ABCC1 p.Pro235Ala
| Predicted by SNAP2: | A: D (59%), C: D (59%), D: D (63%), E: D (71%), F: D (63%), G: D (66%), H: D (66%), I: D (63%), K: D (71%), L: D (66%), M: D (66%), N: D (63%), Q: D (59%), R: D (75%), S: N (66%), T: D (53%), V: D (63%), W: D (66%), Y: D (63%), |
| 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] Mutation of proline residues in the NH(2)-terminal... Biochim Biophys Acta. 2003 Sep 2;1615(1-2):103-14. Ito K, Weigl KE, Deeley RG, Cole SP
Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein, MRP1 (ABCC1): effects on protein expression, membrane localization, and transport function.
Biochim Biophys Acta. 2003 Sep 2;1615(1-2):103-14., [PMID:12948592]
Abstract [show]
The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.
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No. Sentence Comment
50 MRP1-P235/255A was generated by introducing P255A into the MRP1-P235A construct.
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ABCC1 p.Pro235Ala 12948592:50:64
status: NEW107 The seven single (P196A, P205A, P207A, P209A, P235A, P255A, and P272A) and two multiply substituted CL3 mutants (P196/205/207/209A and P235/255A) could also be stably expressed in HeLa cells.
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ABCC1 p.Pro235Ala 12948592:107:46
status: NEW147 GFP-tagged MRP1 proteins are shown in green; nuclei were stained with propidium iodide and are shown in red. (C) HeLa cells stably expressing wild-type MRP1, MRP1-P235A, MRP1-P255A, MRP1-P235/255A, and MRP1-P196/205/207/209A were cultured and processed for immunofluorescence and confocal microscopy as described in panel A.
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ABCC1 p.Pro235Ala 12948592:147:163
status: NEW149 Duplicate immunoblots of the P235A, P255A, P235/255A, and P272A MRP1 mutants probed with MAb QCRL-1 and MAb MRPr1 indicated that detection of the P255A and P235/P255A mutant MRP1 proteins relative to wild-type MRP1 by MAb MRPr1 was lower than that by MAb QCRL-1 (not shown).
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ABCC1 p.Pro235Ala 12948592:149:29
status: NEW151 When MRP1-P235A was detected with MAb QCRL-1, the signal intensity was approximately 1.8-fold higher than that for wild-type MRP1, while the signal intensity for MRP1-P235/P255A was approximately 2.3-fold higher (Fig. 5A).
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ABCC1 p.Pro235Ala 12948592:151:10
status: NEW152 When MRP1-P235A was detected with MAb MRPr1, the signal intensity was also higher than that of wild-type MRP1 (approximately 1.4-fold) but the signal intensity for P235/255A was less than that of wild-type MRP1 (approximately 75%) (Fig. 5B).
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ABCC1 p.Pro235Ala 12948592:152:10
status: NEW153 Thus, MRP1-P235A Fig. 5.
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ABCC1 p.Pro235Ala 12948592:153:11
status: NEWX
ABCC1 p.Pro235Ala 12948592:153:94
status: NEW154 Dot blot analyses of MRP1 expression in membranes from HeLa cells expressing CL3 mutants MRP1-P235A, MRP1-P255A, and MRP1-P235/255A.
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ABCC1 p.Pro235Ala 12948592:154:94
status: NEW156 MRP1-P235A and MRP1-P235/255A mutants probed with (A) MAb QCRL-1 and (B) MAb MRPr1.
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ABCC1 p.Pro235Ala 12948592:156:5
status: NEW159 (A and B) Wild-type MRP1 (n); MRP1-P235A (E); MRP1-P235/255A (z).
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ABCC1 p.Pro235Ala 12948592:159:35
status: NEW167 Plasma membrane localization of CL3 MRP1-Pro mutants is unchanged in transfected HeLa cells The changes in immunoreactivity of the MRP1-P255A and MRP1-P235/255A mutant proteins suggested that the Pro255 to Ala substitution introduced a conformation change in CL3.
