ABCMdb

ABCB1 p.Met986Ala

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Publications

PMID: 10331089 [PubMed] Ambudkar SV et al: "Biochemical, cellular, and pharmacological aspects of the multidrug transporter."

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. Login to comment

PMID: 10350482 [PubMed] Dey S et al: "A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol."

No. Sentence Comment

127 In a recent study, seven amino acid residues, L975, V981, F983, M986, V988, Q990, and V991, in the putative TM 12 of human Pgp were substituted individually by alanine (L975A, V981A, F983A, M986A, V988A, Q990A, and V991A). Login to comment
133 Consistent with the previous report, all the mutant Pgp`s (L975A, V981A, F983A, M986A, V988A, Q990A, and V991A) were expressed on the cell surface at a comparable level to that of wild-type (WT) Pgp (Figure 1A, upper panel). Login to comment
141 Human osteosarcoma (HOS) cells infected with vTF7-3 were transfected with either pTM1 (control) (s), pTM1-MDR1 (wild type) (-), pTM1-MDR1-L975A (L975A) (‚‚‚), pTM1-MDR1-V981A (V981A) (- - -), pTM1-MDR1-F983A (F983A) (- -), pTM1-MDR1-M986A (M986A) (thick dashes), pTM1-MDR1-V988A (V988A) (-‚‚-), pTM1-MDR1-Q990A (Q990A) (-‚-), or pTM1-MDR1-V991A (V991A) (-‚‚‚-) plasmid DNA. Login to comment
143 (A, upper panel) Cells were subjected to FACS analysis after staining with human Pgp external epitope-specific monoclonal antibody MRK-16 (14), as described under Experimental Procedures. (A, lower panel) Total cell lysates were prepared from each cell type, and immunoblot analysis with Pgp-specific monoclonal antibody C219 was performed as described under Experimental Procedures. (B) Similar to section A; cells were infected with vTF7-3, and transfected with either pTM1 (control) (s), pTM1-MDR1 (wild type) (-), pTM1-MDR1-L975A (L975A) (‚‚‚), pTM1-MDR1-V981A (V981A) (- - -), pTM1-MDR1-F983A (F983A) (- -), pTM1-MDR1-M986A (M986A) (thick dashes) pTM1-MDR1-V988A (V988A) (-‚‚-), pTM1-MDR1-Q990A (Q990A) (-‚-), or pTM1-MDR1-V991A (V991A) (-‚‚‚-) plasmid DNAs. Login to comment
158 In cells expressing mutants V981A (Figure 2A,B) and M986A (data not shown), the level of intracellular accumulation of Bodipy Fl-verapamil was effectively increased by both isomers of flupentixol, but not to the same extent as that of the cells expressing wild-type Pgp. Login to comment
159 This suggested that inhibition of drug transport by cis(Z)- and trans(E)- flupentixol in V981A and M986A was not complete, indicating a moderate contribution of these residues to the interaction with flupentixol. Login to comment
239 Although drug transport was inhibited by both cis(Z)- and trans(E)-flupentixol in V981A and M986A (data not shown), inhibition was not complete, FIGURE 6: Effect of trans(E)-flupentixol and cyclosporin A on [125I]IAAP labeling of the wild-type Pgp and F983A. Login to comment
246 It is to be noted that in all three mutants (F983A, V981A, and M986A) the inhibitory effect of both isomers (cis and trans) of flupentixol was affected. Login to comment

PMID: 9819232 [PubMed] Hafkemeyer P et al: "Contribution to substrate specificity and transport of nonconserved residues in transmembrane domain 12 of human P-glycoprotein."

