Research article summary (published 27 Sep 2009):

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

Full 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 approximately 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.

Author information

Author/s: Hou, Yue-xian (YX); Li, Chang-Zhong (CZ); Palaniyandi, Kanagaraj (K); Magtibay, Paul M (PM); Homolya, Laszlo (L); Sarkadi, Balazs (B); Chang, Xiu-bao (XB);

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, USA.

Grants: CA89078 (Agency:NCI NIH HHS)

Journal and publication information

Publication Type: In Vitro; Journal Article; Research Support, N.I.H., Extramural

Journal: Biochemistry (Biochemistry), published in United States. (Language: eng)

Reference: 2009-Sep; vol 48 (issue 38) : pp 9122-31

Dates: Created 2009/09/22; Completed 2009/10/09; Revised 2009/10/30;

PMID: 19691360, status: MEDLINE (last retrieval date: 11/2/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

ATP-Binding Cassette Transporters - chemistry; genetics; metabolism Adenosine Triphosphatases - chemistry; genetics; metabolism Adenosine Triphosphate - metabolism Amino Acid Substitution Animals Antimetabolites, Antineoplastic - pharmacokinetics Bacterial Proteins - chemistry; genetics; metabolism Base Sequence Biological Transport, Active - genetics Catalytic Domain - genetics Cell Line Cricetinae

DNA Primers - genetics Dimerization Drug Resistance, Multiple Drug Resistance, Neoplasm Humans Kinetics Luminescent Proteins - chemistry; genetics; metabolism Methotrexate - pharmacokinetics Mutagenesis, Site-Directed Neoplasm Proteins - chemistry; genetics; metabolism Protein Structure, Quaternary Recombinant Fusion Proteins - chemistry; genetics; metabolism

Associated Chemicals: ABCG2 protein, human (0) ; ATP-Binding Cassette Transporters (0) ; Antimetabolites, Antineoplastic (0) ; Bacterial Proteins (0) ; DNA Primers (0) ; Luminescent Proteins (0) ; Neoplasm Proteins (0) ; Recombinant Fusion Proteins (0) ; yellow fluorescent protein, Bacteria (0) ; Adenosine Triphosphate (56-65-5) ; Methotrexate (59-05-2) ; Adenosine Triphosphatases (EC 3.6.1.-)

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