ABCMdb

PMID: 12450376

Ren XQ, Furukawa T, Aoki S, Sumizawa T, Haraguchi M, Nakajima Y, Ikeda R, Kobayashi M, Akiyama S
A positively charged amino acid proximal to the C-terminus of TM17 of MRP1 is indispensable for GSH-dependent binding of substrates and for transport of LTC4.
Biochemistry. 2002 Dec 3;41(48):14132-40., 2002-12-03 [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC1 p.Arg1249Ala Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Login to comment 72 ABCC1 p.Arg1249Ala ABCC1 p.Arg1222Met MRP1 constructs encoding R1222M and R1249A were generated in a PCR using the forward primers 5'-CATG- CACAGCCTCAGTGCTG-3' and 5'-GCGATGTCATCT- GAAATGGAAACC-3', respectively (bold denotes mismatched bases encoding the mutations). Login to comment 73 ABCC1 p.Arg1222Met An EcoRV site (underlined) was created by silent mutation (bold) in the R1222M construct using the reverse primer 5'-GATATC- ACCGCAAACAGGGCAGC-3'. Login to comment 78 ABCC1 p.Arg1222Met The EcoRV-KpnI fragment from the construct encoding R1222M was cloned into the multiple cloning site II of the pFastBac DUAL plasmid between the SmaI and KpnI sites. Login to comment 165 ABCC2 p.Arg1257Ala The R1257A (TM17) mutant of human MRP2 showed reduced transport activity of GSH-bound meth- ylfluorescein (33). Login to comment 168 ABCC1 p.Arg1249Ala To investigate the role of charged amino acids in GSH-dependent photolabeling of azido AG-A to MRP1, we replaced the arginine at position 1249 which is proximal to the C-terminus of TM17 of MRP1 with Ala (R1249A). Login to comment 169 ABCC1 p.Arg1249Ala ABCC1 p.Arg1222Met Arginine at position 1222 in the azido AG-A-photolabeled MRP11-1222 fragment was replaced with Met, and the functions of MRP1 R1222M were compared with those of MRP1 R1249A. Login to comment 190 ABCC1 p.Arg1249Ala ABCC1 p.Arg1222Met Figure 6A shows that the expression level of MRP1 R1222M was comparable to that of wild-type MRP1, whereas the expression level of MRP1 R1249A was higher than that of wild-type MRP1. Login to comment 192 ABCC1 p.Arg1222Met As shown in Figure 6B, MRP1 R1222M transported LTC4 as efficiently as wild-type MRP1. Login to comment 193 ABCC1 p.Arg1249Ala However, the transport activities of MRP1 R1249A were considerably reduced, suggesting that Arg1249 was important for the LTC4 transporting activity of MRP1. Login to comment 195 ABCC1 p.Arg1222Met As shown in Figure 6C, GSH stimulated [125 I]azido AG-A binding to MRP1 R1222M as efficiently as wild-type MRP1. Login to comment 196 ABCC1 p.Arg1249Ala The level of photolabeling of MRP1 R1249A with [125 I]azido AG-A was increased only 1.6-fold in the presence of 10 mM GSH compared to a 10-fold increase observed with wild-type MRP1. Login to comment 198 ABCC1 p.Arg1249Ala ABCC1 p.Arg1249Ala To test the ability of MRP1 R1249A to confer drug resistance, wild-type MRP1 and the MRP1 R1249A mutant cDNAs were cloned into the mammalian expression vector pCIneo and transfected into pig kidney LLC-PK1 cells. Login to comment 202 ABCC1 p.Arg1249Ala Transfection of the pCIneo empty vector or the MRP1 R1249A mutant resulted in no VCR resistant clones. Login to comment 203 ABCC1 p.Arg1249Ala ABCC1 p.Arg1249Ala However, limited dilution of G418 resistant mass populations transfected with the MRP1 R1249A mutant in the medium without VCR resulted in several clones expressing MRP1 R1249A (Figure 7A). Login to comment 205 ABCC1 p.Arg1249Ala As shown in Figure 7B, the MRP1 R1249A mutant impaired the ability to confer resistance to VCR on LLC-PK1 cells. Login to comment 206 ABCC1 p.Arg1249Ala ABCC1 p.Arg1249Ala ABCC1 p.Arg1249Ala To determine if the inability of the R1249A mutant to confer drug resistance might be due to an inability of the mutant protein to reach the cell membrane, we assessed the cellular localization of MRP1 R1249A by immunofluorescence using MRPm6 mAb. As shown in Figure 7C, the MRP1 R1249A mutant was localized to the plasma membrane in a manner similar to that of wild-type MRP1. Login to comment 207 ABCC1 p.Arg1249Ala Thus, the inability of R1249A to confer drug resistance was not due to impaired trafficking of the protein. Login to comment 225 ABCC1 p.Arg1249Ala ABCC1 p.Arg1222Met Membrane vesicles (25 µg of protein) expressing MRP1 R1222M or MRP1 R1249A as indicated were incubated with 1.37 nM [3H]LTC4 at 37 °C in 50 µL of transport buffer as described in the legend of Figure 3 in the presence or absence of 4 mM ATP at the indicated periods. Login to comment 229 ABCC1 p.Arg1222Met ABCC1 p.Arg1249Met Membrane vesicles (100 µg of protein) expressing MRP1, MRP1 R1249M, and MRP1 R1222M were photolabeled with [125I]azido AG-A in the absence or presence of the indicated concentrations of GSH as described in the legend ofo Figure 2. Login to comment 256 ABCC2 p.Arg1257Ala ABCC2 p.Arg1210Ala It has also been observed that R1210A (TM16) and R1257A (TM17) mutants of human MRP2 showed decreased transport activity with the GSH-methylfluorescein conjugate (33). Login to comment 259 ABCC1 p.Arg1249Ala Mutation of this FIGURE 7: Effect of the R1249A mutation in MRP1 on drug resistance in LLC-PK1 cells. Login to comment 260 ABCC1 p.Arg1249Ala (A) Expression of MRP1 and the MRP1 R1249A mutant in LLC-PK1 cells. Login to comment 261 ABCC1 p.Arg1249Ala Crude membranes (50 µg of protein) prepared from LLC-PK1 cells transfected with an expression vector encoding MRP1 or MRP1 R1249A, or with a control empty vector (pCIneo), were analyzed on 7.5% SDS-PAGE. Login to comment 264 ABCC1 p.Arg1249Ala LLC-PK1 cells expressing MRP1 (b) or MRP1 R1249A (0), or the cells transfected with an empty vector [pCIneo (O)], were exposed to the indicated concentrations of VCR, and the survival rates were determined. Login to comment 268 ABCC1 p.Arg1249Ala Indirect immunofluorescent staining of MRP1 and MRP1 R1249A expressed in LLC-PK1 cells was carried out with the MRPm6 mAb and an FITC-conjugated secondary antibody. Login to comment 271 ABCC1 p.Arg1249Ala arginine 1249 to alanine almost completely impaired the LTC4 transport activity of MRP1 and abrogated the ability of MRP1 to confer VCR resistance. Login to comment 273 ABCC1 p.Arg1249Ala The inability of R1249A to confer VCR resistance was not due to alterations in MRP1 trafficking since the mutant MRP1 was still localized to the plasma membrane. Login to comment