ABCB1 p.Leu65Arg
| Predicted by SNAP2: | A: N (72%), C: N (66%), D: N (53%), E: N (61%), F: N (93%), G: N (53%), H: N (61%), I: N (87%), K: N (57%), M: N (93%), N: N (72%), P: N (57%), Q: N (93%), R: N (66%), S: N (78%), T: N (87%), V: N (87%), W: N (82%), Y: N (78%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, 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] Biochemical, cellular, and pharmacological aspects... Annu Rev Pharmacol Toxicol. 1999;39:361-98. Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM
Biochemical, cellular, and pharmacological aspects of the multidrug transporter.
Annu Rev Pharmacol Toxicol. 1999;39:361-98., [PMID:10331089]
Abstract [show]
Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.
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No. Sentence Comment
47 Table 1 List of mutations in human, mouse, and hamster P-glycoproteins that affect substrate specificitya aa mutation Region Sourceb Reference H61R, F, K, M, W, Y TM 1 Human MDR1 149, 150 ABC20c G64R TM 1 Human MDR1 150 L65R TM 1 Human MDR1 150 aa78-97 EC 1 Human MDR1 151 Q128Hd TM 2 Mouse mdr3 152 R138H IC 1 Mouse mdr3 152 Q139H, R IC 1 Mouse mdr3 152 Q141V IC 1 Human MDR1 15319, Q145H IC 1 Mouse mdr3 152 E155G, K IC 1 Mouse mdr3 152 F159I IC 1 Mouse mdr3 152 D174G IC 1 Mouse mdr3 152 S176G, P IC 1 Mouse mdr3 152 K177I IC 1 Mouse mdr3 152 N179S IC 1 Mouse mdr3 152 N183S/G185V IC 1 Human MDR1 154 G183D IC 1 Mouse mdr3 152 G185V IC 1 Human MDR1 155-157 G187V IC 1 Human MDR1 153 A192T TM 3 Mouse mdr3 152 F204S EC 2 Mouse mdr3 152 W208G EC 2 Mouse mdr3 152 K209E EC 2 Mouse mdr3 152 L210I TM 4 Mouse mdr3 152 T211P TM 4 Mouse mdr3 152 I214T TM 4 Mouse mdr3 152 P223A TM 4 Human MDR1 158 G288V IC 2 Human MDR1 153 I299M, T319S, L322I, TM 5, EC3, Human MDR1 159 G324K, S351N IC 3 F335A TM 6 Human MDR1 19 F335 TM 6 Human MDR1 160 V338A TM 6 Human MDR1 161 G338A, A339P TM 6 Hamster PGY1 162, 163 A339P TM 6 Hamster PGY1 163 G341V TM 6 Human MDR1 161 K536R, Q N-NBD Human MDR1 164 ERGA → DKGT N-NBD Mouse mdr3 165 aa 522-525 T578C N-NBD Mouse mdr3 165 (Continued) G830V IC 4 Human MDR1 P866A TM 10 Human MDR1 158 F934A TM 11 Mouse mdr3 166 G935A TM 11 Mouse mdr3 166 I936A TM 11 Mouse mdr3 166 F938A TM 11 Mouse mdr3 166 S939A TM 11 Mouse mdr3 166 S939F TM 11 Mouse mdr3 167, 168 S941F TM 11 Mouse mdr1 167, 168 T941A TM 11 Mouse mdr3 166 Q942A TM 11 Mouse mdr3 166 A943G TM 11 Mouse mdr3 166 Y946A TM 11 Mouse mdr3 166 S948A TM 11 Mouse mdr3 166 Y949A TM 11 Mouse mdr3 166 C952A TM 11 Mouse mdr3 166 F953A TM 11 Mouse mdr3 166 F983A TM 12 Human MDR1 169 L975A, V981A, F983A TM 12 Human MDR1 169 M986A, V988A, Q990A, TM 12 Human MDR1 169 V991A V981A, F983A TM 12 Human MDR1 169 L975A, F983A TM 12 Human MDR1 169 L975A, V981A TM 12 Human MDR1 169 F978A TM 12 Human MDR1 19 a aa,amino acid; EC, extracellular loop; IC, intracellular loop; TM,transmembrane domain; NBD, nucleotide binding/utilization domain.