No. Sentence Comment

98 Alanine mutants were arranged in such a way that three alanine mutations were putatively located in the outer plasma membrane leaflet (L975A, V981A, and F983A) and four in the inner leaflet (M986A, V988A, Q990A, and V991A), assuming an R-helical structure to TM 12 (Figure 1b). Login to comment
121 Substitution of the four nonconserved amino acid residues (M986A, V988A, Q990A, and V991A) in the carboxy-terminal half of TM 12 had no effect on transport of calcein-AM, rhodamine 123, and bodipy-taxol. Login to comment
126 The V981-F983 double mutant was Table 1: Properties of Wild-Type and Mutant P-glycoproteinsa drug transporta cell surface expressionc rhodamine daunomycin bodipy-verapamil calcein-AM bodipy-taxol wild-type ++++ ++++ ++++ ++++ ++++ ++++ L975A ++++ ++++ ++++ ++++ ++++ ++++ V981A ++++ ++++ ++++ ++++ ++++ ++++ F983A ++++ ++++ ++++ ++++ ++++ ++++ M986A ++++ ++++ n.d. ++++ ++++ n.d. V988A ++++ ++++ n.d. ++++ ++++ n.d. Q990A ++++ ++++ n.d. ++++ ++++ n.d. V991A ++++ ++++ n.d. ++++ ++++ n.d. L975A, V981A, F983A ++++ no transport no transport ++ ++ ++ M986A, V988A, Q990A, V991A ++++ ++++ +++ ++ ++++ ++++ V981A, F983A ++++ + no transport ++ ++ +++ L975A, F983A ++++ + ++ ++++ +++ ++++ L975A, V981A ++++ ++ no transport ++++ +++ ++++ a Symbols are noted as follows: ++++, wild-type activity; ++, impaired activity; +, residual activity; and n.d., not determined. b Drug transport was determined by FACS. Login to comment
130 Mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A, - -), the quadruple mutant in the carboxy-terminal half of TM 12 (M986A-V988A-Q990A-V991A, ‚‚‚), the double mutants L975A-V981A (- - -), L975A-F983A (-‚‚-), and V981A-F983A (-‚-). Login to comment
141 Mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A) and the quadruple mutant (M986A-V988A-Q990A-V991A) (s). Login to comment
146 The quadruple mutant, M986A-V988A-Q990A-V991A, in the carboxy-terminal half still retained a substantial ability to bind IAAP. Login to comment
159 ATP binding was equivalent for wild-type MDR1 and the MDR1 mutants L975A-V981A-F983A as well as for M986A-V988A-Q990A-V991A (Figure 7). Login to comment
174 (a) pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), and mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A) and the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A). Login to comment
188 pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A), the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A), WT (wild-type human MDR1) in the presence of 250 µM EDTA and without MgCl2 to assess the specificity of the 170 kDa band representing Pgp as indicated with the arrow. Login to comment
193 (Middle panel) Vanadate-induced [R-32P]-8-azido-ADP labeling was performed at 37 °C. pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A), and the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A). Login to comment
215 Changing all four amino acid residues M986A, V988A, Q990A, or V991A in the carboxy-terminal part of TM 12 still allows almost normal transport function of Pgp. Login to comment
228 The double mutants showed reduced drug transport suggesting that a change in any two of these three amino Table 2: Properties of Wild-Type and Mutant P-Glycoproteinsa photoaffinity labelinga ATP bindingb ATPase activityc wild-type ++++ ++++ ++++ L975A +++ n.d. +++ V981A +++ n.d. +++ F983A +++ n.d. +++ M986A n.d. n.d. n.d. V988A n.d. n.d. n.d. Q990A n.d. n.d. n.d. V991A n.d. n.d. n.d. L975A,V981A, F983A + ++++ + M986A, V988A, Q990A, V991A ++ ++++ ++ V981A, F983A +++ n.d. +++ L975A, F983A +++ n.d. +++ L975A, V981A +++ n.d. +++ a Symbols are noted as follows: ++++, wild-type activity; ++, impaired activity; +, residual activity; and n.d. not determined. b Photoaffinity labeling was done in HeLa crude membrane preparations using [125 I]iodoarylazidoprazosin. Login to comment

PMID: 22360349 [PubMed] Mandal D et al: "Evidence for modulatory sites at the lipid-protein interface of the human multidrug transporter P-glycoprotein."

No. Sentence Comment

28 The desired nucleotide replacements were confirmed by Big-Dye version 3.1 (BD Sciences) nucleotide sequencing of the MDR1 open reading frame in recombinant plasmids pKM2-MDR1- V715A, -F716A, -I719A, -G723A, -I764A, -I765A, -I768A, -F770A, -L772A, -T837A, -I840A, -I864A, -I867A, -A871F, -T945A, -M948A, -F983A, -M986A, -V988A, -Q990A, and -V991A. Login to comment
102 A comparable level of basal binding of [125 I]IAAP to wild-type Pgp and Pgp mutants M948A, F983A, V988A, M986A, and Q990A (Figure 2B) rules out the possibility that the reduced level of stimulation in the mutants is due to loss of [125 I]IAAP binding and not stimulation per se. To investigate whether M948, Q990, F983, M986, and V988 are part of a general module involved in the regulation of substrate binding or are molecular components specific to cis- (Z)-flupentixol, we studied the effect of a structurally unrelated Pgp modulator, cyclosporin A, on binding of [125 I]IAAP to all 21 mutants. Login to comment
110 Stimulation of ATP Hydrolysis by cis-(Z)-Flupentixol and Verapamil cis-(Z)-flupentixol-mediated stimulation verapamil-mediated stimulation maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark WT 7.3 ± 0.4 6.8 ± 1.5 5.6 ± 0.19 6.6 ± 1.04 V715A 6.2 ± 0.2 5.0 ± 0.9 6.4 ± 0.37 16.3 ± 3.3 V716A 5.1 ± 0.2 4.7 ± 1.1 NDb NDb a I719A 4.6 ± 0.08 4.7 ± 0.4 5.8 ± 0.26 6.5 ± 1.31 G723A 5.3 ± 0.3 10.93 ± 2.7 5.4 ± 0.1 11.6 ± 0.88 I764A 4.5 ± 0.4 4.1 ± 2 3.2 ± 0.26 27.9 ± 8.4 I765A 4.96 ± 0.2 2.3 ± 0.47 NDb NDb a I768A 4.8 ± 0.2 1.2 ± 0.43 4.8 ± 0.19 8.5 ± 1.4 F770A 6.6 ± 0.6 10.9 ± 3.9 8.6 ± 0.59 13.2 ± 3.2 L772A 3.6 ± 0.2 2.96 ± 0.9 3.2 ± 0.11 9.5 ± 1.6 T837A 1.1 ± 0.06 NDb a 2.6 ± 0.1 3.1 ± 0.9 I840A 4.0 ± 0.3 25.6 ± 6.5 6.9 ± 0.2 13.5 ± 0.5 I864A 0.6 ± 0.05 NDb a 4.1 ± 0.25 0.9 ± 1.04 I867A 10.2 ± 1.3 72.3 ± 18.5 10.2 ± 0.52 30.7 ± 3.1 A871F NDb NDb NDb NDb a T945A 5.0 ± 0.3 13.7 ± 3.1 6.4 ± 0.15 6.9 ± 0.71 M948A NDb NDb a 6.8 ± 1.5 44.4 ± 12.4 F983A 4.95 ± 0.36 19.59 ± 5.0 6.1 ± 0.38 6.6 ± 1.8 M986A 3.8 ± 0.3 2.6 ± 1.5 7.7 ± 0.76 30.0 ± 8.6 V988A 3.0 ± 0.2 21.4 ± 7.6 6.5 ± 0.31 16.9 ± 2.9 Q990A 1.8 ± 0.4 18.9 ± 2.6 a 7.7 ± 0.58 12.9 ± 3.7 V991A 3.9 ± 0.2 21.1 ± 5.0 5.9 ± 0.59 21.9 ± 7.2 a Mutants with <2-fold maximal stimulation (<25% of the wild-type level). Login to comment
224 Substitutions F983A, M986A, and V988A completely abrogate stimulation of substrate binding (Figure 2) with only modest effects on ATP-site stimulation (Figure 4), which indicates that the two modulatory events are not tightly coupled to each other. Login to comment

