ABCMdb

ABCB1 p.Ile186Asn

Predicted by SNAP2:	A: D (71%), C: D (59%), D: D (91%), E: D (85%), F: D (66%), G: D (85%), H: D (85%), K: D (91%), L: N (78%), M: N (87%), N: D (85%), P: D (91%), Q: D (85%), R: D (85%), S: D (80%), T: D (71%), V: N (53%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] New horizon of MDR1 (P-glycoprotein) study. Drug Metab Rev. 2005;37(3):489-510. Mizutani T, Hattori A
New horizon of MDR1 (P-glycoprotein) study.
Drug Metab Rev. 2005;37(3):489-510., [PMID:16257832]

Abstract [show]
MDR1 (once P-glycoprotein, now referred to as ABCB1) plays a role as a blood-brain barrier, preventing drug absorption into the brain, and is known to confer multiple drug resistance in cancer chemotherapy. MDR1 is composed of two repeated fragments, and there are six transmembrane domains (TMD) on the N-terminal of each repeat and a nucleotide (ATP) binding domain (NBD) on the C-terminal. These two repeats are dependent but cooperate as one functional molecule, with one pocket for excreting drugs. The 12 TM domains form a funnel facing the outside of cells, and NBD is in cytosol as a dimer. One NBD is composed of the Walker A, Q-loop, ABC-signature and the Walker B for phosphate binding of nucleotide. This tertiary structure of MDR1 is suggested from the structure of the NBD of histidine permease (HisP), clarified by x-ray crystallography. On the model of HisP, the NBD positions described above make a functional domain, and the same NBD structure is found on many other ABC transporters. An experiment with MDR1 gene knockout mice showed the high plasma AUC of drugs in mdr null mice [mdr1a(-/-)] and a high level in the brain, indicating that MDR1 has an efflux function (prevention of absorption) in the intestinal lumen and acts as a barrier of drug uptake in the brain, as well as has the function of urinary and biliary excretion of drugs. The transcription of MDR1 is dependent on two sites; the promoter site (-105/-100)(-245/-141) and the enhancer site (-7864/-7817). Autoantibody from autoimmune hepatitis patients weakly reacted with the extracellular peptide (aa314-aa328 between TM5 and 6) of MDR1 on the outside of the cell membrane, and did not react with peptides in the NBD and in the membrane-spanning region in TM5. There is an ambiguity about the function of MDR1 as GlcCer translocase.

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167 MDR1 with mutation G185V-I186N altered the magnitude of drug-induced increases in UIC2 immunoreactivity (Ruth, 2001). Login to comment

[hide] New light on multidrug binding by an ATP-binding-c... Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20. Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW
New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20., [PMID:16545467]

Abstract [show]
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

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58 Although mutation of only one of these residues (L975A, V981A and F983A) has no effect on the phenotype of the protein [20], double mutations either completely inhibit (V981A/F983A and L975A/V981A) or cause 50% inhibition (L975A/F983A) of Table 1. Login to comment
59 Published mutations in human and murine P-glycoprotein that alter drug transport in cells Location of mutation Mutation Refs Mutation Refs Mutation Refs Transmembrane helices H61A and others [14] I214L [60] L868W [59] G64R [15] P223A [65] I936A [21] L65R [15] S224P [60] F938A [21] Q139[H/P/R] [60] I306R [18] S939[A/C/T/Y/W/D/F] [21,22] G141V [17] F335A [16] T941A [21] G185V [61,62] V338A [66] Q942A [21] I186N [61] G338A [67,68] A943G [21] G187V [17] A339P [67,68] Y946A [21] G187E [60] G341A [66] S948A [21] A192T [60] S344[A/T/C/Y] [66] Y949A [21] F200L [60] N350I [19] C952A [21] F204S [60] P709A [65] F953A [21] R206L [60] G830V [17] L975A [20] W208G [60] I837L [23] F978A [16] K209E [60] N839I [23] V981A [20] L210I [60] I862F [19] F983A [20] T211P [60] L865F [19] F978A [16] V213A [60] P866A [65] N988D [59] Intracellular domain T169I [60] K177I [60] G288V [17] R170L [60] E180G [60] A931T [19] L171P [60] G181R [60] F934A [21] T172P [60] G183D [60] G935A [21] S176P [60] D184N [60] NBD D555N [63] K1076M [69] E1197Q [64] D558N [64] D1093N [64] D1203N [64] D592N [64] E1125Q [64] D1237N [64] E604Q [64] S1173A [70] E1249Q [64] Review TRENDS in Pharmacological Sciences Vol.27 No. Login to comment

[hide] Coordinate changes in drug resistance and drug-ind... Biochemistry. 2001 Apr 10;40(14):4332-9. 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., [PMID:11284689]

Abstract [show]
The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. 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. 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. This selection yielded the I186N substitution, adjacent to G185V. 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. 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. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.

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3 This selection yielded the I186N substitution, adjacent to G185V. Login to comment
4 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 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
75 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
85 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 Effects of G185V and I186N Substitutions on Drug Resistance Profiles. Login to comment
87 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 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
94 The etoposide resistance of G185V was further increased by combining it with I186N [G185V/I186N (4)]. Login to comment
95 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 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 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
105 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 Colchicine resistance conferred by G185V/I186N was indistinguishable from the effect of G185V alone. Login to comment
107 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
109 In contrast, I186N increased vinblastine and Taxol resistance relative to that of the wild type. Login to comment
110 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 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 Analysis of the Effects of the G185V and I186N Mutations on Drug-Induced Conformational Transitions of Pgp Using UIC2 ImmunoreactiVity. Login to comment
114 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 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 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
132 Unlike G185V, I186N produced different effects on the fold increase in UIC2 reactivity by different drugs. Login to comment
134 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 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 DISCUSSION The I186N Mutation Increases the Pgp ActiVity. Login to comment
150 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
152 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 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
160 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 Testing the effects of G185V and I186N mutations individually and in combination showed that this is not the case. Login to comment
162 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 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 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 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 G185V and I186N Mutations Change the Parameters of Drug-Induced Conformational Transitions of Pgp. Login to comment
169 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
174 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 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 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
198 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 On the basis of this observation, we hypothesize that the I186N mutation increases the level of debinding of vinblastine. Login to comment
200 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 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 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
207 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 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 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 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 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 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 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 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 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 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 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