ABCC1 p.Pro272Ala
| Predicted by SNAP2: | A: N (87%), C: N (78%), D: N (72%), E: N (82%), F: N (72%), G: N (78%), H: N (93%), I: N (78%), K: N (82%), L: N (78%), M: N (72%), N: N (93%), Q: N (87%), R: N (78%), S: N (87%), T: N (82%), V: N (78%), W: N (82%), Y: N (82%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: N, K: N, L: N, M: N, N: N, Q: N, R: N, S: N, T: N, V: N, 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
47 For substitution of Pro residues in the CL3 loop connecting MSD1 to MSD2, the mutagenic primers were: MRP1-P196A (5V-CA GAT CGC TCA GCC CTG TTC TC-3V), MRP1-P205A (5V-C ATC CAC GAC GCT AAT CCC TG-3V), MRP1-P207A (5V-C GAC CCT AAT GCC TGC CCA GAG-3V), MRP1-P209A (5V-CTA ATC CCT GCG CAG AGT CCA G-3V), MRP1-P235A (5V-C TAC CGC CAG GCC CTG GAG GG-3V), MRP1-P255A (5V-CG GAA CAA GTC GTG GCT GTT TTG G-3V), MRP1-P272A (5V-CT AGG AAG CAG GCG GTG AAG G-3V).
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ABCC1 p.Pro272Ala 12948592:47:408
status: NEWX
ABCC1 p.Pro272Ala 12948592:47:420
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.Pro272Ala 12948592:107:64
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.Pro272Ala 12948592:149:58
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.Pro272Ala 12948592:188:541
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.Pro272Ala 12948592:148:58
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.Pro272Ala 12948592:187:529
status: NEW