Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2

Henriksen, Ulla; Gether, Ulrik; Litman, Thomas

doi:10.1242/jcs.01729

Cited by 60 publications

(57 citation statements)

References 35 publications

(55 reference statements)

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“…We found that Ala substitution of Pro 485 in TM3 significantly reduced efflux of BODIPY-prazosin by 70%, but had no effect on efflux of mitoxantrone and Hoechst 33342 (94 (96,98). However, another study showed that the activity loss caused by mutations of Lys 86 was possibly due to altered subcellular localization and cell surface targeting of BCRP (97). More information about mutations and their effects on function and expression of BCRP can be found in an open access database (http://abcmutations.hegelab.org/).…”

Section: Mutagenesis Analysismentioning

confidence: 79%

Role of the Breast Cancer Resistance Protein (BCRP/ABCG2) in Drug Transport—an Update

Mao

Unadkat

2014

AAPS J

507

387

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Abstract. The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is an ATP-binding cassette (ABC) efflux transporter. It was so named because it was initially cloned from a multidrugresistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents such as mitoxantrone and topotecan. Since its discovery in 1998, the substrates of BCRP have been rapidly expanding to include not only therapeutic agents but also physiological substances such as estrone-3-sulfate, 17β-estradiol 17-(β-D-glucuronide) and uric acid. Likewise, at least hundreds of BCRP inhibitors have been identified. Among normal human tissues, BCRP is highly expressed on the apical membranes of the placental syncytiotrophoblasts, the intestinal epithelium, the liver hepatocytes, the endothelial cells of brain microvessels, and the renal proximal tubular cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds as well as tissue protection against xenobiotic exposure. As a result, BCRP has now been recognized by the FDA to be one of the key drug transporters involved in clinically relevant drug disposition. We published a highly-accessed review article on BCRP in 2005, and much progress has been made since then. In this review, we provide an update of current knowledge on basic biochemistry and pharmacological functions of BCRP as well as its relevance to drug resistance and drug disposition.

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Section: Mutagenesis Analysismentioning

confidence: 79%

Role of the Breast Cancer Resistance Protein (BCRP/ABCG2) in Drug Transport—an Update

Mao

Unadkat

2014

AAPS J

507

387

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“…However, the impact is much less than observed with K86M, a mutation in Walker A that has been reported to retain dimerization with a functional dominant negative effect. (35) Evaluating the effect of a charged residue at the 553 site, we replaced the glycine with glutamic acid (G553E) and transfected HEK 293 cells. In case of the Drosophila white protein, the corresponding, X-ray induced G589E mutation was described as presumably interfering with heterodimerization (22).…”

Section: Resultsmentioning

confidence: 99%

Mutational Studies of G553 in TM5 of ABCG2: A Residue Potentially Involved in Dimerization

et al. 2006

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ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeutic agents such as mitoxantrone, irinotecan, and flavopiridol. With its one transmembrane and one ATP-binding domain, ABCG2 is thought to homodimerize for function. One conserved region potentially involved in dimerization is a 3-amino acid sequence in transmembrane segment 5 (residues 552-554). Mutations in the corresponding residues in the Drosophila white protein (an orthologue of ABCG2) are thought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. The mutant was not detectable on the cell surface and markedly reduced protein expression levels were observed by immunoblot. A deficiency in N-linked glycosylation was suggested by reduction in molecular weight compared to the 72-kDa wild type ABCG2. Similar results were observed with the G553E mutant. Confocal microscopy demonstrated mostly ER localization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild type protein. Despite its altered localization, the G553L and G553E mutants were cross-linked using amine-reactive cross-linkers with multiple arm lengths, suggesting that the monomers are in close proximity but unable to complete normal trafficking. Interestingly, when expressed in Sf9 insect cells, G553L traffics to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, when coexpressed, the mutant interferes with the Hoechst transport activity of the wild type protein. These data show that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove, its involvement in ABCG2 homodimerization.The ATP-binding cassette 1 (ABC) transporter family, with 48 known human members classified in seven subgroups, constitutes one of the largest families of membrane transporters present in all species (1). These proteins are involved in the energy-dependent transport of a † This research was supported [in part] by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.*To whom correspondence should be addressed. Tel: (301) 402-1357, Fax: (301) wide variety of substrates and play an important role in numerous genetic disorders (2), a wellcharacterized example of which is cystic fibrosis, caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) protein (3). ABC transporters are also thought be responsible for the so called multidrug resistance phenomenon in antimicrobial (4) and anticancer (5) therapy; the most extensively studied example of the latter is resistance attributed to P-glycoprotein (P-gp/ABCB1)(6). ABCG2, also called BCRP/MXR/ABCP, is a member of the G subfamily of human ABC transporters, and has been demonstrated to confer resistance to multiple cancer chemotherapeutic agents, such as mitoxantrone, flavopiridol, methotrexate, and the camptothecin derivatives SN-38 and topotecan.(7-10) In addition to a potential role in c...

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“…homodimers through disulfide bond mediated by cysteine 603; however, such covalent intermolecular link does not appear to be prerequisite for BCRP to exert its transport activity (25,26). It remains to be investigated how phosphorylation of Thr-362 affects the intermolecular interaction between BCRP dimers or multimers.…”

Section: Discussionmentioning

confidence: 99%

The 44-kDa Pim-1 Kinase Phosphorylates BCRP/ABCG2 and Thereby Promotes Its Multimerization and Drug-resistant Activity in Human Prostate Cancer Cells

Xie¹,

Xu²,

Linn³

et al. 2008

Journal of Biological Chemistry

174

165

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We previously showed that the 44-kDa serine/threonine kinase Pim-1 (Pim-1L) can protect prostate cancer cells from apoptosis induced by chemotherapeutic drugs (Xie, Y., Xu, K., Dai, B., Guo, Z., Jiang, T., Chen, H., and Qiu, Y. (2006) Oncogene 25, 70 -78). To further explore the mechanisms of Pim-1L-mediated resistance to chemotherapeutic drugs in prostate cancer cells, we employed a yeast two-hybrid screening to identify cellular proteins that were associated with Pim-1L, and we found the ABC transporter BCRP/ABCG2 as one of the potential interacting partners of Pim-1L. We also showed that the expression level of Pim-1L and BCRP was up-regulated in mitoxantrone and docetaxel-resistant prostate cancer cell lines. Pim-1L was co-localized with BCRP on the plasma membrane and induced phosphorylation of BCRP at threonine 362. Knocking-down Pim-1L expression in the drug-resistant prostate cancer cells abolished multimer formation of endogenous BCRP and resensitized the resistant cells to chemotherapeutic drugs suggesting that BCRP phosphorylation induced by Pim-1L was essential for its functionality. This is further corroborated by our finding that the plasma membrane localization and drug-resistant activity of BCRP were compromised by T362A mutation. Our data suggest that Pim-1L may protect prostate cancer cells from apoptosis, at least in part, through regulation of transmembrane drug efflux pump. These findings may provide a potential therapeutic approach by disrupting Pim-1 signaling to reverse BCRP-mediated multidrug resistance.

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Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2

Cited by 60 publications

References 35 publications

Role of the Breast Cancer Resistance Protein (BCRP/ABCG2) in Drug Transport—an Update

Role of the Breast Cancer Resistance Protein (BCRP/ABCG2) in Drug Transport—an Update

Mutational Studies of G553 in TM5 of ABCG2: A Residue Potentially Involved in Dimerization

The 44-kDa Pim-1 Kinase Phosphorylates BCRP/ABCG2 and Thereby Promotes Its Multimerization and Drug-resistant Activity in Human Prostate Cancer Cells

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