ABCMdb

PMID: 10978330

Ryu S, Kawabe T, Nada S, Yamaguchi A
Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2.
J Biol Chem. 2000 Dec 15;275(50):39617-24., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC2 p.Lys578Ala ABCC2 p.Arg1257Ala ABCC2 p.Arg1210Ala ABCC2 p.Lys324Ala ABCC2 p.Lys483Ala Four mutants, K324A (TM6), K483A (TM9), R1210A (TM16), and R1257A (TM17), showed decreased transport activity, and another mutant, K578A (TM11), showed decreased protein expression. Login to comment 7 ABCC2 p.Arg1230Ala Another observation that MRP2 inhibitor, cyclosporine A, failed to inhibit R1230A specifically, indicated the existence of its binding site within TM16. Login to comment 41 ABCC2 p.Ile448Val For mutagenesis, we constructed pCI-neo/ MRP2X, which contains a deletion of XhoI site in the multicloning site of pCI-neo, and pCI-neo/MRP2V, which contains I448V and L653V mutations by overlapping polymerase chain reaction mutagenesis method to create Sse8387I and MluI sites, respectively. Login to comment 42 ABCC2 p.Lys578Ala ABCC2 p.Lys329Ala ABCC2 p.Lys316Ala ABCC2 p.Lys324Ala ABCC2 p.His439Ala ABCC2 p.Lys483Ala ABCC2 p.Arg590Ala Site-directed Mutagenesis-The seven mutants of MRP2, K316A, K324A, K329A, H439A, K483A, K578A, and R590A, were generated by the overlapping polymerase chain reaction method. Login to comment 43 ABCC2 p.Arg1230Ala ABCC2 p.His1042Ala ABCC2 p.Arg1257Ala ABCC2 p.Arg1100Ala ABCC2 p.Arg1210Ala ABCC2 p.Arg1023Ala The other six mutants, R1023A, H1042A, R1100A, R1210A, R1230A, and R1257A, were generated by the method of Kunkel (30). Login to comment 96 ABCC2 p.Lys578Ala Except for K578A, all of the mutants were expressed at nearly the same level when compared with wild type MRP2. Login to comment 112 ABCC2 p.Lys578Ala The experiments Only the expression of K578A was faint in both immunoblot and immunocytochemistry. Login to comment 113 ABCC2 p.Ile448Val GS-MF Excretion of Mutant MRP2 Proteins-First of all, we examined the excretion of GS-MF from COS-7 cells transfected with pCI-neo/MRP2V, which contains conservative amino acid changes as I448V and L653V, or pCI-neo/MRP2X, which contains a small deletion at the multi-cloning site of the vector. Login to comment 117 ABCC2 p.Lys324Ala ABCC2 p.Lys483Ala In K324A and K483A mutants, the excretion of GS-MF decreased about 40% compared with MRP2V. Login to comment 118 ABCC2 p.Lys578Ala As to K578A mutant, GS-MF excretion decreased to the level of control (Fig. 8); however, expression level of this mutant also decreased (Figs. 5 and 6). Login to comment 120 ABCC2 p.Arg1257Ala ABCC2 p.Arg1210Ala In R1210A and R1257A mutants, the excretion of GS-MF decreased approximately to the level of control, but expression level was comparable with MRP2X (Figs. 5 and 6). Login to comment 121 ABCC2 p.Lys578Ala As shown in Fig. 10, we observed with confocal fluorescence microscopy that these mutant MRP2s including K578A were expressed at the cell surface. Login to comment 122 ABCC2 p.Lys578Ala Therefore, the defect in the transport function of these mutants was clearly due to the functional defect except for K578A. Login to comment 123 ABCC2 p.Lys578Ala The decrease in transport activity of K578A was mainly due to the decrease in the expression level. Login to comment 126 ABCC2 p.Arg1230Ala In R1230A mutant, the MRP2-mediated transport activity was not inhibited by CsA, whereas the other active mutants were suppressed on their transport activity up to 50% (Fig. 11). Login to comment 144 ABCC2 p.Ile448Val pCI-neo/MRP2V contains mutations of I448V and L653V, which were located in TM8 and NBD1, respectively. In patients with Dubin-Johnson syndrome, loss of function mutations was occasionally found in NBD1 (12). Login to comment 147 ABCC2 p.Lys578Ala Except for a mutant K578A, the protein expression level of these mutants in COS-7 cell was comparable with that of wild type (Figs. 5 and 6). Login to comment 148 ABCC2 p.Lys578Ala ABCC2 p.Arg1257Ala ABCC2 p.Arg1210Ala Among these mutants, K578A, R1210A, and R1257A were the most influenced by mutation concerning substrate translocating activity FIG. 6. Login to comment 161 ABCC2 p.Lys578Ala As to K578A, the excretion of GS-MF and protein expression was coincidentally lowered (Figs. 5, 6, and 8). Login to comment 163 ABCC2 p.Lys324Ala ABCC2 p.Lys483Ala In K324A and K483A mutants, GS-MF excretion also moderately decreased (Fig. 8). Login to comment 184 ABCC2 p.Arg1230Ala In most active mutants excretion of GS-MF was inhibited by CsA as well as wild type MRP2, whereas in R1230A mutant excretion of GS-MF was not influenced (Fig. 11). Login to comment