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ABCB1 p.Leu65Arg 10331089:47:220
status: NEW[hide] Suppressor mutations in the transmembrane segments... J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11. Loo TW, Bartlett MC, Clarke DM
Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.
J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PMID:17848563]
Abstract [show]
Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.
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No. Sentence Comment
7 The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold.
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ABCB1 p.Leu65Arg 17848563:7:89
status: NEW69 For disulfide cross-linking analysis, the cDNA of mutant L339C(TM6)/ F728C(TM7) (34) was modified to also encode the L65R, T199R, I306R, or F343R mutations.
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ABCB1 p.Leu65Arg 17848563:69:117
status: NEW164 Effect of L65R and T199R Mutations on Maturation of a P-gp Processing Mutant-We then tested whether arginines introduced into TM1 (Leu65 ) (27) or TM3 (Thr199 ) promoted maturation of a P-gp processing mutant.
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ABCB1 p.Leu65Arg 17848563:164:10
status: NEW204 Effect of L65R Mutation on Protease Sensitivity of P-gp-In a previous study (37), we observed that expression of P-gp processing mutants in the presence of drug substrates appeared to convert the protein from a loosely folded conformation to a more compact structure because they became about 100-fold more resistant to trypsin.
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ABCB1 p.Leu65Arg 17848563:204:10
status: NEW229 These results indicate that the L65R mutation converts mutant G251V into a more protease-resistant conformation.
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ABCB1 p.Leu65Arg 17848563:229:32
status: NEW234 For example, the L65R mutation only caused a large reduction (57-fold) in the apparent affinity for vinblastine and little change in affinities for cyclosporin A or rhodamine B.
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ABCB1 p.Leu65Arg 17848563:234:17
status: NEW270 By contrast, the ATPase activity of mutant L65R was activated by treatment with MTS-verapamil (27) but not by MTS-rhodamine (Ͻ2-fold activation after treatment with 2 mM MTS-rhodamine) (data not shown).
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ABCB1 p.Leu65Arg 17848563:270:43
status: NEW[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]
Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.
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No. Sentence Comment
21 It was found that the L65R mutation promoted maturation of P-gp processing mutants such as ⌬Y490 and G251V (9).
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ABCB1 p.Leu65Arg 18596043:21:22
status: NEW37 Printed in the U.S.A. with a L65R substitution was previously found to be particularly efficient (9).
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ABCB1 p.Leu65Arg 18596043:37:31
status: NEW81 We previously observed that introduction of arginines into TM segments such as L65R (TM1), T199R(TM3), I306R(TM5), and F343R(TM6) promoted maturation of P-gp processing mutants (9).
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ABCB1 p.Leu65Arg 18596043:81:79
status: NEW103 Mutants M68R and L65R showed the highest increase in maturation efficiency (85 and 60% mature 170-kDa mature, respectively) (Fig. 2).
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ABCB1 p.Leu65Arg 18596043:103:17
status: NEW109 The position of the L65R mutation that promotes maturation of P-gp processing mutants is shown as a black ball.
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ABCB1 p.Leu65Arg 18596043:109:20
status: NEW116 The position of Leu-65 (L65R mutation promotes maturation of processing mutants) is shaded in black. about 45%.
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ABCB1 p.Leu65Arg 18596043:116:24
status: NEW149 Mutants L65R and M68R yielded mature 170-kDa P-gp as the major product in the absence of drug substrates.
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ABCB1 p.Leu65Arg 18596043:149:8
status: NEW165 TABLE 1 Effects of drug substrates on the maturation of TM1 arginine mutants containing the G251V mutation Mutation (G251V ؉) No drug Cyclosporin A Verapamil Vinblastine Rhodamine None - 111a,c 111 111 11 V52R -a,b 111 111 111 11 V53R 2 - - - - G64R 2 - - - - T55R 2 - - - - L56R 2 - - - - A57R 2 - - - - A58R 2 - - - - I59R 2 - - - - I60R 2 - - - - H61R - 111 1 1 1 G62R 2 - - - - A63R 2 - - - - G64R 1 111 1 1 11 L65R 11 111 11 11 11 P66R 2 1 - - - L67R - 111 111 111 11 M68R 111 111 111 111 111 M69R - 111 - 11 111 L70R - 111 111 111 11 V71R 1 111 111 111 11 F72R 2 1 1 1 1 a Change in the amount of mature (170 kDa) protein in the presence of drug substrate relative to that in the absence of drug substrate.
