Abstract
Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette membrane transport superfamily and is responsible for multidrug resistance in cancer cells. Currently, there are nine known human MRPs. Distinct from many other members of the ATP-binding cassette superfamily, human MRP1 and four other MRPs have an additional membrane-spanning domain (MSD) with a putative extracellular amino terminus. The functional significance of this additional MSD (MSD1) is currently unknown. To understand the role of MSD1 in human MRP1 structure and function, we studied the amino-terminal 33 amino acids. We found that the amino terminus of human MRP1 has two cysteine residues (Cys(7) and Cys(32)) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys(7) residue is critical for the MRP1-mediated drug resistance and leukotriene C(4) transport activity. On the other hand, mutation of Cys(32) reduced only moderately the MRP1 function. The effect of Cys(7) mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate V(max). We also found that mutation of Cys(7) changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in V(max) of LTC(4) transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys(7) residue plays a critical role in maintaining the proper structure and function of human MRP1.
Highlights
Human multidrug resistance-associated protein 1 (MRP1),1 named ABCC1 is a multiple-drug membrane efflux pump that causes multidrug resistance in human cancer chemotherapy [1]
We found that the amino terminus of human Multidrug resistance-associated protein 1 (MRP1) has two cysteine residues (Cys7 and Cys32) that are conserved among the five human MRPs that have MSD1
Expression of Wild Type and Mutant MRP1 in HEK293 Cells —Fig. 1A shows that the two cysteine residues (Cys7 and Cys32) in the amino terminus are conserved among human MRP1, 2, 3, 6, and 7 and mouse mrp1
Summary
Materials—[3H]LTC4 was purchased from PerkinElmer Life Sciences. LTC4, L-1-tosylamido-2-phenyethyl chloromethyl ketone-treated trypsin, peroxidase- and FITC-conjugated goat anti-mouse IgG, peroxidase-conjugated rabbit anti-rat IgG, doxorubicin, colchicine, vincristine, and VP16 (etoposide) were purchased from Sigma. The cells were washed with ice-cold PBS and resuspended in hypotonic lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.4, 1 mM PMSF) at 1 ϫ 106 cells/ml followed by homogenization and centrifugation at 4,000 ϫ g for 10 min. 10-g membrane vesicles were incubated at 23 °C in 120 l of transport buffer (50 mM Tris-HCl, 250 mM sucrose, 0.02% sodium azide, pH 7.4) containing 4 mM ATP or AMP, 10 mM MgCl2, 100 g/ml of creatine kinase, 10 mM creatine phosphate, and 50 nM [3H]LTC4 (0.05 Ci). The cells were probed with primary antibody QCRL-1 (11 g/ml) for 30 min at 4 °C followed by incubation with FITC-conjugated goat anti-mouse IgG F(abЈ) fragment (Sigma) (1:100 dilution) at 4 °C for another 30 min. The cells were resuspended in 1% paraformaldehyde in PBS for 30 min and analyzed by FACS
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