ABCMdb

PMID: 11375986

Leslie EM, Ito K, Upadhyaya P, Hecht SS, Deeley RG, Cole SP
Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1). Requirement for glutathione or a non-sulfur-containing analog.
J Biol Chem. 2001 Jul 27;276(30):27846-54. Epub 2001 May 25., 2001-07-27 [PubMed]

Sentences

No. Mutations Sentence Comment

61 ABCC1 p.Trp1246Ala MRP1 and MRP2 Expression Vectors and Transfections in HEK293T cells-Construction of the expression vector pcDNA3.1(-)-MRP1K encoding wild-type MRP1 (WT-MRP1) and the vector encoding MRP1 with the substitution of Trp1246 3Ala (W1246A-MRP1) have been described previously (32). Login to comment 86 ABCC1 p.Trp1246Ala [3 H]NNAL-O-glucuronide transport assays with membrane vesicles prepared from HEK293T cells transiently transfected with empty vector, wild-type MRP1, mutant W1246A-MRP1, and wild-type MRP2 cDNAs were carried out under the same conditions as described above except incubations were for 15 min. Login to comment 93 ABCC1 p.Trp1246Ala [3 H]LTC4 Transport Studies-To measure LTC4 uptake, assays were carried out at 23 °C in a 50-␮l volume containing vesicle protein prepared from T5 HeLa cells or HEK293T cells transiently transfected with empty vector, wild-type, and W1246A-MRP1 cDNAs (2 ␮g), [3 H]LTC4 (50 nM; 40 nCi), NNK or NNAL or NNAL-O-glucuronide (0, 10, 100 ␮M, respectively) and components as described above for [3 H] NNAL-O-glucuronide transport. Login to comment 145 ABCC1 p.Trp1246Ala To determine whether Trp1246 was also essential for GSH-dependent transport of NNAL-O-glucuronide, transport assays were carried out using membrane vesicles prepared from HEK293T cells transfected with pcDNA3.1(-) (empty vector), WT-MRP1, and W1246A-MRP1 cDNAs. Login to comment 146 ABCC1 p.Trp1246Ala The W1246A-MRP1 mutant and WT-MRP1 proteins were expressed at similar levels in the HEK293T cells as determined by immunoblot analysis of the membrane vesicles (Fig. 8A). Login to comment 147 ABCC1 p.Trp1246Ala Moreover, [3 H]LTC4 uptake by the W1246A-MRP1 mutant was comparable with uptake by WT-MRP1 whereas [3 H]E217betaG uptake by the mutant protein was undetectable, as reported previously (Fig. 8B) (32). Login to comment 148 ABCC1 p.Trp1246Ala In contrast, whereas the addition of GSH markedly stimulated uptake of [3 H]NNAL-O-glucuronide by WT-MRP1, uptake by the MRP1-W1246A mu- FIG. 5. Login to comment 169 ABCC1 p.Trp1246Ala As expected, [3 H]NNAL-O-glucuronide uptake in membrane vesicles from the wild-type, W1246A mutant and vector control-transfected cells in the absence of GSH was extremely low and indistinguishable from one another. Login to comment 179 ABCC1 p.Trp1246Ala GSH-dependent uptake of [3 H]NNAL-O-glucuronide by wild-type and mutant W1246A-MRP1. Login to comment 180 ABCC1 p.Trp1246Ala A, membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (WT-MRP1), mutant MRP1 (W1246A-MRP1), or empty vector (pcDNA3.1(-)) were immunoblotted with the MRP1-specific MAb QCRL-1 as described in "Experimental Procedures." Login to comment 214 ABCC1 p.Trp1246Ala However, a commonality of at least some binding determinants is suggested by the observation that neither E217betaG nor NNAL-O-glucuronide (ϩGSH) were transported by the mutant W1246A-MRP1 (Fig. 8) (32). Login to comment 221 ABCC1 p.Trp1246Ala Consistent with this conclusion is the observation that the W1246A mutant of MRP1 retains its ability to transport LTC4 but does not transport the tobacco-derived glucuronide (Fig. 8) (32). Login to comment