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

doi:10.1074/jbc.273.48.32167

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

From the ^‡Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences and ^§National Institute of Haematology and Immunology, Research Group of the Hungarian Academy of Sciences, H-1113 Budapest, Hungary and ^‖The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

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.

Footnotes

↵* This work has been supported by Dutch Cancer Society Research Grant NKI 98-1794 (to P. B.), an NWO-OTKA grant (to P. B. and B. S.), and in Budapest by grants from COST, OMFB, OTKA (F23662, T22072, T17602, T6348), FKFP, and ETT.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ These authors contributed equally to this work.
↵** A recipient of a Howard Hughes International Scholarship. To whom correspondence should be addressed: National Institute of Haematology and Immunology, 1113 Budapest, Daróczi u. 24, Hungary. Tel./Fax: 36-1-372-4353; E-mail: B.Sarkadi{at}ohvi.hu.
↵2 R. Evers and P. Borst, unpublished data.
↵3 M. Kool, J. Wijnholds, and P. Borst, unpublished data.
Abbreviations:

MDR1 Pgp

human multidrug transporter P-glycoprotein

ABC

ATP binding cassette

DNP-GS

dinitrophenyl S-glutathione

GSH

reduced glutathione

GSSG

oxidized glutathione

ECL

enhanced chemiluminescence

LTC₄

leukotriene C₄

MDCK

Madin-Darby canine kidney

cMOAT

canalicular multispecific organic anion transporter

mAb

monoclonal antibody

MRP

human multidrug resistance (-associated) protein

NEM-GS

N-ethylmaleimideS-glutathione

Sf9 cells

Spodoptera frugiperda ovarian cells

TMD

transmembrane domain

MOPS

4-morpholinepropanesulfonic acid.
- Received August 10, 1998.
- Revision received September 8, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.