ABCMdb

PMID: 24275649

Loo TW, Clarke DM
Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.
J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Login to comment 11 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu ABCB1 p.Tyr1087Phe Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Login to comment 12 ABCB1 p.Leu814Ala ABCB1 p.Phe793Ala ABCB1 p.Leu797Ala ABCB1 p.Leu818Ala Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. Login to comment 64 ABCB1 p.Leu258Ile IH2-IH3 Cross-linking-Cysteine residues were introduced at each position within residues Leu258 to Ile-261 in IH2 and residues Trp803 and Phe804 in IH3 to generate eight different double cysteine mutants in a Cys-less background (40). Login to comment 79 ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala It was Clamping IH2 to IH3 Inhibits P-gp 230 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 1ߦJANUARY 3, found that two mutations in IH3 (W803A and F804A) inhibited maturation of P-gp (Fig. 1, B and C) so that immature 150-kDa P-gp was the major product. Login to comment 80 ABCB1 p.Asp805Ala ABCB1 p.Ser802Ala ABCB1 p.Asp800Ala ABCB1 p.Val801Ser By contrast, the D800A, V801S, S802A, D805A, and D806A mutations in IH3 appeared to have little or no effect on folding as they yielded mature 170-kDa P-gp as their major product. Login to comment 81 ABCB1 p.Phe793Ala ABCB1 p.Leu797Ala Point mutations to the residues flanking IH3 (L797A, Fig. 1B) along with F793A, L814A, and L818A (data not shown) also inhibited maturation so that the 150-kDa protein was the major product. Login to comment 83 ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala Because W803A and F804A mutations in IH3 yielded the immature 150-kDa immature P-gp as the major product, we tested whether the mutants could be rescued with cyclosporine A. Login to comment 85 ABCB1 p.Trp803Ala ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala ABCB1 p.Phe804Ala Accordingly, the A52-tagged mutants W803A and F804A were expressed in HEK 293 cells in the absence or presence of 5 òe;M cyclosporine A, and whole cell SDS extracts were subjected to immunoblot analysis. Fig. 2A shows that expression of either W803A or F804A in the presence of cyclosporine A yielded the mature 170-kDa P-gp as the major product. Login to comment 86 ABCB1 p.Phe793Ala ABCB1 p.Leu797Ala Because the mutations F793A, L797A, L814A, and L818A in the flanking regions of IH3 also inhibited maturation, these mutants were also expressed in the presence of cyclosporine A to test for rescue. Login to comment 88 ABCB1 p.Phe793Ala ABCB1 p.Leu797Ala Mutants F793A, L797A, and L818A were less efficiently rescued as they yielded about equivalent levels of mature 170-kDa and immature 150-kDa P-gp in the presence of cyclosporine A (Fig. 2B). Login to comment 89 ABCB1 p.Trp803Ala ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala ABCB1 p.Phe804Ala To determine whether the W803A and F804A mutants rescued with cyclosporine A were active, the histidine-tagged mutants W803A and F804A were expressed in HEK 293 cells in the presence of cyclosporine A, and P-gp was isolated by nickel-chelate chromatography. Login to comment 101 ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala Drug rescue of processing mutants and activity of IH3 mutants W803A and F804A. Login to comment 102 ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala ABCB1 p.Phe793Ala ABCB1 p.Leu797Ala A52-tagged WT P-gp, mutants W803A and F804A (A), or IH3 flanking mutants (F793A, L797A, L814A, and L818A) (B) were expressed in HEK293cellsintheabsence(afa;)orpresence(af9;)of5òe;M cyclosporineA(Cyclo) for 18 h, and whole cell extracts were subjected to immunoblot analysis. Login to comment 103 ABCB1 p.Trp803Ala ABCB1 p.Phe804Ala The positions of mature (170-kDa) and immature (150-kDa) forms of P-gp are shown. C, verapamil-stimulated ATPase activities of histidine-tagged wild-type P-gp and W803A and F804A mutants. Login to comment 104 ABCB1 p.Phe804Ser ABCB1 p.Trp803Phe ABCB1 p.Phe804Tyr ABCB1 p.Phe804Leu The results are derived