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ABCC1 p.Pro235Ala 12948592:167:160
status: NEW168 To determine whether this change might also affect plasma membrane trafficking of MRP1-P255A, transfected HeLa cells expressing this mutant as well as the MRP1-P235A and MRP1-P235/255A mutants were examined by confocal microscopy as before.
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ABCC1 p.Pro235Ala 12948592:168:48
status: NEWX
ABCC1 p.Pro235Ala 12948592:168:160
status: NEW169 Like wild-type MRP1, MRP1-P255A as well as MRP1-P235A and MRP1-P235/ 255A localized mostly to the plasma membrane of confluent HeLa cells (Fig. 4C).
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ABCC1 p.Pro235Ala 12948592:169:48
status: NEW188 Results were expressed relative to wild-type Table 1 Kinetic parameters of [3 H]LTC4 uptake by membrane vesicles prepared from transfected HeLa cells expressing MRP1-Pro mutants Transfected cell line Km (nM) Vmax (pmol minÀ 1 mgÀ 1 ) Normalized Vmax MSD1 mutants WT-MRP1 128 80 80 P42A 139 35 41 P51A 109 55 50 P42/51A 103 15 48 P69A 142 46 42 P110A 150 73 124 CL3 mutants WT-MRP1 88 57 57 P196A 143 25 52 P205A 63 14 88 P207A 81 75 110 P209A 112 121 85 P196/205/207/209A 90 87 94 P235A 91 45 30 P255A 99 110 67 P235/255A 97 81 35 P272A 80 59 109 The kinetic parameters of [3 H]LTC4 uptake were determined by regression analysis of the Eadie-Hofstee transformed data.
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ABCC1 p.Pro235Ala 12948592:188:491
status: NEW146 GFP-tagged MRP1 proteins are shown in green; nuclei were stained with propidium iodide and are shown in red. (C) HeLa cells stably expressing wild-type MRP1, MRP1-P235A, MRP1-P255A, MRP1-P235/255A, and MRP1-P196/205/207/209A were cultured and processed for immunofluorescence and confocal microscopy as described in panel A.
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ABCC1 p.Pro235Ala 12948592:146:163
status: NEW148 Duplicate immunoblots of the P235A, P255A, P235/255A, and P272A MRP1 mutants probed with MAb QCRL-1 and MAb MRPr1 indicated that detection of the P255A and P235/P255A mutant MRP1 proteins relative to wild-type MRP1 by MAb MRPr1 was lower than that by MAb QCRL-1 (not shown).
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ABCC1 p.Pro235Ala 12948592:148:29
status: NEW150 When MRP1-P235A was detected with MAb QCRL-1, the signal intensity was approximately 1.8-fold higher than that for wild-type MRP1, while the signal intensity for MRP1-P235/P255A was approximately 2.3-fold higher (Fig. 5A).
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ABCC1 p.Pro235Ala 12948592:150:10
status: NEW155 MRP1-P235A and MRP1-P235/255A mutants probed with (A) MAb QCRL-1 and (B) MAb MRPr1.
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ABCC1 p.Pro235Ala 12948592:155:5
status: NEW158 (A and B) Wild-type MRP1 (n); MRP1-P235A (E); MRP1-P235/255A (z).
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ABCC1 p.Pro235Ala 12948592:158:35
status: NEW187 Results were expressed relative to wild-type Table 1 Kinetic parameters of [3 H]LTC4 uptake by membrane vesicles prepared from transfected HeLa cells expressing MRP1-Pro mutants Transfected cell line Km (nM) Vmax (pmol min 1 mg 1 ) Normalized Vmax MSD1 mutants WT-MRP1 128 80 80 P42A 139 35 41 P51A 109 55 50 P42/51A 103 15 48 P69A 142 46 42 P110A 150 73 124 CL3 mutants WT-MRP1 88 57 57 P196A 143 25 52 P205A 63 14 88 P207A 81 75 110 P209A 112 121 85 P196/205/207/209A 90 87 94 P235A 91 45 30 P255A 99 110 67 P235/255A 97 81 35 P272A 80 59 109 The kinetic parameters of [3 H]LTC4 uptake were determined by regression analysis of the Eadie-Hofstee transformed data.
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ABCC1 p.Pro235Ala 12948592:187:479
status: NEW