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ABCB1 p.Leu65Arg 18596043:165:421
status: NEW172 In a previous cross-linking study we found that introduction of L65R change into mutant L339C(TM6)/ F728C(TM7) reduced its apparent affinity for vinblastine by about 60-fold but had little effect on P-gp interactions with cyclosporin A or rhodamine B (9).
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ABCB1 p.Leu65Arg 18596043:172:64
status: NEW183 The G64R, L65R, and M68R mutations preferentially affected P-gp-vinblastine interactions.
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ABCB1 p.Leu65Arg 18596043:183:10
status: NEW208 Effect of TM11 Mutations on Rescue of Mutant G251V Containing the L65R and M68R Changes-One reason that Arg-68 caused the largest increase in maturation of mutant G251V is that the arginine residue may promote packing of the TM segments.
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ABCB1 p.Leu65Arg 18596043:208:66
status: NEW220 Because the L65R mutation also promoted maturation of mutant G251V, we tested whether the Y950A or Y953A mutations would have any effect on maturation of the L65R/G251V mutant.
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ABCB1 p.Leu65Arg 18596043:220:12
status: NEWX
ABCB1 p.Leu65Arg 18596043:220:158
status: NEW[hide] Amino acid substitutions in the first transmembran... FEBS Lett. 1997 Aug 11;413(1):142-6. Taguchi Y, Morishima M, Komano T, Ueda K
Amino acid substitutions in the first transmembrane domain (TM1) of P-glycoprotein that alter substrate specificity.
FEBS Lett. 1997 Aug 11;413(1):142-6., [PMID:9287132]
Abstract [show]
Recently, we showed that the amino acid at position 61 in TM1 of human P-glycoprotein is important in deciding the substrate specificity of this protein. In this work, we investigated whether the amino acids other than His61 in TM1 of P-glycoprotein are also essential in the function of this protein. Nine amino acids residues, from Ala57 to Leu65 in TM1, were independently substituted to Arg, and analyzed the drug resistance of cells stably expressing each of these mutant P-glycoproteins. The mutant P-glycoproteins Ile60 --> Arg, His61 --> Arg, Ala63 --> Arg, Gly64 --> Arg, and Leu65 --> Arg were normally processed and expressed in the plasma membrane. Substrate specificities of mutant P-glycoproteins Gly64 --> Arg and Leu65 --> Arg were quite different from that of the wild type, and similar to that of the His61 --> Arg mutant, while the Ile60 --> Arg and Ala63 --> Arg mutant P-glycoproteins showed similar substrate specificities to that of the wild-type P-glycoprotein, suggesting that not only the amino acid residue at position 61 but also those at position 64 and 65 are also important in deciding the substrate specificity of P-glycoprotein. These three amino acids His61, Gly64, and Leu65 would form a compact region on an alpha-helix arrangement of TM1. These results suggest that a region consisting of His61, Gly64, and Leu65 in TM1 would participate in the formation of the recognition site for substrates of P-glycoprotein.
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No. Sentence Comment
43 Cells transfected with Gly64 -> Arg, or Leu65 -> Arg cDNAs were selected with stepwise increasing concentrations (5, 10, 20 ng/ml) of vinblastine and finally maintained in 20 ng/ ml vinblastine.
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ABCB1 p.Leu65Arg 9287132:43:40
status: NEW55 From cells transfected with the wild type and He60 -> Arg, His61 -> Arg, Ala63 -> Arg, Gly64 -> Arg, or Leu65 -> Arg mutant cDNA, both Vbl- and Col-resistant colonies were obtained (Fig. 1).
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ABCB1 p.Leu65Arg 9287132:55:104
status: NEW58 Noticeably, cells transfected with mutant Gly64 -> Arg or Leu65 -> Arg yielded comparable number of Col-resistant colonies, but much less Vbl-resistant colonies, suggesting that the substrate specificities of these two mutants were altered from that of the wild type.
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ABCB1 p.Leu65Arg 9287132:58:58
status: NEW68 Membrane proteins were obtained from stable transformants maintained in 20 ng/ml vinblastine for mutants Gly64 -»Arg and Leu65 -> Arg, or 30 ng/ml vinblastine for wild type and the other mutants.
