ABCMdb

PMID: 12721111

Ren XQ, Furukawa T, Aoki S, Sumizawa T, Haraguchi M, Che XF, Kobayashi M, Akiyama S
Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.
Br J Pharmacol. 2003 Apr;138(8):1553-61., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC1 p.Arg1202Gly Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C4 (LTC4) transport activity and conferred Vincristine resistance in LLC-PK1 cells. Login to comment 53 ABCC1 p.Arg1202Gly MRP1 constructs encoding R1202G was generated in PCR reaction using the forward primers: 50 -GGGCTCGAGTGT GTGGGCAACTGC-30 (bold denotes a mismatched base encoding the mutation). Login to comment 116 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Gly To investigate the role of the charged amino acid in the azido AG-A photolabelling of MRP1, we replaced the Arg1202 with Gly (R1202G). Login to comment 118 ABCC1 p.Arg1202Gly Figure 4a shows that the expression level of R1202G mutant was comparable to that of wild-type MRP1. Login to comment 119 ABCC1 p.Arg1202Gly The membrane vesicles expressing the wild-type and R1202G MRP1s were analyzed for their abilities to interact with azido AG-A in a GSH-dependent manner. Login to comment 120 ABCC1 p.Arg1202Gly As shown in Figure 4b, wild-type MRP1 was photolabelled with azido AG-A in a GSH-dependent manner; however, the GSH-dependent photolabelling of MRP1 R1202G with [125 I]azido AG-A was greatly reduced (Figure 4b). Login to comment 121 ABCC1 p.Arg1202Gly To test whether the mutation affects the function of MRP1, the LTC4 transport activity of MRP1 R1202G was examined. Login to comment 122 ABCC1 p.Arg1202Gly As shown in Figure 5a, MRP1 R1202G efficiently transported LTC4. Login to comment 123 ABCC1 p.Arg1202Gly The apparent Km values of MRP1 R1202G and wild-type MRP1 for LTC4 were 147 and 117 nM, respectively (Figure 5b). Login to comment 124 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Gly In order to test the ability of R1202G to confer drug resistance, the wild-type MRP1 and the MRP1 R1202G mutant cDNAs were cloned into the mammalian expression vector pCIneo and transfected into LLC-PK1 cells. Login to comment 139 ABCC1 p.Arg1202Gly Transfection of wild-type MRP1, or the MRP1 R1202G mutant, but not the pCIneo empty vector, resulted in drug-resistant colonies within 2 weeks. Login to comment 140 ABCC1 p.Arg1202Gly The drug resistance of stably transfected clones that express MRP1 or MRP1 R1202G (Figure 6a) was further tested in a cytotoxicity assay. Login to comment 141 ABCC1 p.Arg1202Gly As shown in Figure 6b, MRP1 and MRP1 R1202G conferred VCR resistance on LLC-PK1 cells. Login to comment 142 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Gly MRP1 R1202G mutant protein was localized to the plasma membrane in a similar manner to the wild-type MRP1, indicating that the R1202G mutation did not affect the trafficking of the mutant protein (Figure 6c). Login to comment 143 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Gly The fact that the photolabelling of MRP1 with azido AG-A was impaired by the R1202G mutation, but the transport activity and the ability to confer resistance to VCR were retained in this mutant suggests that the R1202G mutation affected only the photolabelling, but not the binding of azido AG-A to MRP1. Login to comment 144 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Gly To investigate whether the MRP1 R1202G mutant can interact with AG-A, the effect of AG-A on [3 H]LTC4 uptake by wild-type MRP1 and the MRP1 R1202G mutant expressed in Sf21 insect cells was examined. Login to comment 145 ABCC1 p.Arg1202Gly In the presence of 2 mM GSH, AG-A inhibited uptake of [3 H]LTC4 in membrane vesicles that express MRP1 R1202G in a dose-dependent manner and the extent of the inhibition was similar to that in membrane vesicles that express wild-type MRP1 (Figure 7). Login to comment 146 ABCC1 p.Arg1202Gly These findings suggest that MRP1 R1202G as well as wild-type MRP1 can interact with AG-A, and Arg1202 is not critical to the binding of AG-A to MRP1, although it affects