Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain.
The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.
Bakos
E
E
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1113 Budapest, Hungary.
Evers
R
R
Szakács
G
G
Tusnády
G E
GE
Welker
E
E
Szabó
K
K
de Haas
M
M
van Deemter
L
L
Borst
P
P
Váradi
A
A
Sarkadi
B
B
D000818:Animals; D016367:Baculoviridae; D020137:Base Pair Mismatch; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004285:Dogs; D005978:Glutathione / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000493:pharmacokinetics; D008301:Maleimides / Q000493:pharmacokinetics; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D004268:DNA-Binding Proteins; D005978:Glutathione; D017997:Leukotriene C4; D008301:Maleimides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D011994:Recombinant Proteins; D014638:Vanadates; C078444:MSH3 protein, human; C054449:N-ethylmaleimide-S-glutathione; C421109:multidrug resistance-associated protein 1