ABCMdb

ABCB1 p.Gly185Val

ClinVar:	c.554G>T , p.Gly185Val D , Pathogenic
Predicted by SNAP2:	A: N (53%), C: D (75%), D: D (85%), E: D (91%), F: D (85%), H: D (91%), I: D (85%), K: D (91%), L: D (80%), M: D (85%), N: D (85%), P: D (91%), Q: D (91%), R: D (91%), S: D (75%), T: D (80%), V: D (75%), W: D (91%), Y: D (91%),
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] Single amino acid (482) variants of the ABCG2 mult... Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63. Ozvegy-Laczka C, Koblos G, Sarkadi B, Varadi A
Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition.
Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63., 2005-02-01 [PMID:15670731]

Abstract [show]
The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

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25 In colchicine selected cells, mutation of Gly 185 to Val (found in the intracellular loop between TM helices 2 and 3) occurred in the overexpressed MDR1 protein [16]. Login to comment
26 The G185V mutant conferred altered basal ATPase activity and altered interaction with substrates, as well as with the inhibitor cyclosporin A [17,18]. Login to comment

[hide] Genetic polymorphisms of ATP-binding cassette tran... Expert Opin Pharmacother. 2005 Nov;6(14):2455-73. Sakurai A, Tamura A, Onishi Y, Ishikawa T
Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications.
Expert Opin Pharmacother. 2005 Nov;6(14):2455-73., [PMID:16259577]

Abstract [show]
Pharmacogenomics, the study of the influence of genetic factors on drug action, is increasingly important for predicting pharmacokinetics profiles and/or adverse reactions to drugs. Drug transporters, as well as drug metabolism play pivotal roles in determining the pharmacokinetic profiles of drugs and their overall pharmacological effects. There is an increasing number of reports addressing genetic polymorphisms of drug transporters. However, information regarding the functional impact of genetic polymorphisms in drug transporter genes is still limited. Detailed functional analysis in vitro may provide clear insight into the biochemical and therapeutic significance of genetic polymorphisms. This review addresses functional aspects of the genetic polymorphisms of human ATP-binding cassette transporters, ABCB1 and ABCG2, which are critically involved in the pharmacokinetics of drugs.

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128 G185V is an acquired mutation. Login to comment
129 N21D M89T N44S H2N F103L E108K N183S G185V I261V S400N R492C A599T L662R R669C V801M A893S/T I829V I849M M986V A999T G1063A P1051A Q1107P W1108R I1145M S1141T V1251I T1256K COOH ATP-binding site ATP-binding site EXTRACELLULAR INTRACELLULAR A80E Figure 2. Login to comment

[hide] High-speed screening of human ATP-binding cassette... Methods Enzymol. 2005;400:485-510. Ishikawa T, Sakurai A, Kanamori Y, Nagakura M, Hirano H, Takarada Y, Yamada K, Fukushima K, Kitajima M
High-speed screening of human ATP-binding cassette transporter function and genetic polymorphisms: new strategies in pharmacogenomics.
Methods Enzymol. 2005;400:485-510., [PMID:16399366]

Abstract [show]
Drug transporters represent an important mechanism in cellular uptake and efflux of drugs and their metabolites. Hitherto a variety of drug transporter genes have been cloned and classified into either solute carriers or ATP-binding cassette (ABC) transporters. Such drug transporters are expressed in various tissues such as the intestine, brain, liver, kidney, and, importantly, cancer cells, where they play critical roles in the absorption, distribution, and excretion of drugs. We developed high-speed functional screening and quantitative structure-activity relationship analysis methods to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide powerful and practical tools for screening synthetic and natural compounds, and the deduced data can be applied to the molecular design of new drugs. Furthermore, we demonstrate a new "SNP array" method to detect genetic polymorphisms of ABC transporters in human samples.

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150 G185V is an acquired mutation. Login to comment

[hide] Human multidrug resistance ABCB and ABCG transport... Physiol Rev. 2006 Oct;86(4):1179-236. Sarkadi B, Homolya L, Szakacs G, Varadi A
Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.
Physiol Rev. 2006 Oct;86(4):1179-236., [PMID:17015488]

Abstract [show]
In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

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495 A spontaneous glycine to valine mutation in the intracellular end of the third transmembrane helix of MDR1/Pgp (G185V) was shown to confer increased colchicine resistance to cells. Login to comment

[hide] Pharmacogenetics/genomics of membrane transporters... Cancer Metastasis Rev. 2007 Mar;26(1):183-201. Huang Y
Pharmacogenetics/genomics of membrane transporters in cancer chemotherapy.
Cancer Metastasis Rev. 2007 Mar;26(1):183-201., [PMID:17323126]

Abstract [show]
Inter-individual variability in drug response and the emergence of adverse drug reactions are main causes of treatment failure in cancer therapy. Recently, membrane transporters have been recognized as an important determinant of drug disposition, thereby affecting chemosensitivity and -resistance. Genetic factors contribute to inter-individual variability in drug transport and targeting. Therefore, pharmacogenetic studies of membrane transporters can lead to new approaches for optimizing cancer therapy. This review discusses genetic variations in efflux transporters of the ATP-binding cassette (ABC) family such as ABCB1 (MDR1, P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2) and ABCG2 (BCRP), and uptake transporters of the solute carrier (SLC) family such as SLC19A1 (RFC1) and SLCO1B1 (SLC21A6), and their relevance to cancer chemotherapy. Furthermore, a pharmacogenomic approach is outlined, which using correlations between the growth inhibitory potency of anticancer drugs and transporter gene expression in multiple human cancer cell lines, has shown promise for determining the relevant transporters for any given drugs and predicting anticancer drug response.

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89 Colchicine-selected cells exhibited Gly185Val substitution, resulting in increased resistance to colchicine but not to other drugs [35]. Login to comment

[hide] Clinical pharmacogenetics and potential applicatio... Curr Drug Metab. 2008 Oct;9(8):738-84. Zhou SF, Di YM, Chan E, Du YM, Chow VD, Xue CC, Lai X, Wang JC, Li CG, Tian M, Duan W
Clinical pharmacogenetics and potential application in personalized medicine.
Curr Drug Metab. 2008 Oct;9(8):738-84., [PMID:18855611]

Abstract [show]
The current 'fixed-dosage strategy' approach to medicine, means there is much inter-individual variation in drug response. Pharmacogenetics is the study of how inter-individual variations in the DNA sequence of specific genes affect drug responses. This article will highlight current pharmacogenetic knowledge on important drug metabolizing enzymes, drug transporters and drug targets to understand interindividual variability in drug clearance and responses in clinical practice and potential use in personalized medicine. Polymorphisms in the cytochrome P450 (CYP) family may have had the most impact on the fate of pharmaceutical drugs. CYP2D6, CYP2C19 and CYP2C9 gene polymorphisms and gene duplications account for the most frequent variations in phase I metabolism of drugs since nearly 80% of drugs in use today are metabolised by these enzymes. Approximately 5% of Europeans and 1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers. CYP2C9 is another clinically significant drug metabolising enzyme that demonstrates genetic variants. Studies into CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and CYP2C9*3 alleles. Extensive polymorphism also occurs in a majority of Phase II drug metabolizing enzymes. One of the most important polymorphisms is thiopurine S-methyl transferases (TPMT) that catalyzes the S-methylation of thiopurine drugs. With respect to drug transport polymorphism, the most extensively studied drug transporter is P-glycoprotein (P-gp/MDR1), but the current data on the clinical impact is limited. Polymorphisms in drug transporters may change drug's distribution, excretion and response. Recent advances in molecular research have revealed many of the genes that encode drug targets demonstrate genetic polymorphism. These variations, in many cases, have altered the targets sensitivity to the specific drug molecule and thus have a profound effect on drug efficacy and toxicity. For example, the beta (2)-adrenoreceptor, which is encoded by the ADRB2 gene, illustrates a clinically significant genetic variation in drug targets. The variable number tandem repeat polymorphisms in serotonin transporter (SERT/SLC6A4) gene are associated with response to antidepressants. The distribution of the common variant alleles of genes that encode drug metabolizing enzymes, drug transporters and drug targets has been found to vary among different populations. The promise of pharmacogenetics lies in its potential to identify the right drug at the right dose for the right individual. Drugs with a narrow therapeutic index are thought to benefit more from pharmacogenetic studies. For example, warfarin serves as a good practical example of how pharmacogenetics can be utilized prior to commencement of therapy in order to achieve maximum efficacy and minimum toxicity. As such, pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and licensed drugs.

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532 Nucleotide change rs number Amino acid change 49T>C rs28381804 F17L 61A>G rs61615398; rs9282564 N21D 131A>G rs1202183 N44S 178A>C rs41315618 I60L 239C>A rs9282565 A80E 266T>C Rs35810889 M89T 431T>C rs61607171 I144T 502G>A rs61122623 V168I 548A>G rs60419673 N183S 554G>T rs1128501 G185V 781A>G rs36008564 I261V 1199G>A rs2229109 S400N 1696G>A rs28381902 E566K 1777C>T rs28381914 R593C 1778G>A rs56107566 R593H 1795G>A rs2235036 A599T 1837G>T rs57001392 D613Y 1985T>G rs61762047 L662R 2005C>T rs35023033 R669C 2207A>T rs41316450 I736K 2398G>A rs41305517 D800N 2401G>A rs2235039 V801M 2485A>G rs2032581 I829V 2506A>G rs28381967 I836V 2547A>G rs36105130 I849M 2677T>A/G rs2032582 S893A/T 2975G>A rs56849127 S992N 3151C>G rs28401798 P1051A 3188G>C rs2707944 G1063A 3262G>A rs57521326 D1088N 3295A>G rs41309225 K1099E 3320A>C rs55852620 Q1107P 3322T>C rs35730308 W1108R 3410G>T rs41309228 S1137I 3421T>A rs2229107 S1141T 3502A>G rs59241388 K1168E 3669A>T rs41309231 E1223D 3751G>A rs28364274 V1251I 3767C>A r35721439 T1256K Data are from NCBI dbSNP (access date: 2 August 2008). Login to comment

[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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No. Sentence Comment
504 Glycine residues have been shown to play important roles for structure and function; the naturally occurring G185V mutation located between the TM2 and TM3 and other glycines such as G141, G830 resulted in increased resistance to colchicine and decreased resistance to vinblastine [5,93,245]. Login to comment

[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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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
490 1996. Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. Mol. Login to comment

[hide] P-Glycoprotein, Multidrug Resistance and Protein K... Oncologist. 1996;1(4):261-268. Fine RL, Chambers TC, Sachs CW
P-Glycoprotein, Multidrug Resistance and Protein Kinase C.
Oncologist. 1996;1(4):261-268., [PMID:10388000]

Abstract [show]
The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-a isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.

