ABCC1 p.Gly5Glu
| Predicted by SNAP2: | A: N (97%), C: N (61%), D: N (93%), E: N (93%), F: N (53%), H: N (87%), I: N (82%), K: N (93%), L: N (87%), M: N (93%), N: N (93%), P: N (82%), Q: N (93%), R: N (93%), S: N (97%), T: N (97%), V: N (87%), W: D (63%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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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
63 Cell Culture-Drug-sensitive human HeLa cells (ATCC) were grown in Dulbecco`s modified Eagle`s medium (high glucose) supplemented TABLE I Oligonucleotides used for epitope insertion by site-directed mutagenesis 1 Arg-4b (HA epitope)c Gly-5 Glu-127 (HA epitope) Arg-128 2 (363)a C TTT TTA ATT CAG CTG GAG AGG AGG AAG GGA GTT CAG (399) Glu-163 (HA epitope) Asp-164 3 (474) G ACA GCC TTA AAA GAG GAT GCC CAG GTG GAC (504) Gln-271 (HA epitope) Pro-272 4 (799) AAG ACTD AGG AAG CAG CCG GTG AAG GTT GTG TA (830) Ser-417 (HA epitope) Thr-419 5 (1233) C AAT TCA GCC AGA AAA TCC ACG GTC GGG GAG ATT G (1270) Asn-461 (HA epitope) Leu-462 6a (1369) CTC CTG TGG CTG AAT CTG GGC CCT TCC GTC (1398) 6b Asn-461 (HA epitope) ϫ 2 Leu-462 Leu-529 (HA epitope) Ala-530 7 (1572) C AAG GAC AAG GTG CTG GCT ATC AGG CAG GAG GA (1604) Asn-574 (HA epitope) Asn-575 8a (1707) G ACC ATT GAC GAG AAC AAT ATC CTG GAT GCC CA (1739) 8b Asn-574 (HA epitope) ϫ 2 Asn-575 9 Trp-653 (HA epitope) Ala-654 (HA epitope)c :Y P Y D V P D Y A S a Nucleotide position in sequence of MRP published by Cole et al. (10; 55).
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ABCC1 p.Gly5Glu 9334225:63:233
status: NEW