ABCMdb

PMID: 18708637

Loo TW, Bartlett MC, Clarke DM
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16., 2008-10-17 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCB1 p.Gly268Val ABCB1 p.Leu1260Ala The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/ A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Login to comment 47 ABCC7 p.Leu1260Ala ABCB1 p.Gly268Val The G268V and L1260A processing mutations were introduced into the L443C(NBD1)/S909C(TMD2) mutant. Login to comment 51 ABCC7 p.Val510Ala ABCC7 p.Val510Cys We also constructed the V510C(NBD1)/A1067C(TMD2) CFTR mutant because position V510C or V510A promoted maturation of Cys-less CFTR (22). Login to comment 107 ABCB1 p.Arg905Cys ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys The locations of positions equivalent to cysteines S473C and R905C in the other half of P-gp (A266C/F1086C) are indicated. Login to comment 108 ABCC7 p.Leu1260Ala ABCB1 p.Gly268Val The locations of processing mutations G268V and L1260A that were used to inhibit maturation of P-gp are indicated as squares. Login to comment 136 ABCC7 p.Leu1260Ala ABCB1 p.Gly268Val To test if NBD-TMD2 interactions differ in the mature and immature forms of P-gp, the G268V (26) or L1260A (38) processing mutations were introduced into mutant L443C(NBD1)/ S909C(TMD2) to inhibit its maturation. Login to comment 140 ABCC7 p.Leu1260Ala ABCB1 p.Gly268Val The G268V mutation is located in the second intracellular loop in TMD1 whereas the L1260A mutation is located at the COOH-end of NBD2. Login to comment 151 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Phe267Cys ABCB1 p.Tyr1087Cys ABCB1 p.Asp1088Cys ABCB1 p.Arg1085Cys Because no cross-linking studies have identified cysteines that could be cross-linked at the TMD1-NBD2 interface, we constructed a series of double cysteine mutants between a cysteine in TMD1 (A266C or F267C) and another in NBD2 (R1085C, F1086C, Y1087C or D1088C) that would be equivalent to L443C(NBD1) and S909C(TMD2), respectively, in each half of P-gp. Login to comment 159 ABCC7 p.Leu1260Ala ABCB1 p.Gly268Val Mutants G268V/L443C(NBD1)/S909C(TMD2) and L1260A/L443C(NBD1)/S909C(TMD2) were expressed in HEK 293 cells in the presence of no drug (None) or 10 ␮m cyclosporin A (ϩ Cyclo). Login to comment 164 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys A, membranes prepared from cells expressing mutant A266C/F1086C were treated with (ϩ) or without (-) 1 mM copper phenanthroline (CuP) for 15 min at 0 °C. Membranes were also treated with drug substrates or ATP plus vanadate as described in the legend to Fig. 4. Login to comment 166 ABCC7 p.Leu1260Ala B, mutant A266C(TMD1)/F1086C(NBD2) containing the L1260A processing mutation was expressed in HEK 293 cells in the absence (no drug) or presence of 10 ␮M cyclosporin A (ϩ Cyclo). Login to comment 170 ABCB1 p.Phe1086Cys The verapamil-stimulated ATPase activities of P-gp mutants containing only the Cys-266 or Cys-1086 mutation showed that the reduced activity was due to the presence of the F1086C change (data not shown). Login to comment 171 ABCC7 p.Leu1260Ala To test if there were structural differences between the mature and immature forms of P-gp at the TMD1-NBD2 interface, the L1260A processing mutation was introduced into mutant A266C(TMD1)/F1086C(NBD2). Login to comment 172 ABCC7 p.Leu1260Ala Mutant L1260A/ A266C(TMD1)/F1086C(NBD2) was expressed in the presence or absence of cyclosporin A and membranes prepared from the cells were treated with or without copper phenanthroline at 0 °C followed by immunoblot analysis. Fig. 6B shows that cross-linking was observed with mature P-gp (right panel) but not with immature P-gp (left panel). Login to comment 179 ABCC7 p.Val510Ala ABCC7 p.Val510Cys It was previously shown that mutations V510C or V510A promoted maturation of Cys-less CFTR (22). Login to comment 180 ABCC7 p.Val510Cys It was possible that V510C could interact with TMD2. Login to comment 199 ABCC7 p.Leu1260Ala ABCB1 p.Gly268Val Processing mutations near the NH2- (G268V) or COOH-end (L1260A) of the molecule disrupted cross-linking between Cys-443(NBD1) and Cys-909(TMD2). Login to comment 202 ABCC7 p.Leu1260Ala Studies on the L1260A/ A266C(TMD1)/F1086C(NBD2) mutant showed that only the mature form of the protein could undergo cross-linking (Fig. 6B). Login to comment 210 ABCC7 p.Leu1260Ala The presence of a processing mutation such as L1260A traps P-gp as immature protein with incomplete NBD-TMD contacts (Fig. 8, left panel). Login to comment 213 ABCB1 p.Phe1086Cys NBD-TMD contacts appear to be critical for function because cross-linking of mutant L443C(NBD1)/S909C(TMD2) (Fig. 3) or introduction of the F1086C mutation at the TMD1/NBD2 interface inhibited activity. Login to comment