ABCMdb

PMID: 11284689

Ruth A, Stein WD, Rose E, Roninson IB
Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein.
Biochemistry. 2001 Apr 10;40(14):4332-9., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCB1 p.Gly185Val The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. Login to comment 2 ABCB1 p.Gly185Val MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. Login to comment 3 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn This selection yielded the I186N substitution, adjacent to G185V. Login to comment 4 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. Login to comment 5 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. Login to comment 21 ABCB1 p.Gly185Val ABCB1 p.Gly185Val The first altered-function mutation to be identified in ABC transporters was the substitution of glycine to valine at position 185 (G185V), adjacent to TM3 of the human MDR1 Pgp (11). Login to comment 31 ABCB1 p.Gly185Val At the same time, G185V decreased cellular resistance to vinblastine and Taxol, compared to that of the wild-type Pgp (11, 12), and altered the sensitivity of Pgp to different inhibitors (13, 14). Login to comment 34 ABCB1 p.Gly185Val Biochemical assays showed that G185V enhances the ability of colchicine and inhibits the ability of vinblastine to stimulate the ATPase activity of Pgp (17, 18). Login to comment 35 ABCB1 p.Gly185Val On the other hand, G185V was found to decrease the level of Pgp binding of a photoactive analogue of colchicine and to increase the level of binding of a vinblastine analogue, suggesting that this mutation changes the transport efficiency by affecting the release of the substrates from Pgp to the outside of the cell (12). Login to comment 38 ABCB1 p.Gly185Val The first functional mutation identified by this selection turned out to affect amino acid 186, which is immediately adjacent to the previously identified G185V substitution. Login to comment 50 ABCB1 p.Gly185Val The procedures for the propagation of retroviral plasmid vector pLMDR1 (23), carrying MDR1 cDNA with a G185V mutation, in the mutD strain of Escherichia coli, transfection of BOSC 23 ecotropic packaging cells (24), retroviral transduction of NIH 3T3 cells, etoposide selection, and PCR-based recovery and recloning of the integrated MDR1 cDNA have been previously described (19). Login to comment 75 ABCB1 p.Ile186Asn Analysis of the FACS results was performed using the SigmaPlot curve-fitting plot program (SISS), to determine the maximum or minimum fluorescence, the concentration of drug that gave half-maximal change in fluorescence (Km), and the Hill number, n. The best-fit regression through the data points was determined using the binding isotherm given by the following equation: RESULTS Isolation of the I186N Mutant Conferring Increased Resistance to Etoposide. Login to comment 76 ABCB1 p.Gly185Val To isolate Pgp mutants that would further increase the resistance to etoposide conferred by the previously described G185V mutant, we have used the strategy of function-based selection of mutagenized retroviral vectors (19). Login to comment 77 ABCB1 p.Gly185Val A retroviral plasmid vector carrying the coding sequence of human MDR1 cDNA with the G185V mutation was propagated in a mutator strain of E. coli, and the resulting randomly mutagenized retrovirus was transduced into murine NIH 3T3 cells. Login to comment 84 ABCB1 p.Gly185Val One of the selected mutants produced elevated etoposide resistance relative to that of the parental G185V MDR1 cDNA. Login to comment 85 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn This mutant was found to carry a single ATT f AAT mutation of codon 186, which resulted in a change of isoleucine to asparagine at the corresponding position (I186N), adjacent to the original G185V substitution. Login to comment 86 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Effects of G185V and I186N Substitutions on Drug Resistance Profiles. Login to comment 87 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn To confirm that the I186N mutation was indeed responsible for elevated etoposide resistance and to investigate how this mutation interacts with G185V, we used site-directed mutagenesis to introduce the I186N substitution into both G185V and the wild-type versions of the MDR1 cDNA. Login to comment 88 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn Retroviral vectors carrying the wild-type MDR1, MDR1 carrying the individual G185V or I186N mutation, and MDR1 with both G185V and I186N mutations (G185V/ I186N) were individually transduced into NIH 3T3 cells. Login to comment 93 ABCB1 p.Gly185Val The etoposide assays (Figure 2A) showed that the G185V (O) substitution increased etoposide resistance relative to cells transduced with the wild-type MDR1 (b), in agreement with our previous report (12). Login to comment 94 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The etoposide resistance of G185V was further increased by combining it with I186N [G185V/I186N (4)]. Login to comment 95 ABCB1 p.Ile186Asn I186N (2) alone produced the same intermediate increase in etoposide resistance as F ) Fmin + [(Fmax - Fmin)Bn ]/(Km n + Bn ) FIGURE 1: Pgp expression in NIH 3T3 cells transduced with wild-type or mutant