ABCC2 p.Thr1543Ala
| Predicted by SNAP2: | A: N (72%), C: N (78%), D: N (61%), E: N (66%), F: N (57%), G: N (66%), H: N (66%), I: N (61%), K: N (82%), L: N (66%), M: N (66%), N: N (78%), P: N (61%), Q: N (87%), R: N (57%), S: N (93%), V: N (66%), W: N (53%), Y: N (57%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, V: N, W: N, Y: N, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Structural requirements for the apical sorting of ... Eur J Biochem. 2002 Apr;269(7):1866-76. Nies AT, Konig J, Cui Y, Brom M, Spring H, Keppler D
Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2).
Eur J Biochem. 2002 Apr;269(7):1866-76., [PMID:11952788]
Abstract [show]
The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin-Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP-MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP-MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells.
Comments [show]
None has been submitted yet.
No. Sentence Comment
138 We therefore deleted the C-terminal three amino acids or substituted threonine with alanine at position 1543.
X
ABCC2 p.Thr1543Ala 11952788:138:69
status: NEW158 Asacontrol,localizationofGFP-MRP2,GFP-MRP2D3, and GFP-MRP2-T1543A was also analyzed in MDCKII cells grown polarized on Transwell filter membranes (Fig. 6).
X
ABCC2 p.Thr1543Ala 11952788:158:59
status: NEW160 GFP-MRP2, GFP-MRP2D3, and GFP-MRP2-T1543A were almost exclusively present in the apical membrane with some GFP fluorescence also present in intracellular compartments.
X
ABCC2 p.Thr1543Ala 11952788:160:35
status: NEW192 Because HepG2 cells endogenously Table 1. Quantitative analysis of the subcellular localization of C-terminally mutated GFP-MRP2 constructs in polarized HepG2 cells. Data are percentages of cells in which the respective localization of recombinant protein was observed as described in Materials and methods. Cells were observed 2 days after transfection. Data are means ±SD of six transient transfections using butyrate-induced cells as described under Materials and methods. Construct % Apical % Vesicles % ER C-Terminal sequence (1516-1545) GFP-MRP2 73 ± 9 18 ± 9 9 ± 5 GSPEELLQIPGPFYFMAKEAGIENVNSTKF GFP-MRP2D3 64 ± 9 13 ± 5 23 ± 9 GSPEELLQIPGPFYFMAKEAGIENVNS GFP-MRP2-T1543A 67 ± 6 16 ± 2 17 ± 6 GSPEELLQIPGPFYFMAKEAGIENVNSAKF GFP-MRP2D15 16 ± 7 17 ± 7 67 ± 14 GSPEELLQIPGPFYF GFP-MRP2D15TKF 21 ± 11 21 ± 7 58 ± 11 GSPEELLQIPGPFYFTKF Fig. 6.
X
ABCC2 p.Thr1543Ala 11952788:192:707
status: NEW193 Localization of GFP-MRP2 (green in A,B), GFP-MRP2D3 (green in C,D), and GFP-MRP2-T1543A (green in E,F) in polarized MDCKII cells.
X
ABCC2 p.Thr1543Ala 11952788:193:81
status: NEW197 The intense yellow color in the x-z planes, due to merging of the green GFP and the red concanavalin A fluorescence, shows that GFP-MRP2, GFP-MRP2D3, and GFP-MRP2-T1543A are almost exclusively localized in the apical membrane.
X
ABCC2 p.Thr1543Ala 11952788:197:163
status: NEW[hide] Identification of the apical membrane-targeting si... J Biol Chem. 2001 Jun 15;276(24):20876-81. Epub 2001 Mar 27. Harris MJ, Kuwano M, Webb M, Board PG
Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT).
J Biol Chem. 2001 Jun 15;276(24):20876-81. Epub 2001 Mar 27., [PMID:11274200]
Abstract [show]
The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules. In these tissues MRP2 specifically localizes to the apical membrane. The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells. The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1. Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells. Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization. In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.
Comments [show]
None has been submitted yet.
No. Sentence Comment
97 The T1543A and K1544A mutants had both apical and basolateral targeting (nonpolarized distribution) with an increase in protein accumulation in intracellular vesicles.
X
ABCC2 p.Thr1543Ala 11274200:97:4
status: NEW109 A, the T1543A mutation produced a nonpolarized distribution of the fusion protein.
X
ABCC2 p.Thr1543Ala 11274200:109:7
status: NEW162 The T1543A mutant did produce a change in targeting compared with the native protein, allowing both basolateral and apical targeting, i.e. nonpolarized targeting, and also an increased accumulation in vesicles, suggesting some instability in the targeting mechanism.
X
ABCC2 p.Thr1543Ala 11274200:162:4
status: NEW