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141 The human MDR1 P-glycoprotein, with a mutation of amino acid glycine 185 to valine, was also expressed in Sf9 cells and used as membrane vesicles. Login to comment

[hide] Identification of P-glycoprotein mutations causing... J Biol Chem. 1999 Jul 16;274(29):20318-27. Vo QD, Gruol DJ
Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport.
J Biol Chem. 1999 Jul 16;274(29):20318-27., 1999-07-16 [PMID:10400654]

Abstract [show]
P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.

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37 The mutation G185V in the human Pgp causes increased colchicine resistance and decreased vinblastine resistance. Login to comment

[hide] Identification of residues in the drug-binding dom... J Biol Chem. 1999 Dec 10;274(50):35388-92. Loo TW, Clarke DM
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
J Biol Chem. 1999 Dec 10;274(50):35388-92., 1999-12-10 [PMID:10585407]

Abstract [show]
The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.

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176 Mutant G185V is an example of a mutation that probably alters P-gp-drug interactions because of structural perturbations. Login to comment
178 Subsequent analysis of this mutant, however, led to the conclusion that G185V alters function of P-gp by affecting the structure of the transporter (49). Login to comment

[hide] Drug selection of MDR1-transduced hematopoietic ce... Gene Ther. 2000 Feb;7(4):348-58. Licht T, Goldenberg SK, Vieira WD, Gottesman MM, Pastan I
Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice.
Gene Ther. 2000 Feb;7(4):348-58., [PMID:10694816]

Abstract [show]
The MDR1 (multidrug resistance) gene, transferred to hematopoietic cells, is expected to protect them from anticancer chemotherapy and may serve as a selectable marker, restoring gene expression in vivo. Appropriate selection strategies, however, need to be established. To investigate whether preselection ex vivo affects chemoresistance, murine bone marrow cells were retrovirally transduced with high-titer or, as a model for suboptimal gene expression, low-titer retroviruses and exposed to daunomycin or colchicine for 48-96 h. Selection significantly increased chemoresistance of clonogenic progenitor cells. In tissue culture, the entire target population was rendered highly drug resistant after MDR1 transfer with high-titer viruses. If transduction was performed under suboptimal conditions, drug selection increased the frequency of chemoresistant colonies up to 40% over the number of unselected cells. Colchicine and daunomycin were equally efficient in increasing drug resistance ex vivo, but colchicine-preselected cells rescued lethally irradiated mice under conditions where daunomycin-selected bone marrow cells failed to do so. Hence, while hematopoietic cells can be protected by MDR1, the selection strategy is critical for repopulation of bone marrow with transduced cells. Preselection in culture before transplantation significantly increased P-gp expression and chemoresistance in vivo in mice reconstituted with transduced bone marrow cells. This study may help to facilitate the use of MDR1 as a selectable marker in gene therapy of the hematopoietic system. Gene Therapy (2000) 7, 348-358.

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175 Materials and methods Generation and titration of retroviral producer cells Construction of plasmid pHaMDR1/A which consists of a full-length human MDR1 cDNA flanked by long-terminal repeats from Harvey sarcoma virus has been reported.52 This MDR1 cDNA contains a point mutation (G185V) which confers preferential resistance to colchicine.45,46 By transfection of pHaMDR1/A into PA317 packaging cells, a virus producing cell clone, designated MA1, was generated. Login to comment

[hide] Effect of ABC transporters on HIV-1 infection: inh... FASEB J. 2000 Mar;14(3):516-22. Lee CG, Ramachandra M, Jeang KT, Martin MA, Pastan I, Gottesman MM
Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter.
FASEB J. 2000 Mar;14(3):516-22., [PMID:10698967]

Abstract [show]
The MDR1 multidrug transporter P-gp (P-glycoprotein) is an efflux pump that extrudes diverse hydrophobic drugs and peptides from cells. Since the entry of HIV-1 into cells involves an initial interaction of the viral gp41 hydrophobic peptide with the plasma membrane, a potential effect of P-gp on HIV-1 infectivity was explored. Virus production was greatly decreased when P-gp was overexpressed at the surface of a continuous CD4(+) human T-leukemic cell line (12D7) infected with HIV-1(NL4-3), a T-tropic molecular clone of HIV-1. P-gp overexpression did not significantly alter the surface expression or distribution of either the HIV-1 receptor CD4 or the coreceptor CXCR4. Reduction of HIV-1 infectivity in P-gp-expressing cells occurred both during the fusion of viral and plasma membranes and at subsequent step(s) in the HIV-1 life cycle.

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82 Similar results were seen in five independent experiments. Figure 3 shows that fusion was greatly reduced in target cells expressing either wt MDR1, MDR1 with an inactivating amino-terminal ATP-utilization site (D555N) mutation that renders the transporter inactive or MDR1 with a single mutation in the substrate binding site (G185V) (18). Login to comment
96 WR: wild-type control; MDR1: wild-type MDR1; D555N: amino-terminal ATP binding site mutant of MDR1; G185V; substrate mutant of MDR1 that changes the substrate specificity; CFTR: cystic fibrosis transmembrane regulator. Login to comment

[hide] Relating multidrug resistance phenotypes to the ki... Eur J Biochem. 2000 Sep;267(17):5355-68. Westerhoff HV, Riethorst A, Jongsma AP
Relating multidrug resistance phenotypes to the kinetic properties of their drug-efflux pumps.
Eur J Biochem. 2000 Sep;267(17):5355-68., [PMID:10951193]

Abstract [show]
The simplest model for pump-mediated multidrug resistance is elaborated quantitatively. The way in which toxicity data should be evaluated to characterize most effectively the drug-efflux pump is then examined. The isotoxic drug dose (D10) depends on too many unrelated properties. The D10 of a cell line taken relative to that of the parental (nonresistant) cell line has been called the relative resistance (RR). This is inappropriate for characterizing the drug pump, as it depends on the extent of amplification of the latter. The reduced RR (RRR) is newly defined as the ratio of the (RR - 1) for one drug to the (RR - 1) for a different drug. This RRR should be independent of both the drug-target affinity and the extent of amplification of the drug pump in cell lines belonging to a family. The RRR depends on the avidities with which the pump extrudes the drugs relative to the passive membrane permeabilities of the latter. In plots of RRR for one drug combination vs. that for a second drug combination, cell lines that have the same pump amplified should cluster, whereas those with amplification of (functionally) different drug-efflux pumps should segregate. Both a set of new experimental data and literature results are discussed in terms of RRR. RRRs discriminate between human MDR1 and mouse mdr1a and mdr1b, between hamster pgp1 and a mutant thereof, as well as between human MDR1 and a mutant thereof. RRRs are not affected by changes in membrane surface area. Our results indicate that RRR may be used to (a) characterize drug-resistance mechanisms and (b) determine which drug-resistance mechanism is operative. Moreover, our analysis suggests that some of the reported phenotypic diversity among multidrug-resistant cell lines may not be due to diversity in the resistance mechanism.

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211 Analysis of the data of Choi et al. [29] indeed led to vastly different RRR values between the wild-type MDR1 transfectant and two transfectants mutated from Gly to Val in position 185 (not shown). Login to comment

[hide] Transmembrane domain (TM) 9 represents a novel sit... Mol Pharmacol. 2001 Aug;60(2):254-61. Song J, Melera PW
Transmembrane domain (TM) 9 represents a novel site in P-glycoprotein that affects drug resistance and cooperates with TM6 to mediate [125I]iodoarylazidoprazosin labeling.
Mol Pharmacol. 2001 Aug;60(2):254-61., [PMID:11455011]

Abstract [show]
The multidrug resistant cell line DC-3F/ADII was obtained by stepwise selection for growth in actinomycin D (ActD). Compared with parental cells, it displays high resistance to ActD and vincristine and low resistance to colchicine and daunorubicin. These cells overexpress a form of P-glycoprotein (Pgp1) containing a double mutation, I837L and N839I, in transmembrane domain (TM) 9; when transfected into DC-3F, this mutation confers the DC-3F/ADII phenotype. We have shown previously that another cell line, DC-3F/ADX, also displays this phenotype and overexpresses a mutant form of Pgp1 containing a double mutation in TM6 (G338A, A339P). Hence, mutations in TM9 and TM6 are independently capable of conferring the same cross-resistance phenotype. The TM6 mutations inhibit the ability of cyclosporin A to reverse cross-resistance and to block labeling of the protein by [125I]iodoarylazidoprazosin (IAAP), whereas the TM9 mutations do not show similar effects. A chimeric protein containing both pairs of mutations confers twice the level of resistance to ActD than expected from the sum of the individual mutations, but it cannot be labeled to detectable levels with [125I]IAAP. Thus, TM9 represents a novel site that cooperates with TM6 to mediate drug resistance and [125I]IAAP labeling.

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172 A similar argument has been proposed (Loo and Clark, 1999) to explain how the G185V mutation in MDR1, which also lies outside of the proposed drug binding pocket, alters the cross-resistance phenotype compared with the wild-type (Choi et al., 1989). Login to comment
183 Unlike the G185V mutation, whose pleiotropic effects on MDR1 function (Ramachandra et al., 1996) were interpreted to be caused by higher order structural perturbations, the TM9 mutations displayed no such effects on Pgp1 function. Login to comment

[hide] Distinct haplotype profiles and strong linkage dis... Pharmacogenetics. 2002 Aug;12(6):437-50. Tang K, Ngoi SM, Gwee PC, Chua JM, Lee EJ, Chong SS, Lee CG
Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations.
Pharmacogenetics. 2002 Aug;12(6):437-50., [PMID:12172212]

Abstract [show]
The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.

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18 It has been reported that cells carrying the F983A change exhibit increased resistance to reversal by flupentixol but not other drugs [6], while cells carrying the G185V change have increased resistance to colchicine but not other drugs [7,8]. Login to comment

[hide] MDR1 genotype-related pharmacokinetics and pharmac... Biol Pharm Bull. 2002 Nov;25(11):1391-400. Sakaeda T, Nakamura T, Okumura K
MDR1 genotype-related pharmacokinetics and pharmacodynamics.
Biol Pharm Bull. 2002 Nov;25(11):1391-400., [PMID:12419946]

Abstract [show]
The multidrug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily of membrane transporters. MDR1 acts as an energy-dependent efflux pump that exports its substrates out of cells. MDR1 was originally isolated from resistant tumor cells as part of the mechanism of multidrug resistance, but over the last decade, it has been elucidated that human MDR1 is also expressed throughout the body to confer intrinsic resistance to the tissues by exporting unnecessary or toxic exogeneous substances or metabolites. A number of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics and pharmacodynamics. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and detected 15 single nucleotide polymorphisms (SNPs). They also indicated that a polymorphism in exon 26 at position 3435 (C3435T), a silent mutation, affected the expression level of MDR1 protein in duodenum, and thereby the intestinal absorption of digoxin. To date, the genotype frequencies of C3435T have been investigated extensively using a larger population and interethnic difference has been elucidated, and a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical studies on MDR1 genotype-related MDR1 expression and pharmacokinetics have also been performed around the world; however, results were not always consistent with Hoffmeyer's report. In this review, published reports are summarized for the future individualization of pharmacotherapy based on MDR1 genotyping. In addition, recent investigations have raised the possibility that MDR1 and related transporters play a fundamental role in regulating apoptosis and immunology, and in fact, there are reports of MDR1-related susceptibility to inflammatory bowel disease, HIV infection and renal cell carcinoma. Herein, these issues are also summarized, and the current status of the knowledge in the area of pharmacogenomics of other transporters is briefly introduced.

