Functional and Structural Consequences of Cysteine Substitutions in the NH2 Proximal Region of the Human Multidrug Resistance Protein 1 (MRP1/ABCC1)
详细信息 查看全文
- 作者：Elaine M. Leslie ; Isabelle J. Lé ; tourneau ; Roger G. Deeley ; and Susan P. C. Cole
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 13, 2003
- 年：2003
- 卷：42
- 期：18
- 页码：5214 - 5224
- 全文大小：597K
- 年卷期：v.42,no.18(May 13, 2003)
- ISSN：1520-4995
文摘
The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membranespanning 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 oncolyticagents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugatedorganic anions, including leukotriene C4 and 17
-estradiol 17-(-D-glucuronide) and certain xenobioticsin 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 residuesat positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Alaand Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organicanion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteinswere also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organicanions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substitutedCys43 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 indicatethat certain Cys residues in the NH2 proximal region of MRP1 can be important for its structure andselected transport activities.
-estradiol 17-(-D-glucuronide) and certain xenobioticsin 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 residuesat positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Alaand Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organicanion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteinswere also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organicanions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substitutedCys43 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 indicatethat certain Cys residues in the NH2 proximal region of MRP1 can be important for its structure andselected transport activities.