ABCMdb

ABCB1 p.Lys1076Met

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Publications

PMID: 12404126 [PubMed] Ruefli AA et al: "P-glycoprotein inhibits caspase-8 activation but not formation of the death inducing signal complex (disc) following Fas ligation."

No. Sentence Comment

109 The mutant P-gp used in the following studies contained double lysine to alanine substitutions at amino acids 233 (K433M) and 1076 (K1076M) within the Walker A motifs, which disrupted nucleotide binding, and ATPase and drug-efflux activity.4,16 Mutant constructs containing double lysine to alanine substitutions at 433(K433M) and 1076(K1076M) amino acids were generated by excising 4.1 kB Eag1 ± Eag1 fragment from clone pUCFVXMDH3 ± Sal(DAUG)-MDR/Neo (provided by Igor Roninson, Chicago University, Chicago, IL) and cloned into MSCV vector. Login to comment

PMID: 15131592 [PubMed] Tainton KM et al: "Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux."

No. Sentence Comment

23 As shown in Figure 1 (top panel), cells transduced with MSCV alone (CEM-GFP), MSCV-MDR1 (CEM-P-gpWT ) and two clones transduced with MSCV- MDR1K433M/K1076M (CEM-P-gpMM-3 and CEM-P-gpMM-32 ) were obtained and expressed equivalent levels of intracellular GFP. Login to comment
26 To confirm that the desired form of P-gp was expressed, the regions encoding K433M and K1076M substitutions were amplified by PCR using cDNA obtained from CEM-P-gpWT , CEM-P-gpMM-3 and CEM-P-gpMM-32 cells and sequenced (data not shown). Login to comment
164 Production of retrovirally transduced cell lines Production of MSCV-MDR1 (wild type) and MSCV-MDR1 (K433 M, K1076M) has been previously described.41 These constructs or MSCV alone were transfected into 293T cells with PEQ and RD114 helper viruses by CaPO4 À precipitation. Login to comment
171 Primers for K1076M were: (forward) ATCCCAGTGCTTCAGGGA and (reverse) CCTTATTCCAAGCGGCTT. Login to comment

PMID: 7665554 [PubMed] Loo TW et al: "Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelate chromatography and characterization of their drug-stimulated ATPase activities."

No. Sentence Comment

95 To determine the contribution of either nucleotide-binding domain to drug-stimulatable ATPase activity, mutations were made to the core amino acids (G432S, K433M, G1075S, and K1076M, respectively) of the homology A consensus sequences. Login to comment
123 Mutants K433M, G1075S, and K1076M had no detectable ATPase activities and are omitted for clarity. Login to comment

PMID: 7706385 [PubMed] Sardini A et al: "Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling."

No. Sentence Comment

36 Plasmids, mutagenesis and transient expression system Plasmids pMDR7, expressing the human MDR1 gene, and pMDR712, expressing the human MDR1 gene mutated in both ATP binding domains (K433M and K1076M), were constructed as previously described (Valverde et al., 1992; Gill et al., 1992). Login to comment

PMID: 8567633 [PubMed] Muller M et al: "Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein."

No. Sentence Comment

38 In the present experiments we used site-directed mutagenesis to alter single amino acids of the human Pgp in the homology A consensus sequences in the NBDs of the NH2-terminal (Lys433 to Met) and/or COOH-terminal (Lys1076 to Met) halves, and applied the cDNA of the spontaneous Gly185 to Val substitution mutant. Login to comment

PMID: 8662768 [PubMed] Goodfellow HR et al: "Protein kinase C-mediated phosphorylation does not regulate drug transport by the human multidrug resistance P-glycoprotein."

No. Sentence Comment

27 The construction of pMDR712, expressing the human MDR1 gene mutated in both ATP-binding domains (K433M and K1076M), has been described previously (Gill et al., 1992). Login to comment

PMID: 9371774 [PubMed] Mechetner EB et al: "P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity."

No. Sentence Comment

22 The mutant Pgps contain K433M and͞or K1076M substitutions in the Walker A motifs; in addition, the cloning procedure resulted in a Q1280A substitution of the C-terminal amino acid in all of the transfected MDR1 cDNA sequences. Login to comment

PMID: 9521779 [PubMed] Urbatsch IL et al: "Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites."

No. Sentence Comment

29 In purified reconstituted human MDR1, mutations at the corresponding positions (K433M, K1076M) abolished verapamil-stimulated ATPase activity (24). Login to comment

PMID: 9553060 [PubMed] Szabo K et al: "Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1. Cooperation of the nucleotide binding domains."