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38 The first report on the polymorphisms of the MDR1 gene was presented in 1989.47) MDR1 was isolated from human normal adrenal glands, and the deduced amino acid sequencing indicated two amino acid substitutions of Gly185Val and Ala893Ser, the latter suggested reflecting a genetic polymorphism. Login to comment

[hide] Pharmacogenetics of MDR1 and its impact on the pha... Pharmacogenomics. 2003 Jul;4(4):397-410. Sakaeda T, Nakamura T, Okumura K
Pharmacogenetics of MDR1 and its impact on the pharmacokinetics and pharmacodynamics of drugs.
Pharmacogenomics. 2003 Jul;4(4):397-410., [PMID:12831320]

Abstract [show]
The multi-drug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. MDR1 was originally isolated from resistant tumor cells as part of the mechanism of multi-drug resistance, but over the last decade, it has been elucidated that human MDR1 is also expressed throughout the body to confer intrinsic resistance to the tissues by exporting unnecessary or toxic exogeneous substances or metabolites. A number of various types of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics and pharmacodynamics. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and indicated that a single nucleotide polymorphism (SNP), C3435T in exon 26, which caused no amino acid change, was associated with the duodenal expression of MDR1 and thereby the plasma concentrations of digoxin after oral administration. Interethnic differences in genotype frequencies of C3435T have been clarified, and, at present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical studies on the effects of C3435T on MDR1 expression and function in the tissues, and also on the pharmacokinetics and pharmacodynamics have been performed around the world; however, there are still discrepancies in the results, suggesting that the haplotype analysis of the gene should be included instead of SNP detection, and the design of clinical trials must be carefully planned to avoid misinterpretations. A polymorphism of C3435T is also reported to be a risk factor for a certain class of diseases such as the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this might also be explained by the effects on MDR1 expression and function. In this review, the latest reports are summarized for the future individualization of pharmacotherapy based on MDR1 genotyping.

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37 Two amino acid substitutions, Gly185Val and Ala893Ser, were detected in MDR1 isolated from normal human adrenal glands, and the latter was suggested to reflect a genetic polymorphism. Login to comment

[hide] Expression of the human multidrug resistance cDNA ... J Biol Chem. 1992 Mar 5;267(7):4854-8. Sarkadi B, Price EM, Boucher RC, Germann UA, Scarborough GA
Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase.
J Biol Chem. 1992 Mar 5;267(7):4854-8., 1992-03-05 [PMID:1347044]

Abstract [show]
Drug-resistant tumor cells actively extrude a variety of chemotherapeutic agents by the action of the multi-drug resistance (MDR1) gene product, the plasma membrane P-glycoprotein. In this report we show that the expression of the human MDR1 gene in cultured Sf9 insect cells via a baculovirus vector generates a high activity vanadate-sensitive membrane ATPase. This ATPase is markedly stimulated by drugs known to interact with the P-glycoprotein, such as vinblastine and verapamil, and the ability of the various drugs to stimulate the ATPase corresponds to their previously observed affinity for this transporter. The drug-stimulated ATPase is not present in uninfected or mock-infected Sf9 cells, and its appearance correlates with the appearance of the MDR1 gene product detected with a monoclonal anti-MDR protein antibody and by labeling with 8-azido-ATP. The drug-induced ATPase requires magnesium ions, does not utilize ADP or AMP as substrates, exhibits a half-maximal activation at about 0.5 mM MgATP, and its maximal activity (about 3-5 mumol/mg MDR protein/min) approaches that of the well characterized ion transport ATPases. These results provide the first direct demonstration of a high capacity drug-stimulated ATPase activity of the human multidrug resistance protein and offer a new and simple assay for the investigation of functional interactions of various drugs with this clinically important enzyme.

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31 The MDR cDNA contains a mutationfrom Gly to Val at amino acid position 185 (33). Login to comment

[hide] [MDR1 genotypes related to pharmacokinetics and MD... Yakugaku Zasshi. 2003 Sep;123(9):773-9. Nakamura T
[MDR1 genotypes related to pharmacokinetics and MDR1 expression].
Yakugaku Zasshi. 2003 Sep;123(9):773-9., [PMID:14513768]

Abstract [show]
The multidrug-resistant transporter encoded by the MDR1 gene belongs to the ATP-binding cassette superfamily of membrane transporters. It is involved not only in the acquisition of multidrug-resistance phenotypes in cancer cells but also in normal tissues such as the brain, kidneys, liver, and intestines. This transporter has the potential to export unnecessary or toxic exogenous substances or metabolites, and in the intestine it is thought to play a role in limiting the oral absorption of a number of structurally unrelated drugs. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and suggested that a single-nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene (C3435T) was associated with a lower level of intestinal MDR1 expression, and thereby with lower plasma concentrations of digoxin after oral administration. At present, over 20 SNPs have been found in the MDR1 gene. Clinical studies on the effects of C3435T on MDR1 expression and function in the tissues, and consequently on the pharmacokinetics, have been performed worldwide. In this review, the latest reports concerning the relationship of MDR1 genotypes with pharmacokinetics and MDR1 expression are summarized. Our experimental results demonstrate the importance of genetic polymorphisms at positions 3435 and 2677 in the MDR1 gene on pharmacokinetics and intestinal MDR1 expression. In the future, haplotype analysis of the MDR1 gene and subsequent classification of subjects are needed for individualized pharmacotherapy based on MDR1 genotyping.

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11 MDR1 遺伝子多型 MDR1 遺伝子多型に関する最初の報告は 1989 年 Kioka らによってなされ,ヒト正常副腎より単離された MDR1 において 2 種類のアミノ酸置換 (Gly185Val, Ala893Ser)を検出し,後者は遺伝的多型を反映することを示唆するものであった．2) この後,数ヵ所の SNP が報告されたが,3,4) 2000 年に 774774 Vol. 123 (2003) 入り,HoŠmeyer らは 188 人の白人を対象として MDR1 遺伝子多型の網羅的スクリーニングを行い, 15 ヵ所の遺伝的多型の存在を報告した．5) HoŠmeyer らの報告で注目を集めたのはエクソン 26 上 3435 位の遺伝子多型(C3435T)であり,C3435T 遺伝子型は,十二指腸における MDR1 タンパクの発現量や典型的 MDR1 基質であるジゴキシンの消化管吸収に影響を及ぼすといった内容であった．その後, C3435T 遺伝子型に関して世界中で大規模母集団に対する C3435T 遺伝子型発現頻度解析が行われ,その発現頻度には人種差が報告されている．5―8) また, Kim らは,健常なヨーロッパ系並びにアフリカ系アメリカ人を対象に MDR1 遺伝子の遺伝子多型解析を行った結果 , 3435 位が CC3435 ( あるいは TT3435 )である者のうち 95% (64%)で 2677 位が GG2677 (TT2677)であることを示唆しており,人種, 性別,年齢を問わず 3435 位と 2677 位の多型が関連していることを示唆した．9) 著者らは,健常な日本人 117 人を対象として遺伝子型発現頻度を検討したところ,3435 位は CC3435 35.0%, CT3435 53.0%, TT3435 12.0%の頻度分布であり,2677 位は GG2677 12.8%, GA2677 23.9%, GT2677 38.5%, AA2677 1.7%, AT2677 13.7%, TT2677 9.4%であった．10) ここで, CC3435 のうち 31.7%が GG2677, TT3435 のうち 71.4% が TT2677 であり,Kim らの報告を支持するものであった．また,2677 位に A を有する者の発現頻度が白人に比べて日本人において高いことを報告した．10) MDR1 遺伝子に関しては,現在までに 27 ヵ所 28 種類の遺伝子多型が報告されており,このうち 7 ヵ所はイントロン上に位置し,11 ヵ所はアミノ酸置換を伴う多型である．4,5,9,11―14) Kim らは,11 種類の遺伝子多型 C-4T, G-1, A61G, A548G, G1199A, C1236T, C1474T, C2650T, G2677T, T3421A 及び C3435T について,MDR11 から MDR18 とそのサブタイプを含む 15 種類の対立遺伝子を定義しており,今後,MDR1 遺伝子型と表現型の相関解析を行っていく上でこの定義の是非が問われてくると思われる．9) 3. Login to comment

[hide] Transition state analysis of the coupling of drug ... J Biol Chem. 2003 Dec 26;278(52):52629-40. Epub 2003 Oct 9. Al-Shawi MK, Polar MK, Omote H, Figler RA
Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein.
J Biol Chem. 2003 Dec 26;278(52):52629-40. Epub 2003 Oct 9., 2003-12-26 [PMID:14551217]

Abstract [show]
ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.

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359 Recently, we showed that the Pgp mutation G185V improves colchicine transport by enhancing its binding to the coupling transition state (62). Login to comment

[hide] P-glycoprotein: from genomics to mechanism. Oncogene. 2003 Oct 20;22(47):7468-85. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM
P-glycoprotein: from genomics to mechanism.
Oncogene. 2003 Oct 20;22(47):7468-85., 2003-10-20 [PMID:14576852]

Abstract [show]
Resistance to chemically different natural product anti-cancer drugs (multidrug resistance, or MDR) results from decreased drug accumulation, resulting from expression of one or more ATP-dependent efflux pumps. The first of these to be identified was P-glycoprotein (P-gp), the product of the human MDR1 gene, localized to chromosome 7q21. P-gp is a member of the large ATP-binding cassette (ABC) family of proteins. Although its crystallographic 3-D structure is yet to be determined, sequence analysis and comparison to other ABC family members suggest a structure consisting of two transmembrane (TM) domains, each with six TM segments, and two nucleotide-binding domains. In the epithelial cells of the gastrointestinal tract, liver, and kidney, and capillaries of the brain, testes, and ovaries, P-gp acts as a barrier to the uptake of xenobiotics, and promotes their excretion in the bile and urine. Polymorphisms in the MDR1 gene may affect the pharmacokinetics of many commonly used drugs, including anticancer drugs. Substrate recognition of many different drugs occurs within the TM domains in multiple-overlapping binding sites. We have proposed a model for how ATP energizes transfer of substrates from these binding sites on P-gp to the outside of the cell, which accounts for the apparent stoichiometry of two ATPs hydrolysed per molecule of drug transported. Understanding of the biology, genetics, and biochemistry of P-gp promises to improve the treatment of cancer and explain the pharmacokinetics of many commonly used drugs.

