ABCMdb

PMID: 15530432

Loo TW, Bartlett MC, Clarke DM
Thapsigargin or curcumin does not promote maturation of processing mutants of the ABC transporters, CFTR, and P-glycoprotein.
Biochem Biophys Res Commun. 2004 Dec 10;325(2):580-5., [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCB1 p.Gly251Val HEK293 cells expressing A52-tagged wild-type or mutant G251V P-gp were grown at 37 °C. Login to comment 58 ABCB1 p.Gly251Val ABCB1 p.Phe804Ala Mutations G251V and F804A are in the intracellular loops of the NH2- and COOH-terminal halves of P-gp, respectively. Login to comment 59 ABCB1 p.Gly722Ala ABCB1 p.Gly300Val The processing mutations G300V (TM5) and G722A (TM7) are in TMD1 and TMD2, respectively. Login to comment 69 ABCB1 p.Pro709Gly ABCB1 p.Pro1194Ala A processing mutation in NBD2 is P1194A, while P709G is in the linker region. Login to comment 70 ABCB1 p.Gly251Val ABCB1 p.Pro709Ala ABCB1 p.Gly722Ala ABCB1 p.Pro1194Ala ABCB1 p.Phe804Ala ABCB1 p.Gly300Val Although the processing mutations (G251V, G300V, DY490, P709A, G722A, F804A, and P1194A) are located in different segments of P-gp, all the mutants could be rescued when expressed with drug substrates such as cyclosporin A. Login to comment 71 ABCB1 p.Gly251Val A representative example is shown with mutant G251V. Login to comment 72 ABCB1 p.Gly251Val HEK cells expressing mutant G251V were grown in the presence of various concentrations of cyclosporin A for 18 h at 37 °C. Login to comment 76 ABCB1 p.Gly251Val To confirm that the cyclosporin A acted as a chemical chaperone to stabilize the mutant G251V protein resulting in decreased turnover, we performed pulse-chase studies (Fig. 2B). Login to comment 77 ABCB1 p.Gly251Val HEK293 cells expressing mutant G251V were incubated with 35 [S]methionine for 20 min. Login to comment 82 ABCB1 p.Gly251Val When synthesis of mutant G251V is carried out in the presence of 10 lM cyclosporin A, however, mature 170 kDa protein was readily detected after 1 h. Login to comment 85 ABCB1 p.Gly251Val ABCB1 p.Gly251Val To determine if the mature 170 kDa protein of mutant G251V P-gp was active after rescue, we expressed the histidine-tagged wild-type or mutant G251V P-gp in HEK293 cells in the presence of 10 lM cyclosporin A for 18 h at 37 °C. Login to comment 88 ABCB1 p.Gly251Val The mature form of mutant G251V was still active since it retained >90% of wild-type P-gp activity (not shown). Login to comment 90 ABCB1 p.Gly251Val ABCB1 p.Pro709Ala ABCB1 p.Gly722Ala ABCB1 p.Pro1194Ala ABCB1 p.Phe804Ala We then compared the abilities of cyclosporin A, thapsigargin, and curcumin to induce maturation of P-gp processing mutants G251V, G300V, DY490, P709A, G722A, F804A, and P1194A. Login to comment 102 ABCB1 p.Gly251Val ABCB1 p.Gly251Val Accordingly, we stably expressed mutant G251V in baby hamster kidney (BHK) cells because it has been reported that curcumin can induce maturation Fig. 2. Effect of cyclosporin A on maturation of P-gp processing mutant G251V. Login to comment 103 ABCB1 p.Gly251Val (A) HEK293 cells expressing the A52 epitope-tagged mutant G251V were incubated for 18 h at 37 °C in the presence of various concentrations of cyclosporin A (Cyclo A). Login to comment 105 ABCB1 p.Gly251Val (B) HEK293 cells were transfected with A52-tagged G251V P-gp cDNA. Login to comment 111 ABCB1 p.Gly251Val The BHK cells expressing mutant G251V were incubated with 10 lM cyclosporin A or various concentrations of thapsigargin (0-10 lM) or curcumin (0-30 lM). Login to comment 112 ABCB1 p.Gly251Val Fig. 3B shows that in the absence of drug substrates, the major product of mutant G251V in BHK cells was the 150 kDa immature protein. Login to comment 138 ABCB1 p.Gly251Val (B) BHK cells stably expressing A52-tagged mutant G251V were incubated for 18 h at 37 °C in the presence of 10 lM cyclosporin A (Cyclo) or various concentrations (lM) of thapsigargin or curcumin. Login to comment