No. Sentence Comment

5 MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. Login to comment
39 Baculovirus transfer vectors were constructed as described earlier (20, 21) by using the human MDR1 cDNA encoding a protein with the following mutants: ⌬aa 78-97, K433M, K1076M, and K433M/K1076M. Login to comment
117 These lysines were replaced by methionines either in the N-terminal ABC domain (K433M), in the C-terminal ABC domain (K1076M), or in both ABC domains (K433M/K1076M). Login to comment
125 Nucleotide trapping in the nucleotide binding site mutants of MDR1. Labeling was performed in Sf9 cell membranes expressing either beta-galactosidase, the wild-type human MDR1, or the nucleotide binding site mutants K433M, K1076M, and K433M/ K1076M. Login to comment
135 As shown, there was no significant azido-ATP trapping in either the K433M or K1076M mutant MDR1 proteins, whereas the corresponding immunoblots ensured that the isolated Sf9 cell membranes contained about equal amounts of the MDR1 variants in all experiments. Login to comment
162 The mutant MDR1 proteins, in which lysines in the first (K433M), second (K1076M), or both nucleotide binding domains are replaced by methionines, were demonstrated to bind MgATP less efficiently at low MgATP concentrations (2-5 ␮M) but similarly to the wild-type MDR1 at concentrations above 10 ␮M MgATP (21). Login to comment

PMID: 8040304 [PubMed] Fanen P et al: "Identification of mutations in the putative ATP-binding domain of the adrenoleukodystrophy gene."

No. Sentence Comment

138 The chloride channel activity of the CFTR mutant S 1255P, which involves the same amino acid position in the NBF as in the ALD mutant R518W, is less sensitive to ATP stimulation (24) and the MDR mutants K433M or K1076M within the same Walker motif are unable to hydrolyze ATP (25). Login to comment

PMID: 16352426 [PubMed] Ambudkar SV et al: "The power of the pump: mechanisms of action of P-glycoprotein (ABCB1)."

No. Sentence Comment

52 Between the Walker A and B sequences is found a linker peptide with the sequence LSGGQ, also known as the C-region or ABC signature sequence, as it is the hallmark of Table 1 - Summary of mutational analysis of conserved residues in nucleotide-binding domains of Pgp Domain Source Residue number Function Reference NBD1 NBD2 A-loop Human Y401A Y1044A No ATP binding/hydrolysis Kim et al. (submitted for publication) Walker A Mouse K429N K1072N Normal ATP binding but no hydrolysis Azzaria et al. (1989) G431A G1073A Human C431 C1074 ATP protects from modification by N-ethylmaleimide Loo and Clarke (1995) Disulfide bond formation between Walker A domains of both NBDs Urbatsch et al. (2001) Human K433M K1076M Decreased ATP-binding Muller et al. (1996) No ATP hydrolysis Szakacs et al. (2000) No vanadate-trapping, but aluminum and beryllium fluoride-induced trapping normal Q-loop Mouse Q471 Q1114 Not essential for ATP hydrolysis but may be involved in communication with drug-substrate sites Urbatsch et al. (2000a) LSGGQ or linker peptide or signature motif Mouse S528A S1173A Normal ATP binding but no hydrolysis Tombline et al. (2004a) Human S532R Decreased cell surface expression Hoof et al. (1994) Human G534C G1179C No ATP hydrolysis Loo et al. (2002) Human G534D Decreased cell surface expression Hoof et al. (1994) No drug resistance Normal cell surface expression Bakos et al. (1997) No ATP hydrolysis Human G534D/V G1179D Interdomain communication Szakacs et al. (2001) Human Q535C Q1180C No ATP hydrolysis Loo et al. (2002) Human K536Q Decreased drug resistance Hoof et al. (1994) LSGGQ or linker peptide or signature motif Human K536R Increased colchicine resistance (normal ATP hydrolysis?) Login to comment

PMID: 16545467 [PubMed] Shilling RA et al: "New light on multidrug binding by an ATP-binding-cassette transporter."

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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

PMID: 11284688 [PubMed] Druley TE et al: "P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation."

No. Sentence Comment

38 The MK, KM, and MM mutants contain amino acid substitutions at either one (KM or MK) or both (MM) conserved lysine residues in the Walker A motifs of the N-terminal or C-terminal NBS, K433M and K1076M, respectively. Login to comment
135 Figure 4B compares the effects of increasing concentrations of colchicine on UIC2 reactivity of LMtk- cell lines transfected with either the wild-type human Pgp (KK-H) or Pgp mutants carrying K433M or K1076M substitutions of the essential lysine residues in the Walker A motifs of the N-terminal (MK-H) or C-terminal (KM-H) NBS, respectively. Login to comment

PMID: 11284687 [PubMed] Druley TE et al: "Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity."