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86 It is important to note that the G185V mutation linked to this SNP in this report has been clearly shown to be responsible for an alteration in drug resistance conferred by P-gp, even when the P-gp carries only the G185V mutation and not the polymorphism. Login to comment

[hide] Improved energy coupling of human P-glycoprotein b... Biochemistry. 2004 Apr 6;43(13):3917-28. Omote H, Figler RA, Polar MK, Al-Shawi MK
Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.
Biochemistry. 2004 Apr 6;43(13):3917-28., 2004-04-06 [PMID:15049699]

Abstract [show]
A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.

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0 Improved Energy Coupling of Human P-glycoprotein by the Glycine 185 to Valine Mutation† Hiroshi Omote, Robert A. Figler, Mark K. Polar, and Marwan K. Al-Shawi* Department of Molecular Physiology and Biological Physics, UniVersity of Virginia Health System, P.O. Box 800736, CharlottesVille, Virginia 22908-0736 ReceiVed August 1, 2003; ReVised Manuscript ReceiVed January 28, 2004 ABSTRACT: A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. Login to comment
3 Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicineand etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. Login to comment
6 Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicineand etoposide-dependent activities. Login to comment
7 To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. Login to comment
9 G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Login to comment
32 1 Abbreviations: Cys(-), cysteine-less P-glycoprotein; DFP, diisopropyl fluorophosphate; DDM, n-dodecyl -D-maltopyranoside; E. coli lipid, ether/acetone-precipitated Escherichia coli lipid; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis( -aminoethyl ether)-N,N,N',N'-tetraacetic acid; G185V, glycine 185 to valine substitution; kcat, turnover number; Km ATP , apparent Michaelis constant for ATP activation; Km D, apparent Michaelis constant for drug activation; Ki, drug inhibition constant; LFER, linear free energy relationship; PC, phosphatidylcholine; Pgp, P-glycoprotein; PMSF, phenylmethanesulfonyl fluoride; PS, phosphatidylserine; SL-verapamil, spin-labeled verapamil; Vb, apparent Vmax for basal ATPase activity; Vd, apparent Vmax for drug-dependent ATPase activity. Login to comment
39 Interestingly, the selected cell lines had a P-glycoprotein mutated at position 185 (glycine 185 to valine, G185V), and this mutation is now believed to be responsible for high colchicine resistance. Login to comment
40 Stein et al. (8) did demonstrate that cells expressing G185V P-glycoprotein had a decreased rate of colchicine uptake when compared to cells with wild-type P-glycoprotein. Login to comment
41 Although details of the improved resistance afforded by G185V P-glycoprotein are still not clear, it is highly correlated with the colchicine transport mechanism. Login to comment
46 To construct a human P-glycoprotein expression plasmid which carries the glycine 185 to valine mutation, part of pSK1.MDR (9) was transferred to a wild-type expression plasmid YEpMDR1HIS (10). Login to comment
112 RESULTS Drug Dependence of G185V ATPase ActiVity. Login to comment
113 Human G185V mutant P-glycoprotein was overexpressed in yeast plasma membranes using our yeast expression system in the presence of 10% glycerol as a chemical chaperone (10). Login to comment
121 Ramachandra et al. (21) previously reported altered kinetics of plasma membrane ATPase activity of cells expressing the G185V mutant Pgp, including a higher basal ATPase activity. Login to comment
123 There were marked differences between wild-type and G185V enzymes. Login to comment
124 Basal activity of G185V Pgp was reduced to 28% of basal wild-type activity (Figure 2 legend) although both enzyme activities were strongly stimulated by colchicine. Login to comment
125 G185V Pgp appeared to be more tightly regulated than wild type (Figure 2A). Login to comment
131 The apparent Km D for etoposide was decreased 4.4-fold by the G185V mutation. Login to comment
132 The apparent specificity constant, kcat/Km D , at saturating colchicine was reduced slightly from 4.0 × 103 to 1.1 × 103 M-1 s-1 by the G185V mutation. Login to comment
134 In contrast, the apparent specificity constant for etoposide was increased from 2.2 × 103 to 1.7 × 105 M-1 s-1 by the G185V mutation. Login to comment
135 Although the apparent specificity constants change in opposite directions by the G185V mutation for colchicine and etoposide, it will be shown later that the same underlying mechanism of improved drug transport applies to both drugs. Login to comment
142 On this basis, it appears that G185V Pgp binds colchicine tightly in the coupling transition state. Login to comment
155 When G185V Pgp etoposideor colchicine-dependent ATPase activities were plotted on Figure 3, they were located on the line for drug- FIGURE 1: Variation of apparent Km ATP as a function of temperature. Login to comment
160 Symbols: shaded O, WT basal (no drug present); 4, WT and 130 µM valinomycin; 3, WT and 130 µM verapamil; O, WT and 2 mM colchicine; shaded thick O, G185V basal; thick O, G185V and 5 mM colchicine. Login to comment
162 This means that etoposide- and colchicine-dependent ATPase activities of the G185V mutant enzyme have a rate-limiting transition state similar to that of other drug-transport-related coupled activities. Login to comment
176 However, the G185V mutation shifted the etoposideand colchicine-dependent activities toward the lower left corner (Figure 4). Login to comment
177 This shows that the G185V mutant enzyme requires fewer bond rearrangements than the wild type to reach the coupling transition state when transporting these two drugs. Login to comment
178 The G185V mutation turns etoposide and colchicine into excellent transport substrates for this enzyme form. Login to comment
185 100% basal ATPase activity values (Vb) were 0.90, 0.25, 0.55, and 0.18 µmol (mg of P-glycoprotein)-1 min-1 for WT, G185V, Cys(-), and G185C/Cys(-) P-glycoproteins, respectively. Login to comment
186 Panel A: O, WT plus colchicine; b, G185V plus colchicine. Login to comment
187 Panel B: 0, WT plus etoposide; 9, G185V plus etoposide; ], Cys(-) plus colchicine; [, G185C/Cys(-) plus colchicine. Login to comment
188 Table 1: Apparent Kinetic Constants of ATPase Activity of WT, Cysteine-less, and G185 Mutant P-glycoproteinsa verapamil valinomycinb colchicine etoposideb enzyme Km D (µM) Vd (U/mg)d Vd/Vb c (fold) Ki (mM) Km D (µM) Vd (U/mg) Vd/Vb (fold) Km D (µM) Vd (U/mg) Vd/Vb (fold) Ki (mM) Km D (µM) Vd (U/mg) Vd/Vb (fold) WT 62 5.7 6.4 0.64 2.0 3.7 4.1 680 2.5 2.8 18 145 1.3 1.4 G185V 64 1.6 6.4 1.0 3.3 1.1 4.2 5800 3.6 15 20 33 1.7 6.8 Cys(-)e 2.1 0.96 1.6 0.87 0.60 1.3 2.3 410 1.4 2.8 30 G185C/Cys(-) 5.3 0.46 2.6 1.5 1.2 0.30 1.7 1900 0.71 3.9 18 a Conditions were as follows: Standard ATPase activities were measured at 37 °C, pH 7.4, and 10 mM Mg‚ATP. Login to comment
199 The intrinsic Gibb`s free energy Table 2: Intrinsic Drug-Transport Rates and Corrected Transition State Thermodynamic Parameters for Steady-State Activity of WT, Cysteine-less, and G185 Mutant P-glycoproteinsa enzyme lipid typeb transport drug transport ratec (s-1) basal rate (Vb) (s-1) stimulationd (Vd/Vb) (fold) ∆Hq (kJ mol-1) T∆Sq (kJ mol-1) ∆Gq (kJ mol-1) WT type 1 none (basal) 1.9 104.8 30.9 73.9 WT type 1 colchicine 2.2 1.2 121.8 48.3 73.5 WT type 1 etoposide 2.1 1.1 159.5 85.8 73.7 G185V type 1 none (basal) 0.77 67.2 -9.0 76.2 G185V type 1 colchicine 6.6 8.6 105.4 (-16.4)e 34.7 (-13.6) 70.7 (-2.8) G185V type 1 etoposide 4.3 5.6 93.1 (-66.4) 21.3 (-64.5) 71.8 (-1.9) Cys(-) type 1 none (basal) 1.6 127.3 53.0 74.3 Cys(-) type 1 colchicine 3.6 2.2 145.6 73.3 72.2 G185C/Cys(-) type 1 none (basal) 2.0 68.0 -5.7 73.3 G185C/Cys(-) type 1 colchicine 5.2 2.6 98.6 (-47.0) 27.3 (-46.0) 71.3 (-0.9) WT type 2 none (basal) 3.7 134.9 62.7 72.2 WT type 2 colchicine 4.3 1.2 136.2 64.4 71.8 G185V type 2 none (basal) 2.9 107.9 35.1 72.8 G185V type 2 colchicine 8.5 2.9 111.9 (-24.3) 41.8 (-22.6) 70.1 (-1.7) WT type 3 none (basal) 2.6 102.4 29.3 73.1 WT type 3 colchicine 5.6 2.1 121.5 50.4 71.1 G185V type 3 none (basal) 5.5 97.6 26.5 71.1 G185V type 3 colchicine 12.2 2.2 104.5 (-17.0) 35.4 (-15.0) 69.1 (-2.0) a Intrinsic values were calculated at 35 °C, pH 7.5, with saturating Mg‚ATP and saturating transport drug if present. Login to comment
210 Large symbols: shaded O, WT basal; O, WT plus colchicine; 0, WT plus etoposide; 4, WT plus valinomycin; 3, WT plus verapamil; shaded thick O, G185V basal activity; thick O, G185V plus colchicine; thick 0, G185V plus etoposide; shaded ], Cys(-) basal; ], Cys(-) plus colchicine; shaded ] (thick line), G185C/Cys(-) basal; ] (thick line), G185C/ Cys(-) plus colchicine. Login to comment
221 Symbols: b, WT with colchicine; 9, WT with etoposide; 2, WT with valinomycin; 1, WT with verapamil; shaded O, G185V with colchicine; shaded 0, G185V with etoposide; [, Cys(-) with colchicine; shaded ], G185C/Cys(-) with colchicine. Login to comment
224 These results confirm that the major effect of the G185V mutation is in the improved catalytic efficiency for etoposide or colchicine transport. Login to comment
227 To ensure that our results and interpretations were valid, we reconstituted wild-type and G185V P-glycoproteins in three types of lipid preparations (see Materials and Methods for details). Login to comment
228 The basal activities for wild-type P-glycoprotein, when assayed using the conditions of Table 1, were 0.9, 1.4, and 1.0 µmol (mg of P-glycoprotein)-1 min-1 for type 1, 2, and 3 lipids, respectively (1.5-fold range), whereas the basal activities for G185V P-glycoprotein were 0.25, 1.2, and 2.2 µmol (mg of P-glycoprotein)-1 min-1 for type 1, 2, and 3 lipids, respectively (9-fold range). Login to comment
229 Thus, it appears that the G185V mutation is more sensitive than WT Pgp to the type of lipid in the membrane and in lipid control of the flux through the uncoupled basal cycle. Login to comment
230 Nevertheless, when the intrinsic thermodynamic parameters were calculated for colchicine-dependent activity of G185V and compared to WT Pgp, there was always improved colchicine transport by the mutation in any lipid type (Table 2). Login to comment
242 In Table 2 it can be seen that the transport rates of etoposide and colchicine were increased by 2and 3-fold, respectively, in type 1 lipids for G185V Pgp compared to WT Pgp. Login to comment
243 Similarly, a 2-fold increase in the rate of colchicine transport by G185V Pgp in type 2 and type 3 lipids was observed (Table 2). Login to comment
250 Colchicine-dependent activities of G185C/Cys(-) and Cys(-) enzymes are shown in Figure 2B and are qualitatively similar to the pattern seen with G185V when compared to wild-type P-glycoprotein (Figure 2A). Login to comment
257 Overall, the situation was quite similar to the G185V mutation compared against wild-type Pgp (Table 1). Login to comment
258 Furthermore, G185C/Cys(-) when compared to the parental Cys(-) enzyme behaved in a fashion similar to G185V compared to wild-type Pgp on the LFER plot (Figure 3). Login to comment
278 The P-glycoprotein G185V mutant was first isolated from highly colchicine-resistant cell lines (6). Login to comment
279 Cell lines having G185V Pgp exhibit higher resistance to colchicine and etopside but a decreased resistance to vinblastine, Taxol, and actinomycin D, suggesting altered drug specificity (7, 32, 33). Login to comment
280 The high resistance to colchicine was thought to have originated from altered kinetics of the mutant enzyme since there was increased colchicine extrusion from cells without a concomitant change in plasma membrane G185V Pgp expression levels (7, 21, 33). Login to comment
281 Thus, G185V Pgp is of great interest for understanding the mechanism of drug specificity and transport. Login to comment
284 On the other hand, Roninson and co-workers reported reduced affinity of G185V to colchicine based on a P-glycoprotein conformation-specific antibody (33). Login to comment
292 First, G185V Pgp has a lowered apparent affinity to colchicine and decreased basal ATPase activity in type 1 lipids (Figure 2A, Table 1). Login to comment

