ABCMdb

PMID: 12948592

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., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC1 p.Pro104Ala All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. Login to comment 46 ABCC1 p.Pro51Ala ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro104Ala ABCC1 p.Pro104Ala ABCC1 p.Pro69Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala ABCC1 p.Pro110Ala For the single substitutions of Pro with Ala in MSD1, the following sense mutagenic primers were used (substituted nucleotides are underlined): MRP1-P42A (5V-C GTG TGG GTG GCT TGT TTT TAC CTC TGG-3V), MRP1-P51A (5V-G GCC TGT TTC GCC TTC TAC TTC C-3V), MRP1-P69A (5V-CAG ATG ACA GCT CTC AAC-3V), MRP1-P104A (5V-C ATA TTC CTG GCC GCA GTG TTT CTG G-3V), MRP1-P110A (5V-GT TTC TGG TCA GCG CAA CTC TCT TGG-3V). Login to comment 47 ABCC1 p.Pro196Ala ABCC1 p.Pro272Ala ABCC1 p.Pro272Ala ABCC1 p.Pro207Ala ABCC1 p.Pro207Ala ABCC1 p.Pro209Ala ABCC1 p.Pro209Ala 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). Login to comment 49 ABCC1 p.Pro196Ala The quadruple MRP1-P196/205/207/209A construct was generated by introducing the P205/207/209A mutations into the MRP1-P196A construct. Login to comment 50 ABCC1 p.Pro235Ala MRP1-P235/255A was generated by introducing P255A into the MRP1-P235A construct. Login to comment 103 ABCC1 p.Pro104Ala Despite multiple attempts, stable HeLa cell lines expressing MRP1-P104A and MRP1-P104/110A could not be established. Login to comment 104 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala Immunoblots of membrane vesicles prepared from the cell lines expressing MSD1 MRP1 mutants containing single (P42A, P51A, P69A and P110A) and double (P42/51A) Pro substitutions detected with MAb QCRL-1 are shown in Fig. 2A. The relative expression levels of the different mutants ranged from < 20% to approximately 150% that of wild-type MRP1. Login to comment 107 ABCC1 p.Pro196Ala ABCC1 p.Pro272Ala ABCC1 p.Pro207Ala ABCC1 p.Pro209Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro205Ala 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 117 ABCC1 p.Pro51Ala ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala Similarly, the singly substituted MRP1-Pro mutants P42A, P51A, P69A, and P110A localized strongly to the plasma membrane, similar to wild-type MRP1 (MRP1-P42A and MRP1-P51A shown in Fig. 4A, middle two panels). Login to comment 141 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala (A) HeLa cells expressing wild-type MRP1, MRP1-P42A, MRP1-P51A, and MRP1-P42/51A were seeded at 1.5 Â 106 cells per well and processed 24 h later for indirect immunofluorescence and confocal microscopy. Login to comment 146 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala 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 147 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro272Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 149 ABCC1 p.Pro272Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala 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 151 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala 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 152 ABCC1 p.Pro235Ala ABCC1 p.Pro235Ala 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). Login to comment 153 ABCC1 p.Pro235Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala Thus, MRP1-P235A Fig. 5. Login to comment 154 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala 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 155 ABCC1 p.Pro235Ala MRP1-P235A and MRP1-P235/255A mutants probed with (A) MAb QCRL-1 and (B) MAb MRPr1. Login to comment 156 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala MRP1-P235A and MRP1-P235/255A mutants probed with (A) MAb QCRL-1 and (B) MAb MRPr1. Login to comment 157 ABCC1 p.Pro255Ala MRP1-P255A and MRP1-P235/255A mutants probed with (C) MAb QCRL-1 and (D) MAb MRPr1. Login to comment 158 ABCC1 p.Pro235Ala (A and B) Wild-type MRP1 (n); MRP1-P235A (E); MRP1-P235/255A (z). Login to comment 159 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala (A and B) Wild-type MRP1 (n); MRP1-P235A (E); MRP1-P235/255A (z). Login to comment 160 ABCC1 p.Pro255Ala (C and D) Wild-type MRP1 (n); MRP1-P255A (E); MRP1-P235/255A (z). Login to comment 161 ABCC1 p.Pro255Ala We next compared the immunoreactivity of the double mutant MRP1-P235/255A and the single mutant MRP1-P255A. Login to comment 162 ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala We next compared the immunoreactivity of the double mutant MRP1-P235/255A and the single mutant MRP1-P255A. Login to comment 163 ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro255Ala 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 166 ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 167 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro235Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro255Ala 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 179 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala Thus, all of the MSD1 Pro mutants retained the ability to transport LTC4 with TM1 mutants P42A, P51A, and P69A showing an approximately 30-50% decrease in relative LTC4 transport efficiency, while P110A (TM3) showed an increase of approximately 50%. Login to comment 180 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala Thus, all of the MSD1 Pro mutants retained the ability to transport LTC4 with TM1 mutants P42A, P51A, and P69A showing an approximately 30-50% decrease in relative LTC4 transport efficiency, while P110A (TM3) showed an increase of approximately 50%. Login to comment 187 ABCC1 p.Pro196Ala ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala ABCC1 p.Pro272Ala ABCC1 p.Pro207Ala ABCC1 p.Pro209Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro205Ala 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 188 ABCC1 p.Pro196Ala ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala ABCC1 p.Pro272Ala ABCC1 p.Pro207Ala ABCC1 p.Pro209Ala ABCC1 p.Pro235Ala ABCC1 p.Pro255Ala ABCC1 p.Pro205Ala 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 192 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala E217hG uptake levels by the four single Pro MSD1 mutants P42A, P51A, P69A, and P110A were 48%, 43%, 95%, and 95% those of wild-type MRP1, respectively. Login to comment 193 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala ABCC1 p.Pro69Ala ABCC1 p.Pro110Ala E217hG uptake levels by the four single Pro MSD1 mutants P42A, P51A, P69A, and P110A were 48%, 43%, 95%, and 95% those of wild-type MRP1, respectively. Login to comment 221 ABCC1 p.Cys7Ala Consistent with this idea are the recent findings of Yang et al. [36] which showed that replacement of Cys7 with Ala caused an almost complete loss of LTC4 transport activity and a change in conformation of the NH2 terminus although expression levels and plasma membrane localization of this mutant MRP1 were not diminished compared to wild-type MRP1. Login to comment 222 ABCC1 p.Cys7Ala Consistent with this idea are the recent findings of Yang et al. [36] which showed that replacement of Cys7 with Ala caused an almost complete loss of LTC4 transport activity and a change in conformation of the NH2 terminus although expression levels and plasma membrane localization of this mutant MRP1 were not diminished compared to wild-type MRP1. Login to comment 241 ABCC1 p.Pro255Ala 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 242 ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro255Ala ABCC1 p.Pro255Ala 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 ABCC1 p.Pro255Ala The change in MAb MRPr1 reactivity caused by the P255A substitution was unexpected. Login to comment 245 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala Models illustrating the predicted differences in the a-helical axis of TM1 in wild-type MRP1 and MRP1-Pro mutants P42A, P51A, and P42/ 51A. Login to comment 246 ABCC1 p.Pro51Ala ABCC1 p.Pro42Ala Models illustrating the predicted differences in the a-helical axis of TM1 in wild-type MRP1 and MRP1-Pro mutants P42A, P51A, and P42/ 51A. Login to comment