Effects of putative catalytic base mutation E211Q on ABCG2-mediated methotrexate transport

Yue Xian Hou, Chang Zhong Li, Kanagaraj Palaniyandi, Paul Magtibay, Laszlo Homolya, Balazs Sarkadi, Xiu-Bao D Chang

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Abstract

ABCG2 is a half-ATP binding cassette (ABC) drug transporter that consists of a nucleotide binding domain (NBD) followed by a transmembrane domain. This half-ABC transporter is thought to form a homodimer in the plasma membrane where it transports anticancer drugs across the biological membranes in an ATP-dependent manner. Substitution of the putative catalytic residue E211 with a nonacidic amino acid glutamine (E211Q) completely abolished its ATPase activity and ATP-dependent methotrexate transport, suggesting that ATP hydrolysis is required for the ATP-dependent solute transport. However, whether one ATP hydrolysis or two ATP hydrolyses in the homodimer of ABCG2 with the NBD·ATP·ATP·NBD sandwich structure is/are required for the ATP-dependent solute transport is not known yet. To address this question, we have made an YFP/ABCG2 fusion protein and expressed this 99 kDa fusion protein alone or along with the 70 kDa E211Q-mutated ABCG2 in BHK cells. Although membrane vesicles prepared from BHK cells expressing YFP/ABCG2 exert higher ATPase activity than that of wt ABCG2, the dATP-dependent methotrexate transport activities of these two proteins are the same. Interestingly, membrane vesicles prepared from BHK cells expressing both YFP/ABCG2 and E211Q-mutated ABCG2 (with a ratio of 1:1) form homodimers and heterodimer and exert 55% of wt ABCG2 ATPase activity that can be further enhanced by anticancer drugs, suggesting that the wt NBD in the heterodimer of YFP/ABCG2 and E211Q may be able to hydrolyze ATP. Furthermore, the membrane vesicles containing both YFP/ABCG2 and E211Q exert ∼79% of wt ABCG2-mediated methotrexate transport activity, implying that the heterodimer harboring YFP/ABCG2 and E211Q may be able to transport the anticancer drug methotrexate across the biological membranes.

Original languageEnglish (US)
Pages (from-to)9122-9131
Number of pages10
JournalBiochemistry
Volume48
Issue number38
DOIs
StatePublished - 2009

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Hou, Y. X., Li, C. Z., Palaniyandi, K., Magtibay, P., Homolya, L., Sarkadi, B., & Chang, X-B. D. (2009). Effects of putative catalytic base mutation E211Q on ABCG2-mediated methotrexate transport. Biochemistry, 48(38), 9122-9131. https://doi.org/10.1021/bi900675v