the photolabelling of azido AG-A. Login to comment 177 ABCC1 p.Arg1202Gly When the membrane vesicles expressing the mutant protein were photolabelled and subsequently digested with trypsin or V8 Figure 4 Effect of R1202G mutation on the GSH-dependent photolabelling of MRP1 with [125 I]azido AG-A. Login to comment 178 ABCC1 p.Arg1202Gly (a) Wild-type MRP1 and MRP1 R1202G were expressed in insect cells. Login to comment 180 ABCC1 p.Arg1202Gly (b) Wild-type MRP1 and MRP1 R1202G membrane vesicles (100 mg of membrane proteins) were photolabelled with [125 I]azido AG-A in the absence or presence of the indicated concentrations of GSH as described in the legend to Figure 2a. Login to comment 182 ABCC1 p.Arg1202Gly Figure 5 Effect of R1202G mutation on ATP-dependent [3 H]LTC4 transport. Login to comment 183 ABCC1 p.Arg1202Gly (a) Time course of ATP-dependent transport of [3 H]LTC4 by wild-type MRP1 (square) and MRP1 R1202G (triangle) mutant. Login to comment 184 ABCC1 p.Arg1202Gly Membrane vesicles (50 mg) expressing wild-type MRP1 or MRP1 R1202G were incubated with 1.37 nM [3 H]LTC4 at 371C in 50 ml transport buffer as described in the legend to Figure 1b in the presence or absence of 4 mM ATP at the indicated periods. Login to comment 187 ABCC1 p.Arg1202Gly (b) Determination of the Km values of MRP1 wild type (square) and MRP1 R1202G (triangle) for [3 H]LTC4. Login to comment 205 ABCC1 p.Arg1202Gly On the other hand, if one of the trypsin digestion sites is located in the cytoplasmic portion Figure 6 Effect of R1202G mutation on drug resistance in LLC-PK1 cells. Login to comment 206 ABCC1 p.Arg1202Gly (a) Expression of wild-type MRP1 and the MRP1 R1202G mutant in LLC-PK1 cells. Login to comment 207 ABCC1 p.Arg1202Gly Crude membranes (50 mg of protein) prepared from LLC-PK1 cells transfected with either expression vectors encoding wild-type MRP1, MRP1 R1202G, or a control empty vector (pCIneo) were analyzed on 7.5% SDS - PAGE. Login to comment 210 ABCC1 p.Arg1202Gly LLC-PK1 cells expressing either wild-type MRP1 (square), MRP1 R1202G (triangle), or transfected with an empty vector (rhombus) were exposed to the indicated concentrations of VCR and the survival rate was determined by MTT assay as described under Materials and methods. Login to comment 213 ABCC1 p.Arg1202Gly Indirect immunofluorescent staining of wild-type MRP1 and MRP1 R1202G expressed in LLC-PK1 cells was carried out with the MRPm6 mAb and a FITC-conjugated secondary antibody. Login to comment 215 ABCC1 p.Arg1202Gly Both wild-type MRP1 and MRP1 R1202G were localized in the plasma membrane of LLC-PK1 cells. Login to comment 217 ABCC1 p.Arg1202Gly Figure 7 Effect of AG-A on [3 H]LTC4 transport by wild-type MRP1 and MRP1 R1202G. Login to comment 218 ABCC1 p.Arg1202Gly ATP-dependent [3 H]LTC4 uptake into MRP1 and MRP1 R1202G membrane vesicles from Sf21 insect cells was measured in the presence of 2 mM GSH and 10, 30 or 100 mM of AG-A as indicated. Login to comment 231 ABCC2 p.Arg1210Ala R1210A (TM16) mutant of human MRP2 showed reduced transport activity of GSH methylfluorescein (Ryu et al., 2000). Login to comment 233 ABCC1 p.Arg1202Gly Therefore, we replaced the arginine at 1202 that is proximate to the N-terminus of TM16 of MRP1 with Gly (R1202G) to investigate the role of the charged amino acid in the region where GSH-dependent azido AG-A photolabelling site resides. Login to comment 234 ABCC1 p.Arg1202Gly GSH-dependent photolabelling of the mutant MRP1 R1202G with [125 I]azido AG-A was considerably lower than that of wild-type MRP1. Login to comment 235 ABCC1 p.Arg1202Gly The impaired GSH-dependent photolabelling of MRP1 R1202G may be attributable to the impaired binding of MRP1 to AG-A, and thus the GSH-dependent photolabelling with azido AG-A was lowered. Login to comment 241 ABCC1 p.Arg1202Gly Alternatively, the azido AG-A photolabelling site may reside in the region nearby Arg1202 that is proximate to the N-terminus of TM16, and the replacement of Arg1202 with Gly may affect the photolabelling of this site. Login to comment