[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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75 The colchicine-selected cells exhibited a Gly185Val substitution in P-glycoprotein, resulting in increased resistance to colchicine but no apparent effect on sensitivity to adriamycin and vinblastine. Login to comment

[hide] Pharmacogenomics of drug transporters: a new appro... Biol Pharm Bull. 2004 Jul;27(7):939-48. Ishikawa T, Onishi Y, Hirano H, Oosumi K, Nagakura M, Tarui S
Pharmacogenomics of drug transporters: a new approach to functional analysis of the genetic polymorphisms of ABCB1 (P-glycoprotein/MDR1).
Biol Pharm Bull. 2004 Jul;27(7):939-48., [PMID:15256718]

Abstract [show]
In the 21st century, emerging genomic technologies (i.e., bioinformatics, functional genomics, and pharmacogenomics) are shifting the paradigm of drug discovery research and improving the strategy of medical care for patients. In order to realize the personalized medicine, it is critically important to understand molecular mechanisms underlying inter-individual differences in the drug response, namely, pharmacological effect vs. side effect. Evidence is now accumulating to strongly suggest that drug transporters are one of the determinant factors governing the pharmacokinetic profile of drugs. Effort has been made to identify genetic variation in drug transporter genes. In particular, genetic variations of the human ABCB1 (P-glycoprotein/MDR1) gene have been most extensively studied. Hitherto more than fifty single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms in the ABCB1 gene have been reported. However, at the present time, information is still limited with respect to the actual effect of those genetic polymorphisms on the function of ABCB1. In this context, we have undertaken functional analyses of ABCB1 polymorphisms. To quantify the impact of genetic polymorphisms on the substrate specificity of ABCB1, we have developed a high-speed screening system and a new structure-activity relationship (SAR) analysis method. This review addresses functional aspects of the genetic polymorphism of ABCB1 and provides the standard method to evaluate the effect of polymorphisms on the function.

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64 A single amino acid substitution, Gly185Val, in the human ABCB1 protein was found to cause an altered pattern of drug resistance in cell lines transfected with the ABCB1 cDNA carrying this mutation.21) It is suggested that the amino acid at position 185 is involved in colchicines and verapamil but 940 Vol. 27, No. 7 Fig. 2. Login to comment
79 For this purpose, the cDNA of ABCB1 was cloned from the human liver cDNA library, and several variant forms (i.e., N21D, N44S, F103L, G185V, S400N, A893S, A893T, M986V) were prepared by site-directed mutagenesis (see Fig. 4A for primers). Login to comment
94 The variant forms (i.e., N21D, N44S, F103L, G185V, S400N, A893S, A893T, M986V) exhibited the verapamil-enhanced ATPase activity, as did the wild type of ABCB1. Login to comment
97 The variant G185V (acquired mutation) was found to have the highest Vmax value, which was followed by N21D (Table 1). Login to comment
118 Kinetic Parameters of the Wild Type and SNP Variants of ABCB1 Variant Km Vmax (mM) (nmol/min/mg protein) Wild type 2.190Ϯ0.150 13.14Ϯ1.95 N21D 0.502Ϯ0.126 45.26Ϯ11.33 N44S 0.580Ϯ0.148 31.03Ϯ4.65 F103L 1.100Ϯ0.078 36.34Ϯ8.33 G185V 0.831Ϯ0.102 56.76Ϯ6.76 S400N 0.327Ϯ0.025 13.74Ϯ2.08 A893S 0.441Ϯ0.042 17.24Ϯ6.72 A893T 0.904Ϯ0.244 10.77Ϯ1.35 M986V 0.419Ϯ0.062 22.69Ϯ6.84 The wild type and variants of ABCB1 were then expressed it in Sf9 cells using the pFASTBAC1 vector and recombinant baculoviruses. Login to comment
125 Verapamil-Enhanced ATPase Activity in the Plasma Membrane Fraction of Sf9 Cells Closed circles, ABCB1 (G185V)-expressing Sf9 cells; open circles, control Sf9 cells. Login to comment

[hide] Pharmacogenetics of drug transporters and its impa... Curr Top Med Chem. 2004;4(13):1385-98. Sakaeda T, Nakamura T, Okumura K
Pharmacogenetics of drug transporters and its impact on the pharmacotherapy.
Curr Top Med Chem. 2004;4(13):1385-98., [PMID:15379652]

Abstract [show]
Most drug responses are determined by the interplay of several gene products that influence pharmacokinetics and pharmacodynamics, i.e., drug metabolizing enzymes, drug transporters, and drug targets. With the sequencing of the human genome, it has been estimated that approximately 500-1200 genes code for drug transporters. Concerning the effects of genetic polymorphisms on pharmacotherapy, the best characterized drug transporter is the multidrug resistant transporter P-glycoprotein/MDR1, the gene product of MDR1. Little such information is available on other drug transporters. MDR1 is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily, and is expressed mainly in intestines, liver, kidneys and brain. A number of various types of structurally unrelated drugs are substrates for MDR1, and their intestinal absorption, hepatobiliary secretion, renal secretion and brain transport are regulated by MDR1. The first investigation on the effects of MDR1 genotypes on pharmacotherapy was reported in 2000: a silent single nucleotide polymorphism (SNP), C3435T in exon 26, was found to be associated with the duodenal expression of MDR1, and thereby the plasma concentration of digoxin after oral administration. At present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical investigations on the association of MDR1 genotypes with the expression and function of MDR1 in tissues, and with pharmacokinetics and pharmacodynamics have mainly focused on C3435T; however, there are still discrepancies in the results, suggesting that the haplotype of the gene should be analyzed instead of a SNP. C3435T is also reported to be a risk factor for a certain class of diseases including the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this also might be explained by the effects on MDR1 expression and function. In this review, the latest reports on the effects of genetic polymorphisms of MDR1 on pharmacotherapy are summarized, and the pharmacogenetics of other transporters is briefly introduced.

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84 Two amino acid substitutions, Gly185Val and Ala893Ser, were detected in MDR1 isolated from normal human adrenal glands, and the latter was suggested to reflect a genetic polymorphism. Login to comment

[hide] Functional evaluation of ABCB1 (P-glycoprotein) po... Drug Metab Pharmacokinet. 2004 Feb;19(1):1-14. Ishikawa T, Hirano H, Onishi Y, Sakurai A, Tarui S
Functional evaluation of ABCB1 (P-glycoprotein) polymorphisms: high-speed screening and structure-activity relationship analyses.
Drug Metab Pharmacokinet. 2004 Feb;19(1):1-14., [PMID:15499164]

Abstract [show]
Evidence is accumulating to strongly suggest that drug transporters are one of the determinant factors governing the pharmacokinetic profile of drugs. Effort has been made to identify genetic variation in drug transporter genes. In particular, genetic variations of the human ABCB1 (MDR1) gene have been most extensively studied. Hitherto more than fifty single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms in the ABCB1 gene have been reported. However, at the present time, information is still limited with respect to the actual effect of those genetic polymorphisms on the function of ABCB1. In this context, we have undertaken functional analyses of ABCB1 polymorphisms. To quantify the impact of genetic polymorphisms on the substrate specificity of ABCB1, we have developed a high-speed screening system and a new structure-activity relationship (SAR) analysis method. This review addresses functional aspects of the genetic polymorphism of ABCB1 and provides the standard method to evaluate the effect of polymorphisms on the function.

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59 A single amino acid substitution, Gly185Val, in the human ABCB1 protein was found to cause an altered pattern of drug resistance in cell lines transfected with the ABCB1 cDNA carrying this mutation.21) It is suggested that the amino acid at position 185 is involved in colchicines and verapamil but not in vinblastine bindingWtransport. Login to comment
78 For this purpose, the cDNA of ABCB1 was cloned from the human liver cDNA library, and several variant forms (i.e., N21D, N44S, F103L, G185V, S400N, A893S, A893T, M986V) were prepared by site-directed mutagenesis (see Fig. 2A for primers). Login to comment
86 The variant forms (i.e., N21D, N44S, F103L, G185V, S400N, A893S, A893T, M986V) exhibited the verapamil-enhanced ATPase activity, as did the wild type of ABCB1. Login to comment
89 The variant G185V (acquired mutation) was found to have the highest Vmax value, which was followed by N21D (Table 2). Login to comment
105 Kinetic parameters of the wild type and SNP variants of ABCB1 Variant Km Vmax (mM) (nmolWminWmg protein) Wild type 2.190±0.150 13.14±1.95 N21D 0.502±0.126 45.26±11.33 N44S 0.580±0.148 31.03±4.65 F103L 1.100±0.078 36.34±8.33 G185V 0.831±0.102 56.76±6.76 S400N 0.327±0.025 13.74±2.08 A893S 0.441±0.042 17.24±6.72 A893T 0.904±0.244 10.77±1.35 M986V 0.419±0.062 22.69±6.84 The wild type and variants of ABCB1 were then expressed it in Sf9 cells using the pFASTBAC1 vector and recombinant baculoviruses. Login to comment

[hide] Gene therapy with drug resistance genes. Cancer Gene Ther. 2006 Apr;13(4):335-45. Zaboikin M, Srinivasakumar N, Schuening F
Gene therapy with drug resistance genes.
Cancer Gene Ther. 2006 Apr;13(4):335-45., [PMID:16211086]

Abstract [show]
A major side effect of cancer chemotherapy is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease. A second use of drug-resistance genes is for coexpression with a therapeutic gene in HSCs to provide a selection advantage to gene-modified cells. In this review, we discuss several drug resistance genes, which are well suited for in vivo selection as well as other newer candidate genes with potential for use in this manner.