PMID: 11428917 [PubMed] Hrycyna CA et al: "Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance."

No. Sentence Comment

27 List of mutations in human, mouse and hamster P-gp`s that affect substrate specificity f aaa Mutation Regionb Sourcec Reference aa 78-97 EC 1 human MDR1 78 (ABC20)d Q128He TM 2 mouse mdr3 79 R138H IC 1 mouse mdr3 79 Q139H, R IC 1 mouse mdr3 79 G141V IC 1 human MDR1 25,80 Q145H IC 1 mouse mdr3 79 E155G, K IC 1 mouse mdr3 79 F159I IC 1 mouse mdr3 79 D174G IC 1 mouse mdr3 79 S176F, P IC 1 mouse mdr3 79 K177I IC 1 mouse mdr3 79 N179S IC1 mouse mdr3 79 N183S/G185V IC 1 human MDR1 81 G183D IC1 mouse mdr3 79 G185V IC 1 human MDR1 82-84 G187V IC 1 human MDR1 80 A192T TM 3 mouse mdr3 79 F204S EC 2 mouse mdr3 79 W208G EC 2 mouse mdr3 79 K209E EC 2 mouse mdr3 79 L210I TM 4 mouse mdr3 79 T211P TM 4 mouse mdr3 79 I214T TM 4 mouse mdr3 79 P223A TM 4 human MDR1 85 K285T IC 2 human MDR1 1 G288V IC 2 human MDR1 80 I299M, T319S, L322I, TM 5, EC3, IC 3 human MDR1 86 G324K, S351N V334 TM 6 human MDR1 1 F335A TM 6 human MDR1 25 F335 TM 6 human MDR1 87 V338A TM 6 human MDR1 88 G338A, A339P TM 6 hamster PGY 1 89,90 A339P TM 6 hamster PGY 1 90 G341V TM 6 human MDR1 88 K536R,Q N-NBD human MDR1 91 ERGA→DKGT N-NBD mouse mdr3 92 (aa 522-525) T578C N-NBD mouse mdr3 92 G812V IC 4 human MDR1 80 G830V IC 4 human MDR1 25,80 P866A TM 10 human MDR1 85 F934A TM 11 mouse mdr3 93 G935A TM 11 mouse mdr3 93 I936A TM 11 mouse mdr3 93 F938A TM 11 mouse mdr3 93 S939A TM 11 mouse mdr3 93 S939F TM 11 mouse mdr3 94,95 S941F TM 11 mouse mdr1 94,95 T941A TM 11 mouse mdr3 93 Q942A TM 11 mouse mdr3 93 Table 1-continued aaa Mutation Regionb Sourcec Reference A943G TM 11 mouse mdr3 93 Y946A TM 11 mouse mdr3 93 S948A TM 11 mouse mdr3 93 Y949A TM 11 mouse mdr3 93 C952A TM 11 mouse mdr3 93 F953A TM 11 mouse mdr3 93 F983A TM 12 human MDR1 96 L975A, V981A, F983A TM 12 human MDR1 96 M986A, V988A, TM 12 human MDR1 96 Q990A, V991A V981A, F983A TM 12 human MDR1 96 L975A, F983A TM 12 human MDR1 96 L975A, V981A TM 12 human MDR1 96 F978 TM 12 human MDR1 1 F978A TM 12 human MDR1 25 a aa, amino acid. Login to comment