ABCMdb

ABCB1 p.Lys536Gln

Predicted by SNAP2:	A: D (85%), C: D (80%), D: D (95%), E: D (91%), F: D (91%), G: D (91%), H: D (85%), I: D (85%), L: D (91%), M: D (85%), N: D (91%), P: D (95%), Q: D (85%), R: D (63%), S: D (80%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: 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] Functional implications of genetic polymorphisms i... Pharm Res. 2004 Jun;21(6):904-13. Pauli-Magnus C, Kroetz DL
Functional implications of genetic polymorphisms in the multidrug resistance gene MDR1 (ABCB1).
Pharm Res. 2004 Jun;21(6):904-13., [PMID:15212152]

Abstract [show]
The multidrug resistance (MDR1) gene product P-glycoprotein is a membrane protein that functions as an ATP-dependent efflux pump, transporting exogenous and endogenous substrates from the inside of cells to the outside. Physiological expression of P-glycoprotein in tissues with excretory or protective function is a major determinant of drug disposition and provides a cellular defense mechanism against potentially harmful compounds. Therefore, P-glycoprotein has significant impact on therapeutic efficacy and toxicity as it plays a key role in absorption of oral medications from the intestinal tract, excretion into bile and urine, and distribution into protected tissues such as the brain and testes. There is increasing interest in the possible role of genetic variation in MDR1 in drug therapy. Numerous genetic polymorphisms in MDR1 have been described, some of which have been shown to determine P-glycoprotein expression levels and substrate transport. Furthermore, some of these polymorphisms have an impact on pharmacokinetic and pharmacodynamic profiles of drug substrates and directly influence outcome and prognosis of certain diseases. This review will focus on the impact of genetic variation in MDR1 on expression and function of P-glycoprotein and the implications of this variation for drug therapy and disease risk.

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118 Functional Impact in vitro of MDR1 Variants Amino acid change Functional effect of the variant allele Reference Val185Ser Increased colchicine resistance [30] ⌬Phe335 Decreased resistance to vinca alkaloids; no resistance to dactinomycin [31] Lys536Gln, Gly534Asp, Lys536Arg, Ser532Arg, ⌬Tyr490 Defective RNA processing [33] Ala893Ser Acquired overexpression of one allele in drug-resistant cells [20] Ala893Ser Decreased digoxin efflux [19] Asn21Asp, Phe103Leu, Ser400Ala, Ala893Ser, Ala893Thr No effect on P-glycoprotein cell surface expression and substrate specificity [69] Ala893Ser No difference in calcein-AM transport [27] Ala893Ser/Thr No difference in transport of verapamil, digoxin, viblastine and cyclosporine A [35] 3435 polymorphisms were analyzed separately, with AUC values being highest for individuals carrying the reference alleles. Login to comment

[hide] Cystic fibrosis-type mutational analysis in the AT... J Biol Chem. 1994 Aug 12;269(32):20575-83. Hoof T, Demmer A, Hadam MR, Riordan JR, Tummler B
Cystic fibrosis-type mutational analysis in the ATP-binding cassette transporter signature of human P-glycoprotein MDR1.
J Biol Chem. 1994 Aug 12;269(32):20575-83., 1994-08-12 [PMID:7914197]

Abstract [show]
Members of the ATP-binding cassette transporter superfamily such as the P-glycoproteins (MDR) and the cystic fibrosis transmembrane conductance regulator (CFTR) share conserved sequence motifs in their nucleotide binding fold that are the major targets for CFTR mutations in patients with cystic fibrosis. Cystic fibrosis-type mutations were introduced at analogous positions into the human MDR1 gene. Heterologous expression of wild-type or mutated MDR1 revealed similar mRNA transcript levels in Chinese hamster ovary K1 recipients, but the subsequent processing was defective for all mutations that give rise to severe cystic fibrosis in the case of CFTR. Functional multidrug transporter MDR1, however, was obtained when amino acid substitutions were introduced into a less conserved position of the ATP-binding cassette transporter signature (codon 536 in MDR1). The profile of cross-resistance and chemosensitization was modulated in these codon 536 variants, which suggests that this region is involved in the drug transport function of P-glycoprotein.

