ABCMdb

ABCC1 p.Pro255Ala

Predicted by SNAP2:	A: N (78%), C: N (87%), D: N (78%), E: N (72%), F: D (59%), G: N (66%), H: N (87%), I: N (66%), K: N (82%), L: N (72%), M: N (82%), N: N (87%), Q: N (93%), R: N (78%), S: N (87%), T: N (82%), V: N (78%), W: D (59%), Y: N (53%),
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
107 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. Login to comment
147 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. Login to comment
149 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). Login to comment
151 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). Login to comment
153 Thus, MRP1-P235A Fig. 5. Login to comment
154 Dot blot analyses of MRP1 expression in membranes from HeLa cells expressing CL3 mutants MRP1-P235A, MRP1-P255A, and MRP1-P235/255A. Login to comment
156 MRP1-P235A and MRP1-P235/255A mutants probed with (A) MAb QCRL-1 and (B) MAb MRPr1. Login to comment
157 MRP1-P255A and MRP1-P235/255A mutants probed with (C) MAb QCRL-1 and (D) MAb MRPr1. Login to comment
159 (A and B) Wild-type MRP1 (n); MRP1-P235A (E); MRP1-P235/255A (z). Login to comment
160 (C and D) Wild-type MRP1 (n); MRP1-P255A (E); MRP1-P235/255A (z). Login to comment
162 We next compared the immunoreactivity of the double mutant MRP1-P235/255A and the single mutant MRP1-P255A. Login to comment
163 When detected with MAb QCRL-1, the signal intensity for MRP1-P255A was approximately 1.3-fold higher than that for wild-type MRP1 (Fig. 5C). Login to comment
164 In contrast, the signal intensity of MRP1-P255A detected with MAb MRPr1 was only 50% that of wild-type MRP1 detected with the same MAb (Fig. 5D). Login to comment
165 Overall, these findings suggest that substitution of Pro255 with Ala might introduce a change in the structure of this region of MRP1 such that the MRPr1 epitope is less accessible to the MAb. Login to comment
167 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. Login to comment
168 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. Login to comment
169 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). Login to comment
170 Thus, despite the apparent change in conformation of MRP1 proteins containing the Pro255-Ala substitution, their expression at the plasma membrane remained unchanged. Login to comment
188 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. Login to comment
242 Indeed, substitution of Pro255 with Ala caused a significant decrease in MRP1 reactivity with MAb MRPr1 but not MAb QCRL-1, presumably reflecting some conformational change in this region of the protein. Login to comment
243 Nevertheless, neither the P255A mutant nor the P235/255A double mutant showed major changes in plasma membrane localization or organic anion transport. Login to comment
244 The change in MAb MRPr1 reactivity caused by the P255A substitution was unexpected. Login to comment
146 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. Login to comment
148 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). Login to comment
150 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). Login to comment
161 We next compared the immunoreactivity of the double mutant MRP1-P235/255A and the single mutant MRP1-P255A. Login to comment
166 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. Login to comment
187 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. Login to comment
241 Indeed, substitution of Pro255 with Ala caused a significant decrease in MRP1 reactivity with MAb MRPr1 but not MAb QCRL-1, presumably reflecting some conformational change in this region of the protein. Login to comment