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71 In a recent study Modlich et al.62 demonstrated that leukemogenesis in mice transplanted with marrow mononuclear cells transduced with MDR1 containing retroviral vector correlated with increased number of vector integration sites in Table 1 Transgenes commonly used for in vivo selection of transduced cells Transgene Mutants useful for in vivo selection Mechanism of action Selection agents Advantages Disadvantages DHFR L22Y F31S Provides resistance to antifolates Methotrexate Trimetrexate K Provides high levels of chemoprotection K Relative lack of toxicity from antifolate drugs K Drugs used for selection are not genotoxica K Works on S-phase cells only MDR1 Wild type Membrane pump extrudes drugs Colchicine Actinomycin D Etoposide Taxol Vinblastine K Provides high levels of protection K Cryptic splice sites make transgene unstable in viral vectors K Expressed at high levels in hematopoietic cells K Possible myeloproliferative effect on transduced cells G185V K Drugs used for selection are genotoxica MGMT P140K P140A G156A Removes alkyl groups from the O6 position of guanine and thymidine BCNU TMZ ACNU Dacarbazine Procarbazine K Expressed at low levels in stem cells K Modest levels of chemoprotection K Drugs used for selection are genotoxica GST-pi Wild type Inactivates drugs by conjugating glutathione Adriamycin Cisplatin Cyclophosphamide Melphalan K Provides resistance to different spectrum of drugs K May be useful for improving the effectiveness of other in vivo selection markers (e.g. MRP1) K Provides rather low levels of protection K Drugs used for selection are genotoxica BCNU: 1,3-bis(2-chloroethyl)-1-nitrosourea; TMZ: Temozolomide; ACNU: Chloroethylnitrosourea. Login to comment

[hide] New horizon of MDR1 (P-glycoprotein) study. Drug Metab Rev. 2005;37(3):489-510. Mizutani T, Hattori A
New horizon of MDR1 (P-glycoprotein) study.
Drug Metab Rev. 2005;37(3):489-510., [PMID:16257832]

Abstract [show]
MDR1 (once P-glycoprotein, now referred to as ABCB1) plays a role as a blood-brain barrier, preventing drug absorption into the brain, and is known to confer multiple drug resistance in cancer chemotherapy. MDR1 is composed of two repeated fragments, and there are six transmembrane domains (TMD) on the N-terminal of each repeat and a nucleotide (ATP) binding domain (NBD) on the C-terminal. These two repeats are dependent but cooperate as one functional molecule, with one pocket for excreting drugs. The 12 TM domains form a funnel facing the outside of cells, and NBD is in cytosol as a dimer. One NBD is composed of the Walker A, Q-loop, ABC-signature and the Walker B for phosphate binding of nucleotide. This tertiary structure of MDR1 is suggested from the structure of the NBD of histidine permease (HisP), clarified by x-ray crystallography. On the model of HisP, the NBD positions described above make a functional domain, and the same NBD structure is found on many other ABC transporters. An experiment with MDR1 gene knockout mice showed the high plasma AUC of drugs in mdr null mice [mdr1a(-/-)] and a high level in the brain, indicating that MDR1 has an efflux function (prevention of absorption) in the intestinal lumen and acts as a barrier of drug uptake in the brain, as well as has the function of urinary and biliary excretion of drugs. The transcription of MDR1 is dependent on two sites; the promoter site (-105/-100)(-245/-141) and the enhancer site (-7864/-7817). Autoantibody from autoimmune hepatitis patients weakly reacted with the extracellular peptide (aa314-aa328 between TM5 and 6) of MDR1 on the outside of the cell membrane, and did not react with peptides in the NBD and in the membrane-spanning region in TM5. There is an ambiguity about the function of MDR1 as GlcCer translocase.

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121 Colchicine stimulated ATPase activity of the G185V mutant of MDR1, suggesting a tight regulation of ATPase activity by colchicine and the position of G185 was shown in structure model (Omote et al., 2004). Login to comment
167 MDR1 with mutation G185V-I186N altered the magnitude of drug-induced increases in UIC2 immunoreactivity (Ruth, 2001). Login to comment

[hide] Single nucleotide polymorphisms in human P-glycopr... Expert Opin Drug Deliv. 2006 Jan;3(1):23-35. Dey S
Single nucleotide polymorphisms in human P-glycoprotein: its impact on drug delivery and disposition.
Expert Opin Drug Deliv. 2006 Jan;3(1):23-35., [PMID:16370938]

Abstract [show]
Drug efflux pumps belong to a large family of ATP-binding cassette transporter proteins. These pumps bind their substrate and export it through the membrane using energy derived from ATP hydrolysis. P-glycoprotein, the main efflux pump in this family, is expressed not only in tumour cells but also in normal tissues with excretory function (liver, kidney and the intestine). It has a broad specificity of substrates and plays an important role in drug delivery and disposition. Recently, genetic screening of P-glycoprotein has yielded multiple single nucleotide polymorphisms, which seem to alter transporter function and expression. This review discusses the various polymorphisms of this gene and its impact on drug disposition and diseases.

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301 RAMACHANDRA M, AMBUDKAR SV, GOTTESMAN MM, PASTAN I, HRYCYNA CA: Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. Login to comment

[hide] MDR1 genotype-related pharmacokinetics: fact or fi... Drug Metab Pharmacokinet. 2005 Dec;20(6):391-414. Sakaeda T
MDR1 genotype-related pharmacokinetics: fact or fiction?
Drug Metab Pharmacokinet. 2005 Dec;20(6):391-414., [PMID:16415525]

Abstract [show]
Multidrug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily of membrane transporters. A number of various types of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics. The first investigation of the effects of MDR1 genotypes on pharmacotherapy was reported in 2000; a silent single nucleotide polymorphism (SNP), C3435T in exon 26, was found to be associated with the duodenal expression of MDR1, and thereby the plasma concentration of digoxin after oral administration. In the last 5 years, clinical studies have been conducted around the world on the association of MDR1 genotype with MDR1 expression and function in tissues, and with the pharmacokinetics and pharmacodynamics of drugs; however, there are still discrepancies in the results on C3435T. In 1995, a novel concept to predict in vivo oral pharmacokinetic performance from data on in vivo permeability and in vitro solubility has been proposed, and this Biopharmaceutical Classification System strongly suggested that the effects of intestinal MDR1 on the intestinal absorption of substrates is minimal in the case of commercially available oral drugs, and therefore MDR1 genotypes are little associated with the pharmacokinetics after oral administration. This review summarizes the latest reports for the future individualization of pharmacotherapy based on MDR1 genotyping, and attempts to explain discrepancies.

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27 In 1989, the ˆrst report on the polymorphisms of the MDR1 gene was presented by Kioka et al.30) Two amino acid substitutions, Gly185Val and Ala893Ser, were detected in MDR1 isolated from normal human adrenal glands, and the latter was suggested to re‰ect a genetic polymorphism. Login to comment

[hide] Comparison of species differences of P-glycoprotei... Mol Pharm. 2006 Jan-Feb;3(1):78-86. Xia CQ, Xiao G, Liu N, Pimprale S, Fox L, Patten CJ, Crespi CL, Miwa G, Gan LS
Comparison of species differences of P-glycoproteins in beagle dog, rhesus monkey, and human using Atpase activity assays.
Mol Pharm. 2006 Jan-Feb;3(1):78-86., [PMID:16686372]

Abstract [show]
P-glycoprotein (P-gp) is a transmembrane efflux transporter which possesses many important functions in drug absorption, disposition, metabolism, and toxicity. The ultimate goal of investigating drug interactions between P-gp and drug molecules in early drug discovery is to understand the contribution of P-gp to the pharmacokinetic and pharmacodynamic properties of drug candidates and to project drug-drug interaction (DDI) potentials in humans. Understanding species differences in P-gp activities further helps the prediction of P-gp-mediated drug disposition and DDI in humans from preclinical pharmacokinetics data. The objective of the present study is to investigate the species difference in P-gp activities, via P-gp ATPase assays, using rhesus monkey Mdr1, beagle dog Mdr1, and human MDR1 expressed insect cell membranes. Twenty-one compounds with diverse chemical structures and different P-gp binding sites were chosen for the ATPase assays. P-gp ATPase binding affinities (alphaKa) and fold increases in P-gp ATPase activities (beta) of P-gp substrates were determined. Consistent with the gene and amino acid similarity, the binding affinities of test compounds to rhesus monkey P-gp were much closer to those of human P-gp than beagle dog P-gp. This is the first study which investigates the ligand affinities of P-gp from three different species. The result of this study provides an example of how to use membrane P-gp ATPase assays to evaluate interspecies P-gp differences.

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88 (17) Rao, U. S. Mutation of glycine 185 to valine alters the ATPase function of the human P-glycoprotein expressed in Sf9 cells. J. Biol. Chem. 1995, 270, 6686-6690. nology, Liaoning, China) or transferred electrophoretically to PVDF membrane. Login to comment

[hide] The remarkable transport mechanism of P-glycoprote... J Bioenerg Biomembr. 2005 Dec;37(6):489-96. Al-Shawi MK, Omote H
The remarkable transport mechanism of P-glycoprotein: a multidrug transporter.
J Bioenerg Biomembr. 2005 Dec;37(6):489-96., [PMID:16691488]

Abstract [show]
Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that utilizes the energy of ATP hydrolysis to pump toxic xenobiotics out of cells. P-glycoprotein employs a most unusual molecular mechanism to perform this drug transport function. Here we review our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling, and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised to place our results within a proper structural context.

