ABCMdb

PMID: 14622130

Jodoin J, Demeule M, Fenart L, Cecchelli R, Farmer S, Linton KJ, Higgins CF, Beliveau R
P-glycoprotein in blood-brain barrier endothelial cells: interaction and oligomerization with caveolins.
J Neurochem. 2003 Nov;87(4):1010-23., [PubMed]

Sentences

No. Mutations Sentence Comment

74 ABCB1 p.Phe37Ala The mutated DNA was subcloned into the mammalian expression vector pcineo (Promega) to generate plasmid F37A-MDR1. Login to comment 75 ABCB1 p.Tyr42Ala ABCB1 p.Trp45Ala The MDR1 codons for Tyr42 and Trp45 were replaced with Ala codons by Quikchange protocol (Stratagene, La Jolla, CA, USA) using the primer 5'-TCAATGTTTCGCGCTTCGAATGCGCTTGACAAGT- TG-3' and a primer with the reverse complement sequence. Login to comment 206 ABCB1 p.Phe37Ala In order to further study the interaction between P-gp and caveolin-1, two P-gp proteins mutated in the caveolin-binding motif were produced: YWAA-MDR1 and F37A-MDR1. Login to comment 207 ABCB1 p.Tyr42Ala ABCB1 p.Trp45Ala In the first P-gp mutant, Tyr42 and Trp45 were replaced with Ala residues (YWAA-MDR1). Login to comment 208 ABCB1 p.Phe37Ala ABCB1 p.Phe37Ala In the other mutant, Phe37 was replaced with Ala (F37A-MDR1); nucleotide changes in the mutant genes were confirmed by sequencing. Login to comment 210 ABCB1 p.Phe37Ala Immunodetection of P-gp in Cos-7 cells transiently transfected with the wild-type (WT)-MDR1, YWAA-MDR1, and F37A-MDR1 showed that WT-MDR1 and P-gp mutants were expressed to the same extent (Fig. 7a). Login to comment 214 ABCB1 p.Phe37Ala Figure 7(b) shows that relative fluorescence increased fourfold in WT-MDR1-, YWAA-MDR1-, and F37A-MDR1-expressing cells as compared to untransfected and f/s-MDR1-transfected cells. Login to comment 217 ABCB1 p.Phe37Ala Compared with WT-MDR1-transfected cells, the interaction between P-gp and caveolin-1 is reduced by 27% and 48% in cells expressing YWAA-MDR1 and F37A-MDR1, respectively (Fig. 8b). Login to comment 221 ABCB1 p.Phe37Ala ABCB1 p.Phe37Ala In YWAA-MDR1and F37A-MDR1-transfected cells, Hoechst 33342 accumulation is lower than in WT-MDR1-transfected cells, indicating that mutation of caveolin-binding motif present in MDR1 P-gp affected its transport activity (Fig. 8b). Login to comment 222 ABCB1 p.Phe37Ala We also observed that P-gp is mainly located in caveolae in Cos-7 cells transfected with either WT-MDR1, YWAA-MDR1 or F37A-MDR1 (Fig. 8c). Login to comment 248 ABCB1 p.Phe37Ala (a) P-gp and caveolin-1 were immunodetected by western blot analysis in Cos-7 cells untransfected (cells were exposed to FuGENE 6 transfection reagent) or transiently transfected with WT-MDR1, YWAA-MDR1, F37A-MDR1, and f/s-MDR1 (n ¼ 4). Login to comment 270 ABCB1 p.Phe37Ala (a) Cos7 cells untransfected or transiently transfected with WT-MDR1, YWAA-MDR1, F37A-MDR1, and f/s-MDR1 were lysed in lysis buffer and cell lysate (500 lg of protein) was immunoprecipitated (IP) with 3 lg of mAb directed against P-gp (C219) followed by immunodetection (ID) of caveolin-1 (n ¼ 3). Login to comment 309 ABCB1 p.Phe37Ala Western blot and FACS analysis show that P-gp and caveolin-1 were expressed at constant levels in WT-MDR1-, YWAA-MDR1-, and F37A-MDR1-transfected cells, excluding the possibility of variation in protein expression. Login to comment