     

Photoaffinity probes for the alpha 1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein 2-[4-(4-azido-3-[125I]iodobenzoyl) piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline [( 125I]iodoaryl azidoprazosin). [125I] Iodoaryl azidoprazosin, which classically has been used to identify the alpha 1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine greater than actinomycin D greater than doxorubicin greater than colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.     

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.