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137 The mutation G185V increases the strength of colchicine and etoposide interaction with the transition state and improves the transport of these drugs by reducing the level of failed transport (Omote et al., 2004). Login to comment

[hide] Function-altering SNPs in the human multidrug tran... PLoS Genet. 2007 Mar 9;3(3):e39. Jeong H, Herskowitz I, Kroetz DL, Rine J
Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay.
PLoS Genet. 2007 Mar 9;3(3):e39., 2007-03-09 [PMID:17352537]

Abstract [show]
The human ABCB1 (MDR1)-encoded multidrug transporter P-glycoprotein (P-gp) plays a major role in disposition and efficacy of a broad range of drugs including anticancer agents. ABCB1 polymorphisms could therefore determine interindividual variability in resistance to these drugs. To test this hypothesis we developed a Saccharomyces-based assay for evaluating the functional significance of ABCB1 polymorphisms. The P-gp reference and nine variants carrying amino-acid-altering single nucleotide polymorphisms (SNPs) were tested on medium containing daunorubicin, doxorubicin, valinomycin, or actinomycin D, revealing SNPs that increased (M89T, L662R, R669C, and S1141T) or decreased (W1108R) drug resistance. The R669C allele's highly elevated resistance was compromised when in combination with W1108R. Protein level or subcellular location of each variant did not account for the observed phenotypes. The relative resistance profile of the variants differed with drug substrates. This study established a robust new methodology for identification of function-altering polymorphisms in human multidrug transporter genes, identified polymorphisms affecting P-gp function, and provided a step toward genotype-determined dosing of chemotherapeutics.

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68 The cloned cDNA carried the G185V SNP of ABCB1, and therefore site-directed mutagenesis was used to restore it to the most common allele, referred to as the ABCB1 reference allele in the Pharmacogenetics of Membrane Transporters dataset (pJR2703) (http://pharmacogenetics.ucsf.edu or http://www. Login to comment

[hide] Mechanism of multidrug recognition by MDR1/ABCB1. Cancer Sci. 2007 Sep;98(9):1303-10. Epub 2007 Jun 30. Kimura Y, Morita SY, Matsuo M, Ueda K
Mechanism of multidrug recognition by MDR1/ABCB1.
Cancer Sci. 2007 Sep;98(9):1303-10. Epub 2007 Jun 30., [PMID:17608770]

Abstract [show]
MDR1/ABCB1, a member of the ABC group of proteins, is clinically important because it is not only involved in multidrug resistance in cancer but also affects the pharmacokinetic properties of various drugs. The most puzzling feature of MDR1 is that it recognizes and transports such a wide variety of substrates. In the present review, the function of MDR1 is compared with that of other ABC proteins, particularly MDR2/ABCB4, to understand the mechanism of drug recognition and transport by MDR1. MDR2, the amino acid sequence of which has 86% similarity to that of MDR1, excretes phosphatidylcholine and cholesterol in the presence of bile salts. ABCA1 transfers phospholipids, preferentially phosphatidylcholine, and cholesterol to lipid-free apoA-I to generate pre-beta-HDL, and ABCG1 excretes phospholipids, preferentially sphingomyelin, and cholesterol. Cholesterol also binds directly to MDR1 and modulates substrate recognition by MDR1. Cholesterol may fill the empty space of the drug-binding site and aid the recognition of small drugs, and facilitates the ability of MDR1 to recognize compounds with various structures and molecular weights. Eukaryote ABC proteins may retain similar substrate binding pockets and move bound substrates in an ATP-dependent manner. The prototype of eukaryote ABC proteins might be those involved in membrane lipid transport.

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26 (2,15,16) A few years later, human MDR1 was isolated from the adrenal and it was found that cDNA, isolated from KB-C2.5, was associated with a Gly-to-Val substitution at position 185, in the predicted cytoplasmic loop between TM2 and TM3. Login to comment

[hide] ABC proteins protect the human body and maintain o... Biosci Biotechnol Biochem. 2011;75(3):401-9. Epub 2011 Mar 7. Ueda K
ABC proteins protect the human body and maintain optimal health.
Biosci Biotechnol Biochem. 2011;75(3):401-9. Epub 2011 Mar 7., [PMID:21389634]

Abstract [show]
Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

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15 Human MDR1 cDNA, now also called ABCB1 by the HUGO (Human Gene Organization) Nomenclature Committee, was isolated from a multidrug-resistant KB carcinoma cell line, KB-C2.5, selected for its resistance to colchicines, in 1986,1,2) and was found to code for P-glycoprotein,3) a surface glycoprotein reported to be overexpressed in drug-resistant Chinese hamster ovary cell mutants.4) Overexpression of human and mouse MDR1 conferred resistance to many drugs, including Vinca alkaloids, anthracyclines, epipodophyllotoxins, and taxol.5-7) A few years later, Kioka isolated human MDR1 cDNA from the adrenal, and found that cDNA isolated from KB-C2.51,2) was associated with a Gly-to-Val substitution at position 185 in the predicted cytoplasmic loop between TM2 and TM3.8) This mutation increased resistance to colchicine and decreased resistance to vinblastine.8,9) MDR1 is a 1,280 amino-acid protein with two symmetrical halves connected by a short linker region.1) Each half consists of six transmembrane -helices (TM) followed by a nucleotide binding domain (NBD),10) in which ATP is hydrolyzed to energize transport (Fig. 1). Login to comment

[hide] An update on ABCB1 pharmacogenetics: insights from... Pharmacogenomics J. 2011 Oct;11(5):315-25. doi: 10.1038/tpj.2011.16. Epub 2011 May 31. Wolf SJ, Bachtiar M, Wang J, Sim TS, Chong SS, Lee CG
An update on ABCB1 pharmacogenetics: insights from a 3D model into the location and evolutionary conservation of residues corresponding to SNPs associated with drug pharmacokinetics.
Pharmacogenomics J. 2011 Oct;11(5):315-25. doi: 10.1038/tpj.2011.16. Epub 2011 May 31., [PMID:21625253]

Abstract [show]
The human ABCB1 protein, (P-glycoprotein or MDR1) is a membrane-bound glycoprotein that harnesses the energy of ATP hydrolysis to drive the unidirectional transport of substrates from the cytoplasm to the extracellular space. As a large range of therapeutic agents are known substrates of ABCB1 protein, its role in the onset of multidrug resistance has been the focus of much research. This role has been of particular interest in the field of pharmacogenomics where genetic variation within the ABCB1 gene, particularly in the form of single nucleotide polymorphisms (SNPs), is believed to contribute to inter-individual variation in ABCB1 function and drug response. In this review we provide an update on the influence of coding region SNPs within the ABCB1 gene on drug pharmacokinetics. By utilizing the crystal structure of the mouse ABCB1 homolog (Abcb1a), which is 87% homologous to the human sequence, we accompany this discussion with a graphical representation of residue location for amino acids corresponding to human ABCB1 coding region SNPs. Also, an assessment of residue conservation, which is calculated following multiple sequence alignment of 11 confirmed sequences of ABCB1 homologs, is presented and discussed. Superimposing a 'heat map' of residue homology to the Abcb1a crystal structure has permitted additional insights into both the conservation of individual residues and the conservation of their immediate surroundings. Such graphical representation of residue location and conservation supplements this update of ABCB1 pharmacogenetics to help clarify the often confounding reports on the influence of ABCB1 polymorphisms on drug pharmacokinetics and response.

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97 The classic E8/554G4T (G185V) polymorphism that influences drug specificity resides in close 3D proximity to two other non-synonymous polymorphisms in a region that is less evolutionary conserved Although research into ABCB1 polymorphisms appears to focus on E13/1236C4T, E22/2677G4T/A and E27/ 3435C4T, it is important to note that a number of additional non-synonymous SNPs have been associated with drug pharmacokinetics, drug response, protein expression and disease progression. Login to comment
99 One of these SNPs, E8/554G4T (#ns7 in Figure 2b) confers the classic amino-acid substitution G185V that was reported more than 20 years ago to confer changes in drug specificity of vinblastine and colchicine.39 This glycine residue was suggested to have a pivotal role in transmitting conformational changes between the catalytic sites and the drug binding sites.40 Combining molecular dynamics simulations with atomic detail homology modeling, it was further proposed that this improved efflux involves a reduction in non-polar van der Waals forces, which would otherwise secure colchicine to its binding site near or at residue 185. Login to comment
106 It would be interesting to evaluate if these two closeby SNPs also have similar roles in influencing conformational changes between the catalytic sites and the drug binding sites or influence the role of G185V. Login to comment
125 This Figure 4 Location and conservation of (a) E8/554G4T (G185V), (b) E12/1199G4A (S400N), (c) E26/3151C4G (P1051A), (d) E27/3322T4C (W1108R), (e) E27/3421T4A (S1141T), (f) E29/3751G4A (V1251I). Login to comment

[hide] Functional complementation of the ste6 gene of Sac... Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8921-5. Volkman SK, Cowman AF, Wirth DF
Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum.
Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8921-5., 1995-09-12 [PMID:7568044]

Abstract [show]
The pfmdr1 gene has been associated with a drug-resistant phenotype in Plasmodium falciparum, and overexpression of pfmdr1 has been associated with mefloquine- and halofantrine-resistant parasites, but little is known about the functional role of pfmdr1 in this process. Here, we demonstrate that the pfmdr1 gene expressed in a heterologous yeast system functions as a transport molecule and complements a mutation in ste6, a gene which encodes a mating pheromone a-factor export molecule. In addition, the pfmdr1 gene containing two mutations which are associated with naturally occurring chloroquine resistance abolishes this mating phenotype, suggesting that these genetic polymorphisms alter this transport function. Our results support the functional role of pfmdr1 as a transport molecule in the mediation of drug resistance and provide an assay system to address the nature of this transport function.

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130 One possible explanation for these results (19) is the presence of a mutation in the MDR1 gene which resulted in a change from glycine to valine at amino acid position 185. Login to comment

[hide] Expression of retroviral vectors containing the hu... Blood. 1995 Jul 15;86(2):491-501. Sorrentino BP, McDonagh KT, Woods D, Orlic D
Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice.
Blood. 1995 Jul 15;86(2):491-501., 1995-07-15 [PMID:7605985]

Abstract [show]
Transfer of the human multidrug resistance 1 (MDR1) gene to hematopoietic stem cells offers an approach to overcome the myelosuppression caused by a number of antineoplastic drugs. This study was designed to determine the effect of MDR1 gene transfer on overall P-glycoprotein (P-gp) expression in murine hematopoietic cells. Mice were transplanted with bone marrow cells infected with either of two different MDR1 retroviral vectors. A reverse-transcriptase polymerase chain reaction-based assay was used to quantify expression levels of both endogenous and vector-derived P-gp encoding transcripts in hematopoietic cells of transplanted mice. Expression of both a truncated and full-length MDR1 mRNA species was noted in bone marrow and spleen colony cells. The truncated message resulted from cryptic mRNA splice sites within the MDR1 cDNA and was detected with both vectors. Full-length message levels exceeded those from the endogenous genes in all but one case and roughly approximated that seen in the modestly drug-resistant cell line SW620. We conclude that transfer of MDR1 retroviral vectors resulted in a significant increase in P-gp expression in most cases; however, aberrant splicing of MDR1 transcripts can result in reduced expression of vector-derived P-gp.

