Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1).
The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.
Leslie
Elaine M
EM
Department of Pharmacology & Toxicology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Létourneau
Isabelle J
IJ
Deeley
Roger G
RG
Cole
Susan P C
SP
D018053:Arsenites / Q000494:pharmacology; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D017997:Leukotriene C4 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014750:Vincristine / Q000494:pharmacology
D018053:Arsenites; D003545:Cysteine; D049452:Green Fluorescent Proteins; D017997:Leukotriene C4; D008164:Luminescent Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011993:Recombinant Fusion Proteins; D014750:Vincristine; C421109:multidrug resistance-associated protein 1