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ABCB1 p.Leu65Arg 9287132:68:126
status: NEW74 The drug-resistance profiles of the cells expressing He60 -> Arg, His61 -»Arg, Ala63 -»Arg, Gly64 -> Arg, and Leu65 -> Arg mutant P-glycoproteins were investigated by comparing their relative resistance to Vbl, Col, VP16, and Adr (Fig. 3).
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ABCB1 p.Leu65Arg 9287132:74:120
status: NEW75 The drastic alterations in the drug resistance profile were observed with Gly64 -> Arg or Leu65 -> Arg mutant as well as His61 -»Arg mutant.
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ABCB1 p.Leu65Arg 9287132:75:90
status: NEW82 From each of membrane fraction, a band migrating at about 170 kDa was detected, indicating that He60 -> Arg, His61 -»Arg, Ala63 -»Arg, Gly64 -»Arg, and Leu65 -> Arg mutant P-glycoproteins were normally processed and expressed in the plasma membrane.
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ABCB1 p.Leu65Arg 9287132:82:167
status: NEW89 The mutant P-glycoproteins, in which either He60 , Ala63 , Gly64 , or Leu65 was replaced by Arg were successfully expressed in the plasma membrane of KB3-1 cells and were functional as drug transporters as the His61 mutants.
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ABCB1 p.Leu65Arg 9287132:89:70
status: NEW90 The substrate specificities of Gly64 -»Arg and Leu65 -> Arg mutant P-glycoproteins were quite different from that of the wild type, and similar to that of His61 -»Arg mutant, while He60 and Ala63 -> Arg mutant P-glycoproteins showed similar substrate specificities to that of the wild-type P-glycoprotein.
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ABCB1 p.Leu65Arg 9287132:90:52
status: NEW92 The similarity among the substrate specificities of Gly64 -> Arg, Leu65 -> Arg, and His61 -> Arg mutant P-glycoproteins suggest that the amino acid residues at position 64 and 65 are also important in deciding the substrate specificity.
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ABCB1 p.Leu65Arg 9287132:92:66
status: NEW106 The substrate specificities of the Gly64 -> Arg and Leu65 -> Arg mutants were similar to, but not exactly same with that of the His61 -> Arg mutant.
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ABCB1 p.Leu65Arg 9287132:106:52
status: NEW107 The cells expressing Gly64 -> Arg or Leu65 -> Arg mutant were more resistant to VP16 than to Vbl, while cells expressing the His61 -* Arg mutant were more resistant to Vbl than to VP16 (Fig. 3).
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ABCB1 p.Leu65Arg 9287132:107:37
status: NEW[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]
Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
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No. Sentence Comment
175 In the N-terminal TMD1 domain, the largest number of arginine mutations predicted to line the drug-binding pocket that inhibited tariquidar rescue were located in TM1 (H61R, G64R, L65R, M68R, M69R, and F72R) and TM5 (F303R, I306R, Y307R, S309R, and Y310R) (Fig. 4, A and E).
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ABCB1 p.Leu65Arg 26507655:175:180
status: NEW193 It was found that 16 of the 28 mutants resembled the G251V/I868R mutant as expression in the presence of 5 òe;M cyclosporine A yielded mature P-gp as the major product in TM1 (H61R, G64R, L65R, M68R, and M69R), TM5 (F303R, I306R, and S309R), TM7 (Q725R and F728R), TM10 (I868R and G872R), TM11 (F942R, T945R, and Q946R), and TM12 (V982R) (Fig. 7).
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ABCB1 p.Leu65Arg 26507655:193:192
status: NEW283 We identified 13 additional arginine mutations (H61R, G64R, L65R, and M68R in TM1; A129R in TM2; I306R and S309R in TM5; F343R in TM6; F942R, T945R, Q946R, and Y950R in TM11; and L975R in TM12) (Fig. 11A) that were not predicted to lie within 4.5-&#c5; of the predicted site 3 tariquidar-binding site (9).
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ABCB1 p.Leu65Arg 26507655:283:60
status: NEW313 Therefore, arginines predicted to lie outside of the tariquidar-binding site in TM1 and TM11 in the docking studies (H61R, G64R, L65R, and M68 in TM1; F942R, T945R, Q946R, and Y950R in TM11) might alter interactions between TM1 and TM11.
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ABCB1 p.Leu65Arg 26507655:313:129
status: NEW