MDR1. Login to comment 97 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The overlapping FACS profiles represent four different NIH 3T3 populations expressing either the wild-type MDR1 or MDR1 mutants G185V, I186N, or G185V/I186N (double mutant). Login to comment 100 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn NIH 3T3 cells were transduced with insert-free LXSN vector (9), with the wild-type MDR1 (b), with G185V (O), with I186N (1), or with G185V/I186N (3). Login to comment 103 ABCB1 p.Gly185Val G185V (Figure 2A). Login to comment 105 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Colchicine assays (Figure 2C) showed that both I186N and G185V increased colchicine resistance relative to the wild-type MDR1, but G185V (a mutation that was originally found in colchicine-selected cells) had a stronger effect. Login to comment 106 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Colchicine resistance conferred by G185V/I186N was indistinguishable from the effect of G185V alone. Login to comment 107 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn A major functional difference between G185V and I186N mutations was observed, however, in the vinblastine (Figure 2D) and Taxol (Figure 2E) assays. Login to comment 108 ABCB1 p.Gly185Val In agreement with our previous reports (11, 12), G185V decreased the resistance to both of these drugs relative to the wild-type MDR1. Login to comment 109 ABCB1 p.Ile186Asn In contrast, I186N increased vinblastine and Taxol resistance relative to that of the wild type. Login to comment 110 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn These opposite effects of the two mutations were balanced out in the G185V/I186N double mutant, which produced vinblastine and Taxol resistances that were indistinguishable from that of the wild type (Figure 2D,E). Login to comment 111 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn These results indicate that I186N increases the ability of MDR1 to confer resistance to all the tested drugs and augments the effect of G185V in providing a high level of etoposide resistance. Login to comment 112 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Analysis of the Effects of the G185V and I186N Mutations on Drug-Induced Conformational Transitions of Pgp Using UIC2 ImmunoreactiVity. Login to comment 114 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn We have used changes in UIC2 reactivity to investigate how G185V and I186N mutations change the effects of transported drugs on the conformation of Pgp. Login to comment 119 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn NIH 3T3 cells were transduced with wild-type MDR1 (b), with G185V (O), with I186N (1), or with G185V/I186N (3). Login to comment 123 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn Measurement of the level of cellular ATP in oligomycin-treated and control untreated samples showed that oligomycin decreased cellular ATP levels to 3.2, 3.3, 3.4, and 2.9% of the control in the wild type, G185V, I186N, and G185V/ I186N, respectively. Login to comment 131 ABCB1 p.Gly185Val The G185V mutation had no effect on UIC2 reactivity in the absence of drugs, but it raised the reactivity in the presence of all the drugs or oligomycin to a level similar to that of MRK16 (Figure 4). Login to comment 132 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Unlike G185V, I186N produced different effects on the fold increase in UIC2 reactivity by different drugs. Login to comment 134 ABCB1 p.Ile186Asn Interestingly, oligomycin reproducibly failed to raise the UIC2 reactivity of I186N Pgp to the maximal level, although a stronger effect was observed when oligomycin was combined with vinblastine or colchicine (Figure 4). Login to comment 135 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The G185V/I186N double mutant showed the same increase in UIC2 reactivity with all three drugs and with oligomycin as I186N alone (Figure 4). Login to comment 143 ABCB1 p.Ile186Asn DISCUSSION The I186N Mutation Increases the Pgp ActiVity. Login to comment 150 ABCB1 p.Ile186Asn In the study presented here, we have identified a novel mutation, I186N, which improves the ability of Pgp to confer resistance to different cytotoxic drugs. Login to comment 151 ABCB1 p.Gly185Val As shown in many previous studies, including those that analyzed the effects of G185V on the accumulation (12) and transport (15) of different drugs, changes in Pgp-mediated drug resistance reflect the corresponding changes in the outward drug pumping. Login to comment 152 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn We also investigated the effects of I186N, together with a previously identified specificity-altering mutation of the adjacent amino acid, G185V, on substrate-induced conformational transitions of the transporter. Login to comment 153 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn This analysis revealed that mutations that affect the ability of Pgp to transport Table 1: Effects of G185V and I186N Mutations on the Relative Resistance and Conformational Effects of Vinblastine, Colchicine, and Etoposidea changes in UIC2 reactivity MDR1 mutant relative resistance fold increase Km (µM) Hill number vinblastine wild type 7.8 2.2 0.24 ( 0.01 