Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1.
Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.
Nagata
K
K
Laboratory of Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan.
Nishitani
M
M
Matsuo
M
M
Kioka
N
N
Amachi
T
T
Ueda
K
K
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D001386:Azides / Q000493:pharmacokinetics / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005047:Etoposide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014357:Trypsin; D014638:Vanadates / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D001386:Azides; D005047:Etoposide; D005978:Glutathione; D019803:Glutathione Disulfide; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; D014357:Trypsin; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate