Abstract
Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C4, 17β-estradiol 17-β-d-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.
Footnotes
This work was supported by the Canadian Institutes of Health Research [Grant MOP-97877] (to R.G.D.).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS:
- MRP
- multidrug resistance protein
- ABC
- ATP-binding cassette
- CFTR
- cystic fibrosis transmembrane conductance regulator
- SUR
- sulfonylurea receptor
- CL3
- cytoplasmic loop 3
- ΔMRP1
- MRP1204–1531
- MSD
- membrane-spanning domain
- TM
- transmembrane
- PM
- plasma membrane
- cysless
- cysteineless
- LTC4
- leukotriene C4
- NBD
- nucleotide binding domain
- HEK
- human embryonic kidney
- ER
- endoplasmic reticulum
- wt
- wild type
- E217βG
- 17β-estradiol 17-β-d-glucuronide
- E13SO4
- estrone-3-sulfate
- aa
- amino acids
- CFP
- cyan fluorescent protein
- mAb
- monoclonal antibody
- PNGase F
- peptide:N-glycosidase F
- Endo H
- endoglycosidase H
- PAGE
- polyacrylamide gel electrophoresis.
- Received January 30, 2012.
- Accepted April 16, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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