2.1 ( 0.2 G185V 5.7 3.9 0.10 ( 0.01 1.2 ( 0.2 I186N 10.8 3.8 0.44 ( 0.02 1.9 ( 0.3 G185V/I186N 8.1 4.2 0.24 ( 0.04 0.9 ( 0.1 colchicine wild type 4.5 2.3 1700 ( 400 0.7 ( 0.1 G185V 15.8 3.6 2400 ( 200 2.0 ( 0.4 I186N 8.6 1.8 5700 ( 1200 1.8 ( 0.2 G185V/I186N 14.6 1.9 4800 ( 500 3.7 ( 1.1 etoposide wild type 4.3 1.7 220 ( 10 2.1 ( 0.2 G185V 9.3 3.8 480 ( 30 2.9 ( 0.6 I186N 9.3 2.5 600 ( 70 3.3 ( 0.2 G185V/I186N 13.1 2.4 730 ( 20 4.2 ( 0.3 a Relative resistance is the ratio of the LD50 of NIH 3T3 cells transduced with the corresponding form of MDR1 to the LD50 of cells transduced with the control vector LXSN. Login to comment 159 ABCB1 p.Gly185Val This residue abuts TM3 and is immediately adjacent to the G185V mutation, which was present in the original MDR1 cDNA used for mutagenesis and which was already known to increase etoposide resistance. Login to comment 160 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn This coincidence raised the possibility that I186N could act by augmenting the effect of G185V and would only be functional in combination with the latter mutation. Login to comment 161 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Testing the effects of G185V and I186N mutations individually and in combination showed that this is not the case. Login to comment 162 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn I186N alone increased etoposide resistance to the same extent as G185V (~2-fold), while combining these two mutations enhanced the ability of Pgp to confer etoposide resistance approximately 3-fold relative to that of the wild-type protein. Login to comment 163 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Another possibility suggested by the proximity of the two mutations was that I186N would affect Pgp function in the same way as G185V. Login to comment 165 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn While G185V increased the resistance to some Pgp-transported drugs, it also decreased the activity of Pgp toward other substrates, whereas I186N increased the Pgp activity toward all five of the tested drugs. Login to comment 166 ABCB1 p.Ile186Asn To the best of our knowledge, I186N is the first reported Pgp mutation with such a uniformly positive effect on the drug transport functions of Pgp. Login to comment 168 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn G185V and I186N Mutations Change the Parameters of Drug-Induced Conformational Transitions of Pgp. Login to comment 169 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn The mechanistic effects of the G185V and I186N mutations were approached by analyzing the effects of drugs on the conformation of Pgp, as measured by increased UIC2 reactivity in the presence of drugs. Login to comment 173 ABCB1 p.Gly185Val This limit of UIC2 reactivity in NIH 3T3 cells was overcome by the mutation G185V, which increased the highest levels of UIC2 reactivity in the presence of all three tested transport substrates and oligomycin to the levels obtained with MRK16. Login to comment 174 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Unlike the G185V mutation, the I186N mutation allowed the maximal level of UIC2 reactivity in the presence of vinblastine, but not in the presence of etoposide, colchicine, or oligomycin. Login to comment 175 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The G185V/I186N double mutant was essentially indistinguishable in this respect from I186N alone, indicating that the restrictions on the UIC2 reactivity shift imposed by I186N are dominant over the "releasing" effect of G185V. Login to comment 177 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Meaningful correlations with drug resistance were observed, however, for the effects of G185V and I186N mutations on the Km and Hill number values for this drug-induced increase in UIC2 reactivity. Login to comment 179 ABCB1 p.Gly185Val This assumption is confirmed by a comparison between the effects of G185V on the Km values for UIC2 reactivity changes and the previously reported effects of this mutation on drug binding to Pgp. Login to comment 180 ABCB1 p.Gly185Val In the study presented here, we observed that G185V increases the Km for colchicine (i.e., decreases its apparent affinity) and decreases the Km for vinblastine (i.e., increases the apparent affinity of this drug). Login to comment 181 ABCB1 p.Gly185Val These findings are in complete agreement with the earlier work (12), where we found that G185V decreases the level of binding of a photoactive colchicine analogue and increases the level of binding of a vinblastine analogue to Pgp. Login to comment 183 ABCB1 p.Gly185Val Since changes in drug binding conferred by G185V were inversely associated with changes in the transport of the corresponding drug, we had suggested that it was debinding at the off site that was affected by this mutation (12). Login to comment 185 ABCB1 p.Gly185Val Despite the results with G185V, we cannot tell a priori whether a higher Km found in other cases should always point to a higher rate of debinding of the substrate at the off site, or whether it may also reflect drug binding at the on site. Login to comment 194 ABCB1 p.Gly185Val G185V, which decreases vinblastine resistance, also decreases