Effects of putative catalytic base mutation E211Q on ABCG2-mediated methotrexate transport.
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.
Hou
Yue-xian
YX
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, USA.
Li
Chang-Zhong
CZ
Palaniyandi
Kanagaraj
K
Magtibay
Paul M
PM
Homolya
Laszlo
L
Sarkadi
Balazs
B
Chang
Xiu-bao
XB
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008727:Methotrexate / Q000493:pharmacokinetics*; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000964:Antimetabolites, Antineoplastic; D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008727:Methotrexate
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