No. Sentence Comment

49 Site-directed mutagenesis was used to generate the MK, KM, and MM mutants, which contain amino acid substitutions at either one (KM or MK) or both (MM) conserved lysine residues in the Walker A motifs of the N-terminal or C-terminal nucleotide-binding sites, K433M and K1076M, respectively. Login to comment

PMID: 11027628 [PubMed] Szakacs G et al: "Transition-state formation in ATPase-negative mutants of human MDR1 protein."

No. Sentence Comment

0 Transition-State Formation in ATPase-Negative Mutants of Human MDR1 Protein Gergely Szaka´cs,* Csilla O¨ zvegy,† E´ va Bakos,*, † Bala´zs Sarkadi,* and Andra´s Va´radi†,1 *National Institute of Haematology and Immunology, Membrane Research Group, Hungarian Academy of Sciences, Daro´czi ut 24, H-1113 Budapest, Hungary; and †Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary Received August 26, 2000 In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Login to comment
26 We have shown earlier that the replacement of a lysine residue with methionine in the catalytic Walker A motif in either ABC domains of the human MDR1 (K433M and K1076M) eliminated drug-stimulated ATPase activity, while these mutants could still bind ATP [13]. Login to comment
59 The arrow indicates the position of human MDR1 on the blot. Nucleotide Trapping in the Walker A Mutants in the Presence of AlF4 and BeFx: Effects of Drugs Our earlier results showed that the replacement of lysine with methionine in either of the Nor C-terminal Walker A sequences (mutations K433M and K1076M) resulted in the loss of ATPase activity [13], although these mutations did not interfere with MgATP-binding. Login to comment
78 Labeling was performed by the incubation of 100 ␮g of Sf9 cell membranes expressing K433M MDR1 (lanes 1, 2 and 5, 6) or K1076M MDR1 (lanes 3, 4 and 7, 8), at 37°C for 10 min in the presence of 20 ␮M [␣-32 P]-8-azido-ATP and 2 mM MgCl2, as described under Materials and Methods. Login to comment
82 Labeling was performed by the incubation of 200 ␮g of Sf9 cell membranes expressing wild-type MDR1 (lanes 1 and 2), K433M MDR1 (lanes 3 and 4), K1076M MDR1 (lanes 5 and 6), at 37°C for 10 min in the presence of 20 ␮M [␣-32 P]-8-azido-ATP and 2 mM MgCl2. Login to comment
85 The arrows indicate the position of human MDR1 and the tryptic fragments containing the N-terminal and C-terminal halves, as revealed by immunostaining of the same the blot. by AlF4 are formed in both ABC domains of the wild-type, as well as of the K433M and the K1076M mutant variants (reactions with BeFx gave similar results, not shown). Login to comment
89 We have replaced a key lysine of the Walker A motif (GCGKST in both ABC domains) to methionine (K433M and K1076M). Login to comment
108 Drug stimulation of the rate of formation of the occluded nucleotide transitory complexes was preserved in the Walker A mutants, K433M and K1076M (Fig. 2). Login to comment
109 In the double mutant, K433M/K1076M, no nucleotide trapping was detected, regardless of the choice of the stabilizing anion (not shown). Login to comment
132 However, the results of this study show that a unilateral mutation (K1076M, that prevents the vanadate-dependent labeling, but allows labeling in the presence of AlF4 and BeFx), has the same effect on both sites: the C-terminal mutation results in an altered conformation, and this minor alteration of the mutated active center is "propagated" to the other (native) catalytic site. Login to comment

PMID: 9733949 [PubMed] Takada Y et al: "Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein."

No. Sentence Comment

73 Vanadate-induced nucleotide trapping and ATP binding in K433M and K1076M mutant P-glycoproteins To further examine the roles of the two NBFs in the function of P-glycoprotein, we constructed three other mutants, K433M, K1076M, and K433M/ K1076M, in which the lysine residue in the WA motif of either or both NBFs was replaced by methionine. Login to comment
78 Immunoblot analysis (A), vanadate-induced nucleotide trapping (B), and 8-azido-ATP binding (C) of wild-type protein and lysine-to-methionine mutants of P-glycoprotein-S. Lanes: 1, cells not transfected; 2, P-glycoprotein-S; 3, K433M; 4, K1076M; 5, K433M/K1076M. Login to comment
86 As previously reported all these mutants bound ATP, although the binding to the K433M/K1076M double-mutant form appeared substantially reduced (lane 5). Login to comment

PMID: 8549739 [PubMed] Senior AE et al: "The catalytic cycle of P-glycoprotein."

No. Sentence Comment

103 Loo and Clarke [37] extended this approach, showing that the mutations K433M, K1076M, G432S and G1075S in the Homology A sequences of either NBS1 or NBS2 in human Pgp abolish drug-exclusion capability in cells and eliminate ATPase activity in membranes. Login to comment