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110 MRK16 recognizes external epitopes of human MDRl without cross-reactions with rodent P-glycoproteins (Hamada and Tsuruo, 1991).The AY490 and AY490-K536Q transfectants were negative for MRK16 like the parentcontrol line CHO K1 (Fig. 4A).In the S532R and G534D MDRls recombinant cell lines, mixed populations of MRK16 immunoreactive cells were observed (Fig. 4A).Subsequentexposure to colchicine(100,125, 150,or 175ng/ml) increased the proportion of cells with higher antigen density with increasing colchicine concentration (Fig. 4B). Login to comment
112 Homogeneous populations of human P-glycoprotein-expressing cells were observed in thecelllines thatwere carryingwild-type MDRl, K536R MDR1, or K536Q MDRl cDNA (Fig. 4A). Login to comment
113 Phenotype of Multidrug Resistance and Collateral Sensitiuity-Transfection withMDRl vector constructsren- AY490 G534D K536Q 1 ". Login to comment
116 1 , ', k. ,-I wt S532R K536R AY490 I K536Q transcripts in recombinant CHO K1 cells by RTPCR kinetics. Login to comment
122 dered the CHO K1 cells more resistant toadriamycin and colchicine when either wild-type sequence or the codon 536 variants, K536R or K536Q, had beenintroduced(Fig.2).This finding on the G418-preselected cell pool was confirmed by growth inhibitionexperiments withclonal cell lines. Login to comment
130 Cell line Fluorescence Signalnormalized to CHO K1 control CHO K1 8.6 x 10' CHO K1 (MDR1) 1.0 CHO K1 (AY490 MDR1) 6.3 x 10' 0.7 CHO K1 (S532R MDR1)" 4.4 x 104 51 CHO K1 (G534D MDR1)" 2.1 x 105 240 CHO K1 (K536R MDR1) 5.6 x 105 650 CHO K1 (K536Q MDR1) 9.4 x 105 CHO K1 (AY490-K536QMDR1) 2.1 x lo3 1,100 6.5 x 105 750 2.4 a The datarefer to the MRK16-positive subpopulation. Login to comment
141 The strongly photoreactive band of P-glycoprotein was exclusively seeninplasmamembranes from cells exhibiting the multidrug resistance phenotype, i.e. transfectants MDRls K1, K536Q MDRls K1, K536R MDRls K1(Fig. 6, left lane in eachpanel), and theB30 positive control. Login to comment
147 Opentriangles, wild-type MDRls CHO K1; closed cycles, K536R MDRls K1; closed triangles, K536Q MDRls K1; stars, S532R MDRls K1; opencircles, AY490 MDRls K1; closed squares, G534D MDRls K1; bars, AY490-K536QMDRls K1; open squares,CHO K1. Login to comment
148 TABLE111 Multidrug resistance and chemosensitizationof P-glycoprotein-expressingCHO cell clones Cell line ID,, values 0 prt' 1 p!.t" 3PMc" 10PMO nglml Colchicine CHO K1 54 6/13 3/13 5'18 CHO K1 (MDR1) 530 1901320 121110 2'/40 ' CHO K1 (K536Q MDR1) 18015/56 6/26 5'113' CHO K1 (K536R MDR1) 840 4801680 471140 7'1546 Vinblastine CHO K1 12 213 112 1'11 CHO K1 (MDR1) 240 1001180 10170 2'I8 ' CHO K1 (K536Q MDR1) 150 10148 3/14 2'I3 ' CHO K1 (K536R MDR1) 220180/210 15190 2'17 ' CHO K1 18 314 314 3'I3 CHO K1 (MDR1) 280100/230 8170 4'114 CHO K1 (K536Q MDR1) 60 6116 317 3'13' CHO K1 (K536R MDR1) 350 190/270 17/70 3'Ill ' a Concentration of cyclosporin (lefi)/FK506 (right). Login to comment
175 MDR1" Wildt w e AY490 S532RG534D AY490-K536Q K536Q. Login to comment

[hide] Characterization of the human multidrug resistance... Biochem J. 1997 May 1;323 ( Pt 3):777-83. Bakos E, Klein I, Welker E, Szabo K, Muller M, Sarkadi B, Varadi A
Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region.
Biochem J. 1997 May 1;323 ( Pt 3):777-83., 1997-05-01 [PMID:9169612]

Abstract [show]
A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly.

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26 The mutant K536Q (Lys to Gln) was expressed in comparable amounts to the wild-type protein, but conferred decreased drug resistance, while the K536R (Lys to Arg) replacement increased multidrug resistance, with a preferential resistance to colchicine. Login to comment

[hide] The power of the pump: mechanisms of action of P-g... Eur J Pharm Sci. 2006 Apr;27(5):392-400. Epub 2005 Dec 13. Ambudkar SV, Kim IW, Sauna ZE
The power of the pump: mechanisms of action of P-glycoprotein (ABCB1).
Eur J Pharm Sci. 2006 Apr;27(5):392-400. Epub 2005 Dec 13., [PMID:16352426]

Abstract [show]
Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.

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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