ABCMdb

ABCC7 p.Leu1260Ala

CF databases:	c.3779T>G , p.Leu1260Arg (CFTR1) D ,
Predicted by SNAP2:	A: D (71%), C: D (59%), D: D (80%), E: D (75%), F: D (66%), G: D (85%), H: D (80%), I: N (57%), K: D (85%), M: N (93%), N: D (75%), P: D (85%), Q: D (66%), R: D (85%), S: D (75%), T: D (71%), V: D (53%), W: D (80%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] Processing mutations disrupt interactions between ... J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16. 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 [PMID:18708637]

Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. 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. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.

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No. Sentence Comment
47 The G268V and L1260A processing mutations were introduced into the L443C(NBD1)/S909C(TMD2) mutant. Login to comment
108 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 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 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
159 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
166 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
171 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 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
199 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 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 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

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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No. Sentence Comment
57 The red balls show the locations of Trp232 and the processing mutations at positions 251 (G251V), 490 (ΔY490), 709 (P709A), 722 (G722A), and 1260 (L1260A). Login to comment
67 Mutations were introduced into wild-type P-gp or processing mutants containing processing mutations in different domains (G251V in TMD1, ΔY490 in NBD1, P709A in the linker region, G722A in TMD2, or L1260A in NBD2) as described previously (28). Login to comment
91 The G251V or L1260A processing mutations were introduced into Cys-less P-gp containing pairs of cysteines in different domains (L443C(NBD1)/ S909C(TMD2), L531C(NBD1)/C1074(NBD2), or C137(TMD1)/A935C(TMD2)) with or without the W232R mutation. Login to comment
92 The L1260A and L1260A/W232R mutations were also introduced into a Cys-less P-gp containing the A266C(TMD1)/ F1086C(NBD2) mutations. Login to comment
125 The W232R mutation was introduced into mutants P709A (linker region), G722A (TMD2), or L1260A (NBD2). Login to comment
155 To test if the W232R mutation restored domain-domain contacts, it was introduced into the G251V or L1260A processing mutants that also contained pairs of cysteines at the TMD1-TMD2 (C137(TMD1)/A935C(TMD2), NBD1-NBD2 (L531C(NBD1)/C1074(NBD2), or NBD1-TMD2 (L443C(NBD1)/S909C(TMD2) interfaces. Login to comment
156 The G251V and L1260A parents were used because the G251V mutation is in the same domain as W232R (TMD1) whereas L1260A is in another domain (NBD2). Login to comment
158 Introduction of the W232R mutation into all of the G251V (Figure 3A, lanes 3, 7, and 11) or L1260A (Figure 3B, lanes 3, 7, and 11) double-cysteine processing mutants restored maturation. Login to comment
162 Since the L1260A mutation is located in the NBD2 (Figure 1A), we also examined the effects of W232R on NBD2-TMD1 interactions. Login to comment
164 Accordingly, we introduced the W232R mutation into mutant A266C/F1086C/L1260A. Login to comment
166 Figure 3C shows that the W232R mutation promoted maturation of the mutant A266C/F1086C/L1260A (lane 3) and that only the mature protein was cross-linked (lane 4). Login to comment
187 Membranes were prepared from cells expressing P-gp processing mutants G251V ( W232R (A) or L1260A ( W232R (B) that also contained pairs of cysteines in various domains (L443C(NBD1)/S909C(TMD2), L531C(NBD1)/C1074(NBD2), C137(TMD1)/A935C(TMD2)). Login to comment
188 Membranes were also prepared from cells expressing the L1260A processing mutant ( W232R containing cysteines in TMD1 and NBD2 (A266C/F1086C) (C). Login to comment

[hide] Insertion of an arginine residue into the transmem... J Biol Chem. 2006 Oct 6;281(40):29436-40. Epub 2006 Aug 22. Loo TW, Bartlett MC, Clarke DM
Insertion of an arginine residue into the transmembrane segments corrects protein misfolding.
J Biol Chem. 2006 Oct 6;281(40):29436-40. Epub 2006 Aug 22., [PMID:16926162]

Abstract [show]
Deletion of Phe-508 (DeltaF508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (DeltaY490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gp-drug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drug-binding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress DeltaY490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature DeltaY490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of DeltaY490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule.

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No. Sentence Comment
81 The I306R mutation was introduced into mutants containing a processing mutation in the TM segments (G300V in TM5, G722A in TM7), the loops connecting the TM segments (G251V in intracellular loop 2, F804A in intracellular loop 3), the linker region (P709A), and NBD2 (L1260A) (Fig. 1A). Login to comment
83 The I306R mutation was more efficient, however, in promoting maturation of mutants G251V, G300V, P709A, and G722A than mutants F804A and L1260A. Login to comment
100 A, immunoblot analysis of whole cell extracts of HEK 293 cells expressing the A52-tagged P-gp processing mutants G251V, G300V, P709A, G722A, F804A, and L1260A with or without the I306R mutation. Login to comment

[hide] The Transmission Interfaces Contribute Asymmetrica... J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18. Loo TW, Clarke DM
The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.
J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18., [PMID:25987565]

Abstract [show]
P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.

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No. Sentence Comment
317 For example the CFTR N1303K (54) and P-gp L1260A (55) NBD2 mutations inhibit maturation of the proteins. Login to comment