ABCC2 p.Gly418Arg
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (91%), E: D (91%), F: D (91%), H: D (91%), I: D (85%), K: D (91%), L: D (91%), M: D (85%), N: D (80%), P: D (91%), Q: D (85%), R: D (91%), S: D (75%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Topology mapping of the amino-terminal half of mul... J Biol Chem. 1997 Oct 17;272(42):26479-87. Kast C, Gros P
Topology mapping of the amino-terminal half of multidrug resistance-associated protein by epitope insertion and immunofluorescence.
J Biol Chem. 1997 Oct 17;272(42):26479-87., [PMID:9334225]
Abstract [show]
The multidrug resistance-associated protein (MRP) is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells. Within the ATP-binding cassette superfamily, MRP belongs to a subgroup of structurally and functionally related proteins that includes the yeast cadmium factor 1 and yeast oligomycin resistance I proteins, and the mammalian sulfonylurea receptors SUR1 and SUR2. Hydropathy analysis of these proteins predicts a unique membrane-associated region at the amino terminus followed by a structural unit composed of 12 transmembrane (TM) domains and two nucleotide-binding domains that is characteristic of eukaryotic ATP-binding cassette transporters. The topology of the membrane-associated regions of MRP remains largely unknown and was investigated. Small hemagglutinin epitopes (YPYDVPDYAS) were inserted in predicted hydrophilic segments of the membrane-associated regions from the amino-terminal half of MRP and these proteins were expressed in HeLa cells, and tested for their capacity to confer etoposide resistance. The polarity of the inserted tags with respect to plasma membrane was then deduced by immunofluorescence in intact and permeabilized cells. Insertion of epitopes at positions 4, 163, 271, 574, and 653 produced functional proteins while insertions at positions 127, 417, 461, and 529 abrogated the capacity of MRP to confer drug resistance. Epitopes inserted at positions 4, 163, and 574 were localized extracellularly, whereas those inserted at positions 271 and 653 revealed an intracellular location. Although a single epitope inserted at position 461 was compatible with MRP function, it was inaccessible to the anti-epitope antibody and two copies of the tag at that site abrogated MRP function. These results indicate that the amino terminus of MRP is extracellular, while the linker segment joining the first and second membrane-associated regions is intracellular as is the first nucleotide-binding domain. Our findings are therefore consistent with a topological model of MRP that contains 5 TM segments in the first membrane-associated region and 6 TM segments in the second membrane region.
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No. Sentence Comment
69 Mass populations of G418R transfectants were harvested after 9 days and subcultured in medium containing the VP-16 (final concentration 250 ng/ml) to select mass populations of drug-resistant cell clones overexpressing the tagged MRP proteins.
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ABCC2 p.Gly418Arg 9334225:69:20
status: ERRONEOUS109 Stable transfectants were selected in G418 and mass populations of G418R colonies were further selected in VP-16 (250 ng/ml).
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ABCC2 p.Gly418Arg 9334225:109:67
status: ERRONEOUS[hide] Epitope insertion favors a six transmembrane domai... Biochemistry. 1998 Feb 24;37(8):2305-13. Kast C, Gros P
Epitope insertion favors a six transmembrane domain model for the carboxy-terminal portion of the multidrug resistance-associated protein.
Biochemistry. 1998 Feb 24;37(8):2305-13., [PMID:9485377]
Abstract [show]
The overexpression of the multidrug resistance protein, MRP, in mammalian cells is associated with pleiotropic resistance to cytotoxic drugs. MRP is an integral membrane protein which belongs to the family of ATP-binding cassette transporters. Secondary structure predictions combined with biochemical analyses suggest that MRP encodes 11 transmembrane (TM) domains in the amino-terminal half of the protein and four or six transmembrane domains in the carboxy-terminal half of the protein. To gain insight into the membrane topology of the carboxy-terminal half of MRP, small, antigenic hemagglutinin (HA) epitopes (YPYDVPDYAS) were inserted within six predicted hydrophilic subfragments of this region (938, 1001, 1084, 1175, 1222, 1295). These epitope-tagged MRP variants were expressed in HeLa cells to evaluate their ability to confer resistance to the drug etoposide (VP-16). Insertion of the HA epitopes at positions 938, 1001, and 1222 resulted in functional proteins, while epitope insertion at positions 1084, 1175, and 1295 abrogated MRP function. The intracellular versus extracellular location of the HA epitopes present in biologically active MRP variants was then established in intact and permeabilized cells by immunofluorescence using an anti-HA antibody. Epitopes inserted at positions 1001 and 1222 were located on the extracellular side of the plasma membrane, while the epitope inserted at position 938 was located intracellularly. These results are consistent with a six TM rather than a four TM domain model for the membrane portion of the carboxy-terminal half of MRP.
Comments [show]
None has been submitted yet.
No. Sentence Comment
65 Mass populations of G418R transfectants were harvested after 9 days and subcultured in medium containing the etoposide VP-16 (final concentration 250 ng/mL) to select mass populations of drug-resistant cell clones overexpressing the tagged MRP proteins.
X
ABCC2 p.Gly418Arg 9485377:65:20
status: NEW105 Stable transfectants were selected in G418, and mass populations of G418R colonies were further selected in VP-16 (250 ng/ mL).
X
ABCC2 p.Gly418Arg 9485377:105:68
status: NEW106 Drug-resistant colonies emerged within 1-2 weeks of selection in populations of G418R cells transfected with wild-type MRP or with MRP constructs 1, 2a, 2b, 5a, and Table 1: Oligonucleotides Used for Epitope Insertion by Site-Directed Mutagenesis 5b.
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ABCC2 p.Gly418Arg 9485377:106:80
status: NEW