the Km value in the UIC2 shift assay. Login to comment 195 ABCB1 p.Gly185Val As discussed above, this observation, together with the previous results (12), suggests that G185V decreases the level of vinblastine debinding at the off site. Login to comment 196 ABCB1 p.Gly185Val Strikingly, G185V also causes an apparent loss of one vinblastine-binding site, as it decreases the Hill number for vinblastine from 2 to 1. Login to comment 197 ABCB1 p.Gly185Val The loss of the binding site could conceivably provide a mechanism for the decreased level of transport of vinblastine by G185V. Login to comment 198 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn In contrast to G185V, the I186N mutation increases the vinblastine resistance, and this effect is associated with an increase in the Km value. Login to comment 199 ABCB1 p.Ile186Asn On the basis of this observation, we hypothesize that the I186N mutation increases the level of debinding of vinblastine. Login to comment 200 ABCB1 p.Ile186Asn The increased Km value of I186N, however, is not associated with a change in the Hill number, which remains at 2, as in the case of the wild type. Login to comment 201 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The Hill number for vinblastine is 1 in the G185V/I186N double mutant, indicating that I186N cannot reverse the loss of a drug-binding site caused by G185V. Login to comment 202 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The double mutant shows an intermediate Km between those of G185V and I186N, which happens to match the Km for the wild-type Pgp, and G185V/I186N shows the same level of vinblastine resistance as wild-type Pgp. Login to comment 205 ABCB1 p.Gly185Val The G185V mutation, which originally arose in a colchicine-selected cell line and which strongly increases colchicine resistance, produces a moderate increase in the Km value for colchicine, in agreement with the previous study (12). Login to comment 207 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn I186N, like G185V, increases resistance to colchicine, and this is accompanied by a higher Km and an increase in the Hill number from 1 to 2. Login to comment 208 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn At the quantitative level, however, I186N provides a stronger increase in the Km value, but G185V confers a larger increase in resistance. Login to comment 209 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn This contradiction raises the question of whether the effect of G185V on colchicine resistance may be due not only to increased debinding but also to some additional factors, or whether an increased colchicine Km (at least for I186N) may reflect changes in both drug binding and debinding. Login to comment 210 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn G185V/ I186N produces a higher Hill number than either mutation alone, suggesting an additive effect of the two mutations on the apparent number of drug-binding sites. Login to comment 211 ABCB1 p.Gly185Val ABCB1 p.Gly185Val ABCB1 p.Ile186Asn ABCB1 p.Ile186Asn The effects of the mutations on the Km value and on drug resistance, however, do not appear to be additive, since the G185V/ I186N double mutant has about the same Km as the I186N mutant but produces the same level of colchicine resistance as the G185V mutant. Login to comment 212 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn In contrast to the complicated colchicine situation, etoposide, the drug that was originally used to select the G185V/ I186N double mutant, provides the most straightforward correlation between drug resistance, on one hand, and the Km and Hill number changes, on the other hand. Login to comment 213 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Both G185V and I186N increase the apparent number of etoposide-binding sites from 2 to 3, while combining these mutations brings this number up to 4. Login to comment 214 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn Similarly, the Km values are increased by either G185V or I186N, and the double mutant exhibits the highest Km value, i.e., the lowest apparent affinity. Login to comment 215 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn The changes in both of these parameters agree with the effects of the mutations on etoposide resistance, which is higher in the G185V or I186N mutant than in the wild type and becomes the highest in the double mutant. Login to comment 216 ABCB1 p.Gly185Val ABCB1 p.Ile186Asn In summary, the observed effects of the G185V and I186N mutations on the transport of individual drugs can be interpreted through the effects of these mutations on the ability of the drugs to alter Pgp conformation, as reflected by the process of the change in UIC2 reactivity. Login to comment 220 ABCB1 p.Ile186Asn These correlations are less apparent for colchicine, since I186N increases the Km value out of proportion to the increase in colchicine resistance, and combining the two mutations does not have an additive effect on the Km for colchicine. We have also found that the changes in the Km and drug resistance were associated in some cases with the corresponding changes in the Hill number, the apparent number of drug-binding sites on Pgp. Login to comment