from three different transfections af9; S.D. Clamping IH2 to IH3 Inhibits P-gp JANUARY 3, 2014ߦVOLUME 289ߦNUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 231 at SEMMELWEIS UNIV OF MEDICINE on December 12, We then tested whether mutating Trp803 to Phe (hydrophobic), Leu (hydrophobic), Ser (hydrophilic), Tyr (aromatic), or Asp (charged) and Phe804 to Leu, Ser, Tyr, Asp, or Trp affected maturation of the protein. Login to comment 106 ABCB1 p.Trp803Leu Replacement of Trp803 with Leu, Ser, or Asp inhibited maturation, although all three mutants could still be rescued with cyclosporine A (Fig. 3, A and B). Login to comment 108 ABCB1 p.Phe804Tyr ABCB1 p.Phe804Tyr ABCB1 p.Phe804Trp ABCB1 p.Phe804Trp Mature 170-kDa P-gp was the major product in mutants F804Y and F804W (Fig. 3, C and D), although the amount of mature protein in the absence of cyclosporine A in mutant F804W was lower than in F804Y (65% versus 85%, respectively). Login to comment 109 ABCB1 p.Phe804Ala ABCB1 p.Phe804Ser ABCB1 p.Phe804Ser ABCB1 p.Phe804Leu Mutating Phe804 to Leu, Ser, or Asp inhibited maturation, and only the F804A or F804S mutants could be rescued with cyclosporine A to yield mature 170-kDa P-gp as the major product (Fig. 3, C and D). Login to comment 114 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys Cross-linking of IH2 and IH3 Inhibits Activity-In a previous cysteine mutagenesis and cross-linking study, we showed that clamping ICL3 (N820C) in close proximity to ICL1 (D175C or D177C) by cross-linking cysteines with short cross-linkers activated P-gp ATPase activity (15, 33). Login to comment 123 ABCB1 p.Trp803Cys ABCB1 p.Phe804Cys It was found that the yield of all the F804C double mutants was significantly lower than the W803C double cysteine mutants (data not shown). Login to comment 125 ABCB1 p.Trp803Cys Therefore, the W803C double mutants were tested for cross-linking with cupric chloride oxidant. Login to comment 128 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys Cross-linked product was observed in all mutants (data not shown), with the greatest amount present in mutant A259C/W803C (about 75%, Fig. 4A). Login to comment 129 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys We then tested whether cross-linking of mutant A259C/ W803C could be inhibited by nucleotides. Login to comment 132 ABCB1 p.Trp803Phe ABCB1 p.Trp803Leu ABCB1 p.Trp803Ser ABCB1 p.Trp803Tyr ABCB1 p.Trp803Asp A52-tagged mutants containing changes to Trp803 (A and B) (W803F (F), W803L (L), W803S (S), W803Y (Y), or W803D (D)) or Phe804 (CandD)(F804L(L),F804S(S),F804Y(Y),F804D(D),orF804W(W))were expressed in HEK 293 cells in the absence (afa;) or presence (af9;) of 5 òe;M cyclosporine A (Cyclo) for 18 h, and whole cell extracts were subjected to immunoblot analysis. Login to comment 137 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys To test the effect of cross-linking on activity, histidine-tagged mutant A259C/W803C was expressed in HEK 293 cells in the presence of cyclosporine A and P-gp isolated by nickel-chelate chromatography. Login to comment 148 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu ABCB1 p.Tyr1087Phe To test whether Tyr1087 was important for folding or mediating drug stimulation of ATPase activity, we tested the effect of Y1087A, Y1087L, and Y1087F mutations. Login to comment 149 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu ABCB1 p.Tyr1087Phe The A52-tagged mutants were expressed in HEK 293 cells in the absence or presence of cyclosporine A, and samples of whole cell SDS extracts were subjected to immunoblot analysis. Fig. 5A shows that the mature 170-kDa protein was the major product in mutant Y1087F, whereas the 150-kDa immature protein was the major product in mutants Y1087A and Y1087L. Login to comment 150 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu Both mutants Y1087A and Y1087L, however, could be rescued when they were expressed in the presence of cyclosporine A (Fig. 5A). Login to comment 151 ABCB1 p.Phe1086Leu These results show that maturation of P-gp was