ABCB1 p.Pro709Gly
| Predicted by SNAP2: | A: N (87%), C: N (93%), D: N (78%), E: N (82%), F: N (93%), G: N (93%), H: N (97%), I: N (93%), K: N (93%), L: N (93%), M: N (93%), N: N (87%), Q: N (87%), R: N (93%), S: N (93%), T: N (93%), V: N (93%), W: N (93%), Y: N (97%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]
Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.
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No. Sentence Comment
519 Loo et al. [256] demonstrated that mutations affecting the processing and targeting of Pgp disrupted interactions between the NBDs such as in DY490, G269V (ICL2), P709G (linker), G722A (TM7) and A841L (TM9).
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ABCB1 p.Pro709Gly 16442101:519:163
status: NEW[hide] The transmembrane domains of the human multidrug r... J Biol Chem. 1999 Aug 27;274(35):24759-65. Loo TW, Clarke DM
The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface.
J Biol Chem. 1999 Aug 27;274(35):24759-65., 1999-08-27 [PMID:10455147]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface.
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29 EXPERIMENTAL PROCEDURES Generation of Deletion Mutants-The cDNAs coding for full-length MDR1 (36), mutant P709G (37), or the N-half and C-half P-gp molecules (14) and containing the epitope for monoclonal antibody A52 at the C-terminal ends were subcloned into the mammalian expression vector pMT21.
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ABCB1 p.Pro709Gly 10455147:29:106
status: NEW43 For expression studies involving drug resistance assays, the cDNAs of the A52-tagged wild-type, mutant P709G, N-half, C-half, and TMD1ϩ2 were also inserted into the Epstein-Barr virus-based vector, pREP4 (Invitrogen Inc.).
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ABCB1 p.Pro709Gly 10455147:43:103
status: NEW137 The cDNAs for the A52-tagged wild-type, and P-gp mutants P709G, N-half, C-half, and TMD1ϩ2 were inserted into the pREP4 vector and transfected into HEK 293(EBNA-1) cells.
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ABCB1 p.Pro709Gly 10455147:137:57
status: NEW138 Mutant P709G was included because it shows similar processing defects as mutant TMD1ϩ2.
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ABCB1 p.Pro709Gly 10455147:138:7
status: NEW139 When mutant P709G is expressed in HEK 293 cells in the absence of drug substrate, the major product is a protein of 150 kDa that is retained in the endoplasmic reticulum as a core-glycosylated intermediate (37).
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ABCB1 p.Pro709Gly 10455147:139:12
status: NEW145 For mutant P709G, the major product in the absence of drug substrate was the 150-kDa (core-glycosylated) protein, while the 170-kDa protein was the major product in the presence of substrate. When N-half (90-kDa) P-gp was co-expressed with C-half P-gp in the presence of substrate, another N-half protein of 115 kDa was present.
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ABCB1 p.Pro709Gly 10455147:145:11
status: NEW155 Fig. 6A shows that the mature forms of wild-type, mutant P709G, N-half, and TMD1ϩ2 P-gps were still present in the cells after treatment with cyclosporin A followed by vinblastine.
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ABCB1 p.Pro709Gly 10455147:155:57
status: NEW170 In mutant P709G or in the half-molecules of P-gp, preincubation of the cells with cyclosporin A greatly increased the relative resistance to vinblastine.
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ABCB1 p.Pro709Gly 10455147:170:10
status: NEW171 The cells expressing mutant P709G that were not pretreated with cyclosporin A showed about an 8-fold increase in resistance to vinblastine relative to the control cells.
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ABCB1 p.Pro709Gly 10455147:171:28
status: NEW195 A, HEK 293(EBNA-1) cells were transfected with pREP4 vector (B, Control) or pREP4 vectors containing cDNAs for full-length wild-type P-gp (Wild), mutant P709G, or TMD1ϩ2, or they were cotransfected with the cDNAs for the N-half and C-half of P-gp (Halves).
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ABCB1 p.Pro709Gly 10455147:195:153
status: NEW[hide] Thapsigargin or curcumin does not promote maturati... Biochem Biophys Res Commun. 2004 Dec 10;325(2):580-5. 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., [PMID:15530432]
Abstract [show]
Misprocessed plasma membrane proteins of CFTR and P-glycoprotein (P-gp) are retained in the endoplasmic reticulum (ER) by molecular chaperones. Depletion of the calcium stores in the ER by the SERCA calcium pump inhibitors thapsigargin or curcumin inhibits these interactions and allows the protein to be trafficked to the plasma membrane [Nat. Med. 8 (2002) 485; Science 304 (2004) 600]. We tested this hypothesis by treating various cell lines expressing misprocessed mutants of CFTR or P-gp with thapsigargin or curcumin. Conversion of the immature core-glycosylated protein to mature product was detected by immunoblot analysis of whole cell extracts. Mature product was not detected in any of the misprocessed mutants. By contrast, all misprocessed P-gp mutants were rescued by the chemical chaperone/drug substrate cyclosporin A in a dose-dependent manner. These results show that thapsigargin or curcumin is not effective in rescuing misprocessed mutants of P-gp and CFTR.
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No. Sentence Comment
69 A processing mutation in NBD2 is P1194A, while P709G is in the linker region.
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ABCB1 p.Pro709Gly 15530432:69:47
status: NEW