ABCMdb

ABCB1 p.Gly1073Cys

Predicted by SNAP2:	A: D (91%), C: D (75%), D: D (91%), E: D (95%), F: D (95%), H: D (91%), I: D (91%), K: D (95%), L: D (95%), M: D (91%), N: D (85%), P: D (95%), Q: D (91%), R: D (95%), S: D (85%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] The "LSGGQ" motif in each nucleotide-binding domai... J Biol Chem. 2002 Nov 1;277(44):41303-6. Epub 2002 Sep 10. Loo TW, Bartlett MC, Clarke DM
The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence.
J Biol Chem. 2002 Nov 1;277(44):41303-6. Epub 2002 Sep 10., 2002-11-01 [PMID:12226074]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) family of transport proteins, actively transports many cytotoxic compounds out of the cell. ABC transporters have two nucleotide-binding domains (NBD) and two transmembrane domains. The presence of the conserved "signature" sequence (LSGGQ) in each NBD is a unique feature in these transporters. The function of the signature sequences is unknown. In this study, we tested whether the signature sequences ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) in P-gp are in close proximity to the opposing Walker A consensus nucleotide-binding sequences ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1). Pairs of cysteines were introduced into a Cys-less P-gp at the signature and "Walker A" sites and the mutant P-gps were subjected to oxidative cross-linking. At 4 degrees C, when thermal motion is low, P-gp mutants (L531C(Signature)/C1074(Walker A) and C431(Walker A)/L1176C(Signature) were cross-linked. Cross-linking inhibited the drug-stimulated ATPase activities of these two mutants. Their activities were restored, however, after addition of the reducing agent, dithiothreitol. Vanadate trapping of nucleotide at the ATP-binding sites prevented cross-linking of the mutants. These results indicate that the signature sequences are adjacent to the opposing Walker A site. They likely participate in forming the ATP-binding sites and are displaced upon ATP hydrolysis. The resulting conformational change may be the signal responsible for coupling ATP hydrolysis to drug transport by inducing conformational changes in the transmembrane segments.

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No. Sentence Comment
58 The complete cross-linking results between the cysteines in the NBD1 terminal signature sequence (531 LSGGQ535 ) and the cysteines in the NBD2 Walker A site (1070 GSSGCGKS1077 ) are shown in Table I. Residues G1073C and C1074 in the NBD2 Walker A site and L531C and S532C in the NBD1 signature sequence appear to be closest, since mutants L531C/G1073C, L531C/C1074, and S532C/G1073C were cross-linked when treated with oxidant at 4 °C. Login to comment
59 The cysteines in other mutants (e.g. L531C/S1072C, S532/S1072C, and G533C/G1073C) must be further apart, since cross-linked product was only observed at either 21 or 37 °C. Login to comment
66 Representative cross-linking results of two (NBD1 signature/NBD2 Walker A) mutants (L531C/G1073C and L531C/C1074) are shown in Fig. 2. Login to comment
72 The other mutants (Table I) such as L531C/G1073C (Fig. 2) showed no detectable inhibition of cross-linking when preincubated with ATP plus vanadate. Login to comment
74 This appears to be the case for mutants such as L531C/G1073C that showed no inhibition of cross-linking after treatment with ATP plus vanadate. Login to comment
89 CP, copper phenanthroline TABLE I Cross-linking between residues in the NBD1 signature sequence and in the NBD2 Walker A site L531C (21%)a S532C (38%) G533C (91%) G534C (4%) Q535C (0%) 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C G1070C (0%) -b - - - - - - - - - - - - - - S1071C (85%) - - *c - - - - - - - - ϩ - - ϩ S1072C (23%) - ϩc ϩϩd - ϩϩ ϩϩ - - ϩ - - ϩ - - - G1073C (4%) ϩ ϩϩ ϩϩ ϩ ϩϩ ϩϩ - ϩ ϩϩ - - - - - - C1074 (103%) ** ** **d,e - * * - - * - - - - - - G1075C (0%) - - ϩϩ - - ϩ - - - - - - - - - K1076C (12%) - - * - - - - - - - - - - - - S1077C (0%) - - - - - - - - - - - - - - - a Activity of the single cysteine mutant relative to Cys-less P-gp. b No cross-linked product detected in SDS-PAGE. c Relatively weak cross-linking (Ͻ50% of P-gp cross-linked). Login to comment
112 Membranes prepared from HEK 293 cells expressing mutants L531C/ G1073C, L531C/C1074, or L1176C/C431 were preincubated for 10 min at 37 °C in the presence (ϩ) or absence (-) of ATP plus vanadate. Login to comment
131 Hydrolysis of ATP was essential, since inhibition of cross-linking was not observed in the presence of the non-hydrolyzable ATP analog AMP.PNP (data not shown) or in the inactive mutants (e.g. mutant L531C/G1073C; Fig. 2). Login to comment

[hide] Validation of in vitro cell models used in drug me... Toxicol Appl Pharmacol. 2006 Feb 15;211(1):1-10. Epub 2005 Jun 21. Brandon EF, Bosch TM, Deenen MJ, Levink R, van der Wal E, van Meerveld JB, Bijl M, Beijnen JH, Schellens JH, Meijerman I
Validation of in vitro cell models used in drug metabolism and transport studies; genotyping of cytochrome P450, phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines.
Toxicol Appl Pharmacol. 2006 Feb 15;211(1):1-10. Epub 2005 Jun 21., [PMID:15975613]

Abstract [show]
Human cell lines are often used for in vitro biotransformation and transport studies of drugs. In vivo, genetic polymorphisms have been identified in drug-metabolizing enzymes and ABC-drug transporters leading to altered enzyme activity, or a change in the inducibility of these enzymes. These genetic polymorphisms could also influence the outcome of studies using human cell lines. Therefore, the aim of our study was to pharmacogenotype four cell lines frequently used in drug metabolism and transport studies, HepG2, IGROV-1, CaCo-2 and LS180, for genetic polymorphisms in biotransformation enzymes and drug transporters. The results indicate that, despite the presence of some genetic polymorphisms, no real effects influencing the activity of metabolizing enzymes or drug transporters in the investigated cell lines are expected. However, this characterization will be an aid in the interpretation of the results of biotransformation and transport studies using these in vitro cell models.

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No. Sentence Comment
56 (2000), was used to determine the *6 (G211A), *7 (T1456G), *27 (C686A), *28 (A(TA)6TAA to A(TA)7TAA), *33 (A(TA)6TAA to A(TA)5TAA), *34 (A(TA)6TAA to A(TA)8TAA) and *29 (C1099G) polymorphisms in the UGT1A1 gene. Login to comment
57 The UGT2B7 polymorphisms A-268G in the promoter region, A386G in exon 1, *2 coding a change of T801A and C802T in exon 2 and G1073C in exon 4 were determined according to the methods described by Bhasker et al. Login to comment
171 W W W W *2 56 normal M H H H G1073C 61 ? Login to comment
169 W W W W *2 56 normal M H H H G1073C 61 ? Login to comment