more sensitive to changes to Tyr1087 compared with Phe1086 because the F1086L mutation did not inhibit maturation (16). Login to comment 153 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu It was found that the Y1087A and Y1087L mutations reduced activity by over 90% (Fig. 5B). Login to comment 154 ABCB1 p.Tyr1087Phe Mutant Y1087F showed about one-third of wild-type P-gp ATPase activity. Login to comment 156 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys Cross-linking of A259C (IH2) to W803C (IH3) inhibits activity. Login to comment 157 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys A, membranes prepared from cells expressing histidine-tagged mutant A259C/ W803C were treated without (afa;) or with (af9;) 0.5 mM CuCl2 for 10 min at 0 &#b0;C in the absence (None) or presence of 5 mM ATP, adenosine 5b18;-(beta,ॹ- imino)triphosphate(AMP.PNP),orADP.Thereactionswerestoppedbyaddition of SDS sample buffer containing no reducing agent, and samples were then subjected to immunoblot analysis. Login to comment 158 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys The positions of the cross-linked (X-link) and mature (170-kDa) P-gps are indicated. B, the amount of cross-linked protein relative to total P-gp (170-kDa plus 150-kDa P-gp) was quantitated from three different transfections af9; S.D. C, the histidine-tagged mutant A259C/ W803C was isolated by nickel-chelate chromatography, reconstituted with lipid, and treated without (afa;) or with (af9;) 0.2 mM CuCl2 for 10 min at 20 &#b0;C. The reaction was stopped with 1 mM EDTA. Login to comment 164 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu ABCB1 p.Tyr1087Phe A, A52-tagged Y1087A (A), Y1087F (F), and Y1087L (L) mutants were expressed in the absence (afa;) or presence (af9;) of 5 òe;M cyclosporine A (Cyclo). Login to comment 166 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu ABCB1 p.Tyr1087Phe The positions of mature (170-kDa) and immature (150-kDa) P-gps are indicated. B, histidine-tagged WT, Y1087A (A), Y1087F (F), and Y1087L (L) P-gp were expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment 167 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu Mutants Y1087A and Y1087L were expressed in the presence of cyclosporine A to promote maturation. Login to comment 171 ABCB1 p.Tyr1087Ala ABCB1 p.Tyr1087Leu Mutations of Tyr1087 to Ala or Leu severely reduced both maturation and activity of P-gp, and even the small change of replacing Tyr with Phe caused about a 70% reduction in activity. Login to comment 185 ABCB1 p.Phe793Ala ABCB1 p.Leu797Ala In support of the prediction that Trp803 and Phe804 form a hydrophobic pocket with these residues, alanine-scanning mutagenesis of the 793-797 and 814-818 regions showed that only the F793A, L797A, L814A, and L818A mutations inhibited maturation (Fig. 2B). Login to comment 199 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys In support of these predictions, we found that clamping IH2 to IH3 by oxidative cross-linking of mutant A259C/W803C or mutations to Tyr1087 inhibited activity. Login to comment 201 ABCB1 p.Phe1086Ala Although we showed previously that mutation of the adjacent Phe1086 to Ala also inhibited activity (16), the activity of P-gp was much more sensitive to changes to Tyr1087 . Login to comment 202 ABCB1 p.Phe1086Leu For example, mutation of Phe1086 to Leu, Phe, or Trp yielded P-gps with activities similar to the wild-type protein. Login to comment 203 ABCB1 p.Tyr1087Leu ABCB1 p.Tyr1087Phe Mutation of Tyr1087 to Leu severely inhibited activity, whereas the conservative Y1087F change caused about a 70% reduction in activity. Login to comment 209 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys Cross-linking of A259C (blue) in ICL2 to W803C (green) in ICL3 inhibited activity. Login to comment 212 ABCB1 p.Asp805Cys In support of this prediction, it has been reported recently that a P-gp mutant containing the D805C mutation reduced verapamil-stimulated ATPase activity by about 70% but had no effect on the Km for ATP (49). Login to comment