Effects of ATP depletion and phosphate analogues on P-glycoprotein conformation in live cells.

P-glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes conformational changes in the presence of substrates/modulators, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2-shift was elicited by certain modulators (e.g. cyclosporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E. & Szabó Jr, G. (2001) Eur. J. Biochem. 268, 2416-2420]. To further study the relationship between UIC2-shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro-aluminate) that trap nucleotides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp-expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in response to direct modulation of the ATP-binding site, confirming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.     

Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity

The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus alpha-toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UIC2 reactivity; this effect was potentiated by vanadate, a nucleotide-trapping agent. The Hill number for the nucleotide-induced conformational transition was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly diminished the overall effect of nucleotides. Vinblastine reversed the decrease in the UIC2 reactivity brought about by all the nucleotides, including nonhydrolyzable analogues; this effect of vinblastine was blocked by vanadate. These data indicate that UIC2-detectable conformational changes of Pgp are driven by binding and debinding of nucleotides, that nucleotide hydrolysis affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are consistent with a conventional E1/E2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.     

P-Glycoprotein conformational changes detected by antibody competition.

Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.     

Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody

The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the “UIC2 shift”. Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r = 0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.     

P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation

The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine induces a conformational change in Pgp, evidenced by increased reactivity with a conformation-sensitive monoclonal antibody UIC2, in all the tested cell lines. In contrast, colchicine increases the UIC2 reactivity in only some of the cell lines. In those lines where colchicine alone did not affect UIC2 reactivity, this drug was, however, able to reverse the vinblastine-induced increase in UIC2 reactivity. The magnitude of the increase in UIC2 reactivity in the presence of saturating concentrations of colchicine correlates with the relative ability of Pgp to confer colchicine resistance in different cell lines, suggesting the existence of some cell-specific factors that have a coordinate effect on the ability of colchicine to induce conformational transitions and to be transported by Pgp. Colchicine, like vinblastine, reverses the decrease in UIC2 reactivity produced by nonhydrolyzable nucleotides, but unlike vinblastine, it does not reverse the effect of ATP at a high concentration. Colchicine, however, decreases the Hill number for the effect of ATP on the UIC2 reactivity from 2 to 1. Colchicine increases the UIC2 reactivity and reverses the effect of ATP in ATPase-deficient Pgp mutants, but not in the wild-type Pgp expressed in the same cellular background, suggesting that ATP hydrolysis counteracts the effects of colchicine on the Pgp conformation.     

The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells in vitro almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).     

Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein

The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.     

Allosteric modulation of human P-glycoprotein. Inhibition of transport by preventing substrate translocation and dissociation

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 ([(125)I]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.     

Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates

The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.     

(+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone, a derivative of (+/-)-praeruptorin A, reverses P-glycoprotein mediated multidrug resistance in cancer cells

P-glycoprotein (Pgp) is an ATP-driven membrane exporter for a broad spectrum of hydrophobic xenobiotics. Pgp-overexpression is a common cause of multidrug resistance (MDR) in cancer cells and could lead to chemotherapeutic failure. Through an extensive herbal drug screening program we previously showed that (+/-)-praeruptorin A (PA), a naturally existing pyranocumarin isolated from the dried root of Peucedanum praeruptorum Dunn., re-sensitizes Pgp-mediated MDR (Pgp-MDR) cancer cells to cancer drugs. A number of PA derivatives were synthesized and one of these, (+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone (DCK), was more potent than PA or verapamil in the reversal of Pgp-MDR. In Pgp-MDR cells DCK increased cellular accumulation of doxorubicin without affecting the expression level of Pgp. In Pgp-enriched membrane fractions DCK moderately stimulated basal Pgp-ATPase activity, suggesting some transport substrate-like function. However, DCK also inhibited Pgp-ATPase activity stimulated by the standard substrates verapamil or progesterone with decreased V(max)s but K(m)s were relatively unchanged, suggesting a primarily non-competitive mode of inhibition. While the binding of substrates to active Pgp would increase the reactivity of the Pgp-specific antibody UIC2, DCK decreased UIC2 reactivity. These results suggest that DCK could bind simultaneously with substrates to Pgp but perhaps at an allosteric site and thus affect Pgp-substrate interactions.     

