Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.     

Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.     

PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter

The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.     

High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells

Expression of multidrug resistance ABC transporters has been suggested as a functional marker and chemoprotective element in early human progenitor cell types. In this study we examined the expression and function of the key multidrug-ABC transporters, ABCB1, ABCC1 and ABCG2 in two human embryonic stem (HuES) cell lines. We detected a high level ABCG2 expression in the undifferentiated HuES cells, while the expression of this protein significantly decreased during early cell differentiation. ABCG2 in HuES cells provided protection against mitoxantrone toxicity, with a drug-stimulated overexpression of the transporter. No significant expression of ABCB1/ABCC1 was found either in the undifferentiated or partially differentiated HuES cells. Examination of the ABCG2 mRNA in HuES cells indicated the use of selected promoter sites and a truncated 3' untranslated region, suggesting a functionally distinct regulation of this transporter in undifferentiated stem cells. The selective expression of the ABCG2 multidrug transporter indicates that ABCG2 can be applied as a marker for undifferentiated HuES cells. Moreover, protection of embryonic stem cells against xenobiotics and endobiotics may depend on ABCG2 expression and regulation.     

Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers

Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.     

N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane

The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa plasma membrane protein that can confer multidrug resistance to cells in culture when overexpressed. Both transiently and stably expressed ABCG2 are glycosylated, and treatment with peptide N-glycosidase F reduces the apparent molecular mass on SDS-PAGE gels to approximately 60 kDa. Sequence analysis revealed three potential N-linked glycosylation sites in human ABCG2 at amino acids 418, 557, and 596. Site-directed mutagenesis experiments, in which each Asn was changed to Gln independently, revealed that only asparagine 596 is N-linked glycosylated. These data provide the first direct identification of the modified residue in ABCG2 and evidence for the localization of loop 5 to the extracellular space, previously only predicted from hydropathy analysis. Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 (N596Q) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives. Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein. The ABCG2 (N596Q) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2. Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated ( approximately 2-fold) ATPase activities of ABCG2 (N596Q) were affected compared to glycosylated ABCG2. Although subtle defects in transporter trafficking and function may exist, these data taken together suggest that N-glycosylation at arginine 596 is not essential for the expression, trafficking to the plasma membrane, or the overall function of ABCG2.     

Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells.

ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer. Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated membrane preparations. ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein. ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N-ethylmaleimide and cyclosporin A. Verapamil had no effect, while Fumitremorgin C, reversing ABCG2-dependent cancer drug resistance, strongly inhibited this ATPase activity. The functional expression of ABCG2 in this heterologous system indicates that no additional partner protein is required for the activity of this multidrug transporter, probably working as a homodimer. We suggest that the Sf9 cell membrane ATPase system is an efficient tool for examining the interactions of ABCG2 with pharmacological agents.     

ABCG transporters: structure, substrate specificities and physiological roles

The ATP-binding cassette (ABC) transporter superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides. The G subfamily of ABC transporters consists of half-transporters, which oligomerise to form the functional transporter. While ABCG1, ABCG4 and ABCG5/8 are involved in the ATP-dependent translocation of steroids and, possibly, other lipids, ABCG2 (also termed the breast cancer resistance protein) has been identified as a multidrug transporter that confers resistance on tumor cells. Evidence will be summarized suggesting that ABCG2 can also mediate the binding/transport of non-drug substrates, including free and conjugated steroids. The characterization of the substrate specificities of ABCG proteins at a molecular level might provide further clues about their potential physiological role(s), and create new opportunities for the modulation of their activities in relation to human disease.     

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.     

乳腺癌耐药蛋白的研究进展

乳腺癌耐药相关蛋白(BCRP/ABCG2)是新近发现的ATP结合盒(Adenosine triphosphate-binding cassette,ABC)膜转运蛋白超家族成员。它作为细胞膜上的药物排出泵,可以将一系列细胞毒药物转运至胞外从而介导肿瘤细胞多药耐药。在很多血液肿瘤和实体瘤中均检测到ABCG2表达。ABCG2在肿瘤的多药耐药上发挥重要作用。本文对ABCG2的发现、基因表达特征、与造血干细胞分化的关系、转运底物及其耐药逆转和临床意义等方面的研究进展进行综述。     

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.     

Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

Background

BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.

Aim

The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes.

Methods and Results

Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.

Conclusions

Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.     

Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface

The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.     

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.     

Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the anti-estrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [3H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.     

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.     

Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2

ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.     

半分子转运蛋白ABCG2的肿瘤多药耐药性研究及其应用

肿瘤常对临床上传统使用的多种化学治疗显示其内源性或获得性的药物耐受性即多药耐药性(multidrug resistance,MDR)．这种多药耐药性主要是由一类称为ABC(ATP-binding cassette)转运体蛋白超家族的跨膜蛋白引起的,它们结合并利用水解ATP提供的能量来转运药物,导致肿瘤细胞呈现抗药性．半分子转运蛋白ABCG2是近年来才发现的可归于ABC转运体大家族中的一个新成员,在肠、肝、胎盘和血脑屏障等部位大量表达,与全分子转运蛋白如P-gp (P-glycoprotein)和多药耐药蛋白(multi-drug resistance protein,MRP)相似,可以主动地把具有不同化学结构和作用于细胞内不同靶位点的化疗药物泵出胞外,从而引起肿瘤对多种抗癌药物(包括最新开发的药物)产生抗性．最近的一些十分有趣的研究还表明,ABCG2可能与干细胞分化状态和保护干细胞发育过程中免受周围环境的影响有关,而且还发现,它在侧群骨髓和神经干细胞内大量存在,因此,ABCG2可能在基因治疗中作为选择性的蛋白质标记正受到研究者们的极大关注．同时,ABCG2的单核苷酸多态性影响其结合并转运不同的底物/药物．鉴于ABCG2在肿瘤抗药性研究中的重要性以及它的一些新功能的初步研究表明,对ABCG2的生物学功能和作用机理以及在医学实践中的应用研究必将受到更大的关注．主要阐述了半分子ABC转运蛋白ABCG2的发现、重要的生化特性和生理功能及其相关的新研究进展和问题．