Functional Multidrug Resistance Protein (MRP1) Lacking the N-terminal Transmembrane Domain*

Abstract

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.