Methoxylation of 3',4'-aromatic side chains improves P-glycoprotein inhibitory and multidrug resistance reversal activities of 7,8-pyranocoumarin against cancer cells

The overexpression of P-glycoprotein (Pgp), an ATP-driven membrane exporter of hydrophobic xenobiotics, is one of the major causes of multidrug resistance (MDR) in cancer cells. Through extensive screening we have found that the extracts of Peucedanum praeruptorum Dunn. and one of the major components (+/-)-praeruptorin A (PA) may reverse Pgp-mediated multidrug resistance. Studies on novel PA derivatives have shown that (+/-)-3'-O,4'-O-dicinnamoyl-cis-khellactone (DCK) is more active than PA or verapamil and is a non-competitive inhibitor of Pgp. Here, we report that methoxylation of the cinnamoyl groups on DCK may further enhance its bioactivity. The structure-activity relationship is demonstrated by comparing two new pyranocoumarins (+/-)-3'-O,4'-O-bis(3,4-dimethoxycinnamoyl)-cis-khellactone (DMDCK) and (+/-)-3'-O,4'-O-bis(4-methoxycinnamoyl)-cis-khellactone (MMDCK). While the co-existence of 3- and 4-methoxy groups on cinnamoyl remarkably enhanced the Pgp-inhibitory activity, the lone existence of the 4-methoxy group on cinnamoyl reduced the activity. Contrary to DCK, DMDCK promoted the binding of UIC2 antibody to Pgp which signifies a conformational change of Pgp similar to that induced by transport substrates. While DCK moderately stimulated the basal Pgp-ATPase activity, DMDCK inhibited the activity. A pharmacophore search with verapamil-based template revealed that four functional groups of DMDCK could be simultaneously involved in interaction with Pgp whereas for DCK or MMDCK only three groups were involved. It is speculated that the additional 3-methoxy group on cinnamoyl allows DMDCK to interact more efficiently with Pgp substrate site(s). If DMDCK was tightly bind to Pgp substrate site(s) the complexes could be inactive with regard to transportation and ATP hydrolysis could also be inhibited.     

Targeting of multidrug-resistant human ovarian carcinoma cells with anti-P-glycoprotein antibody conjugates

A monoclonal antibody (mAb) to P-glycoprotein (Pgp), UIC2, is used as a targeting moiety for N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer/drug [(meso chlorin e(6) mono(N-2-aminoethylamide) (Mce(6)) or doxorubicin (DOX)] conjugates to investigate their cytotoxicity towards the Pgp-expressing human ovarian carcinoma cell line A2780/AD. The binding, internalization, and subcellular trafficking of a fluorescein labeled UIC2 targeted HPMA copolymer are studied and show localization to the plasma membrane with limited internalization. The specificity of the UIC2-targeted HPMA copolymer/drug conjugates are confirmed using the sensitive cell line A2780 that does not express Pgp.     

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.     

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein

Dihydro-β-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-β-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-β-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-β-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-β-agarofurans.     

Drug transport by reconstituted P-glycoprotein in proteoliposomes. Effect of substrates and modulators, and dependence on bilayer phase state.

The P-glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a 'hydrophobic vacuum cleaner', and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis--Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mM, close to the K(m) for ATP hydrolysis, and the K(m) for TMR was 0.3 microM. TMR transport was inhibited in a concentration-dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.     

Raft and cytoskeleton associations of an ABC transporter: P-glycoprotein.

BACKGROUND: A novel flow cytometric assay has been described in an accompanying report (Gombos et al., METHODS: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents. RESULTS: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping. CONCLUSIONS: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.     

In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity

While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.     

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.     

2'-Fluoroarabino- and arabinonucleic acid show different conformations, resulting in deviating RNA affinities and processing of their heteroduplexes with RNA by RNase H

2'-Deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA) paired to RNA are substrates of RNase H. The conformation of the natural DNA/RNA hybrid substrates appears to be neither A-form nor B-form. Consistent with this, the conformations of FANA and ANA were found to be intermediate between the A- and B-forms. However, FANA opposite RNA is preferred by RNase H over ANA, and the RNA affinity of FANA considerably exceeds that of ANA. By investigating the conformational boundaries of FANA and ANA residues in crystal structures of A- and B-form DNA duplexes at atomic resolution, we demonstrate that FANA and ANA display subtle conformational differences. The structural data provide insight into the structural requirements at the catalytic site of RNase H. They also allow conclusions with regard to the relative importance of stereoelectronic effects and hydration as modulators of RNA affinity.     

Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [¹²⁵I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.