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38 Construction of the Harvey murine sarcoma-based MDRl vector and isolation of a ecotropic producer line has been previously described.`*The MDRl cDNA used in the Harvey-based vector contains a point mutation resulting in a substitution of valine for glycine at codon 185. Login to comment

[hide] Rapid purification of human P-glycoprotein mutants... J Biol Chem. 1995 Sep 15;270(37):21449-52. Loo TW, Clarke DM
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.
J Biol Chem. 1995 Sep 15;270(37):21449-52., 1995-09-15 [PMID:7665554]

Abstract [show]
P-glycoprotein containing 10 tandem histidine residues at the COOH end of the molecule was transiently expressed in HEK 293 cells and purified by nickel-chelate chromatography. The purified protein had an apparent mass of 170 kDa, and its verapamil-stimulated ATPase activity in the presence of phospholipid was 1.2 mumol/min/mg of P-glycoprotein. We then characterized P-glycoprotein mutants that exhibited altered drug-resistant phenotypes and analyzed the contribution of the two nucleotide binding folds to drug-stimulated ATPase activity. Mutation of residues in either nucleotide binding fold abolished drug-stimulated ATPase activity. The pattern of drug-stimulated ATPase activities of mutants, which conferred increased relative resistance to colchicine (G141V, G185V, G830V) or decreased relative resistance to all drugs (F978A), correlated with their drug-resistant phenotypes. By contrast, the ATPase activity of mutant F335A was significantly higher than that of wild-type enzyme when assayed in the presence of verapamil (3.4-fold), colchicine (9.1-fold), or vinblastine (3.7-fold), even though it conferred little resistance to vinblastine in transfected cells. These results suggest that both nucleotide-binding domains must be intact to couple drug binding to ATPase activity and that the drug-stimulated ATPase activity profile of a mutant does not always correlate with its drug-resistant phenotype.

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66 In this study, we also included for comparison mutant G185V, which was recently shown by Rao (1995) to have increased verapamiland colchicine-stimulated ATPase activities (2and 3.3-fold, respectively). Login to comment
69 The maximal verapamil-stimulated ATPase activities of mutants G141V, G185V, and G830V were all slightly increased (1.4-1.7-fold) relative to that of wild-type enzyme (Fig. 2). Login to comment
81 Wild-type (E) and mutants G141V (å), G185V (Ⅺ), G830V (q), F335A (f), and F978A (Ç) P-glycoproteins-(His)10 were purified using Ni-NTA spin columns and reconstituted with sheep brain phosphatidylethanolamine. Login to comment
111 For mutants G141V, G185V, G830V, and F978A, the pattern of drug-stimulated ATPase correlated with their relative drug-resistant profiles in transfected cells. Login to comment
131 Acknowledgments-We thank Dr. Randal Kaufman (Boston) for pMT21 and Dr. Michael M. Gottesman (NIH) for the cDNA coding for P-glycoprotein mutant G185V. Login to comment

[hide] The biology of the P-glycoproteins. J Membr Biol. 1995 Jan;143(2):89-102. Leveille-Webster CR, Arias IM
The biology of the P-glycoproteins.
J Membr Biol. 1995 Jan;143(2):89-102., [PMID:7731035]

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204 A naturally occurring mutation of glycine to valine at position 185 in transmembrane domain 3 of human MDR1 changed the specificity of the transporter so that colchicine and etoposide resistance were enhanced while resistance to vinblastine, vincristine and actinomycin D was decreased [48, 104, 150]. Login to comment

[hide] Differential effects of P-glycoprotein inhibitors ... Cancer Res. 1995 Mar 1;55(5):1086-91. Cardarelli CO, Aksentijevich I, Pastan I, Gottesman MM
Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters.
Cancer Res. 1995 Mar 1;55(5):1086-91., 1995-03-01 [PMID:7866993]

Abstract [show]
Multidrug resistance (MDR) may be associated with the expression of the MDR1 gene which encodes the 170-kDa cell surface P-glycoprotein (PGP) acting as an energy-dependent multidrug efflux pump. This pump can be inhibited by a variety of drugs including cyclosporin A, quinidine, and verapamil. Substrate specificity of the MDR1 gene product can be altered by a point mutation at amino acid residue 185 in which valine is substituted for glycine, but the effect of this mutation on inhibition of PGP is unknown. Multidrug-resistant NIH3T3 cells transfected with the MDR1 retroviral vector pHaMDR-1/A (G185) or pHaMDR1/A (V185) expressing comparable levels of PGP were compared for patterns of drug resistance and inhibition of drug resistance by MDR reversing agents. The NIH-MDR-G185 transfectants were somewhat preferentially resistant to daunorubicin, taxol, and vinblastine. The mutant (V185) conferred increased resistance to colchicine. This MDR phenotype in both NIH-MDR-G185- and NIH-MDR-V185-transfected NIH3T3 cells was overcome by the addition of cyclosporin A, quinidine, or verapamil. Verapamil was the most potent of the three agents affecting wild-type PGP. However, specific inhibitors showed different potency with wild-type or mutant transporters, depending on the cytotoxic drug whose resistance was being reversed. For example, cyclosporin A at a concentration of 1 microgram/ml, was a powerful reverser of taxol and colchicine resistance for the mutant drug transporter, but was much less effective for the wild-type transporter. In contrast, verapamil reversed resistance to vinblastine more efficiently for the wild-type transporter than for the mutant transporter. These results suggest that the sensitivity of a multidrug transporter to a reversing agent will depend on the reversing agent, the cytotoxic drug, and the presence or absence of mutations which alter substrate specificity.

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29 The well-characterized mutation of a glycine to valine at residue 185 in a colchicine-selected human KB cell line changes the pattern of resistance by improving the ability of the mutant to pump colchicine and etoposide while decreasing its ability to pump vinblastine and actinomycin D (14, 31). Login to comment
131 The substitution of valine for glycine at position 185 appears to increase interactions of cyclosporin A and quinidine with the trans porter so as to more effectively block transport of colchicine, dauno rubicin, and taxol. Login to comment

[hide] Kinetic evidence suggesting that the multidrug tra... Mol Pharmacol. 1994 Apr;45(4):763-72. Stein WD, Cardarelli C, Pastan I, Gottesman MM
Kinetic evidence suggesting that the multidrug transporter differentially handles influx and efflux of its substrates.
Mol Pharmacol. 1994 Apr;45(4):763-72., [PMID:7910372]

Abstract [show]
A kinetic approach was used to analyze the mechanism by which a substitution of valine for glycine at position 185 in the multidrug transporter alters its substrate specificity so that colchicine and etoposide transport is increased, daunorubicin transport is unchanged, and vinblastine transport is decreased. Time courses for uptake and efflux of colchicine, vinblastine, etoposide, and daunorubicin for NIH/3T3 mouse cells transfected with wild-type (MDR1-G185) and mutant (MDR1-V185) strains of the human mdr1 gene were determined at room temperature in the presence and absence of an energy supply. The initial rate of vinblastine uptake was reduced approximately 5-fold by glucose feeding of ATP-depleted wild-type (MDR1-G185) cells but was only halved in MDR1-V185 transfectants. In contrast, glucose feeding decreased the initial rate of colchicine uptake approximately 4-fold in the MDR1-V185 (mutant) transfectant but not in the MDR1-G185 (wild-type) transfectant. Efflux of colchicine was accelerated > 5-fold in both the MDR1-V185 (mutant) and MDR1-G185 (wild-type) transfectants when glucose was given to raise ATP levels. The effects on initial rates of colchicine uptake accounted semiquantitatively for the increased colchicine resistance of MDR1-V185 (mutant) transfectants. Similar effects were found for etoposide in the MDR-V185 transfectants. Quinidine in the external medium greatly inhibited drug entry rates but had little effect on efflux, whereas verapamil inhibited both uptake and efflux. A possible interpretation of these data is that the multidrug transporter extracts drugs from the external and internal halves of the membrane bilayer by different paths, which are distinguishable by mutation and inhibitors.

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166 GLY 185 VAL 185 1.4 . Login to comment
223 7. tution of valine for glycine at position 185 along the polypeptide chain of P-glycoprotein might be interpreted as having a positive effect on the ability of the mutant to extract colchicine but a negative effect on its ability to extract vinblastine from the outer half of the membrane bilayer. Login to comment
227 The finding that there is little effect of this mutation on the ability of the pump to handle daunorubicin is consistent with our conclusion that it is the process by which drugs enter the cell that is solely affected by the Gly-185 to Val-185 mutation. Login to comment

[hide] Gene transfer of drug resistance genes. Implicatio... Ann N Y Acad Sci. 1994 May 31;716:126-38; discussion 138-43. Gottesman MM, Germann UA, Aksentijevich I, Sugimoto Y, Cardarelli CO, Pastan I
Gene transfer of drug resistance genes. Implications for cancer therapy.
Ann N Y Acad Sci. 1994 May 31;716:126-38; discussion 138-43., 1994-05-31 [PMID:7912913]

Abstract [show]
Two general approaches to the gene therapy of cancer have been proposed: (1) strategies that use exogenous genes to modify cancer cells so that they are less malignant or more susceptible to host defenses or to killing by exogenous agents; and (2) approaches that modify host cells so that they are more effective in eliminating cancer cells or more resistant to agents that are used to treat cancer. In both cases, the development of vectors that encode in vivo selectable phenotypes, such as drug resistance, would be extremely valuable because of the inherent inefficiency of gene transfer and the potential of such vectors to protect normal tissues against toxic agents. To allow the selection of cells in vivo that have been transduced with vectors for gene therapy, we have utilized the human multidrug resistance (MDR1) gene. The product of this gene is a 170,000-dalton glycoprotein known as P-glycoprotein, which acts as an energy-dependent efflux pump for a great many cytotoxic anticancer drugs, including doxorubicin, daunorubicin, etoposide, teniposide, actinomycin D, and taxol. Vectors encoding an MDR1 cDNA are able to transduce many cell types, including bone marrow cells, with high efficiency to allow selection of drug resistance in vitro and in vivo in mouse models. Thus, it should be possible to protect the bone marrow of patients undergoing intensive chemotherapy by transduction of their bone marrow with MDR1 vectors. Furthermore, the ability to select for the presence of the MDR1 cDNA in vivo means that it can be used to introduce otherwise nonselectable genes into the bone marrow for therapy of cancer and other diseases.

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65 Summaryofthe PhenotypeoftheVarionsMutations Shown in FIGURE 3 with Respect to Several MDR Drugs Mutation Phenotype Reference Gly 185Val Col t, VP-16t, Vbl J, Act-D J Choi etd.,198935 Gly 185 Val, Asn 183 Ser Col t, Vbl -, Act-D - Currier et d.,19924'3 Pro 223 Ala Col J, Vbl +,Act-D .1 Loo and Clarke, 19934' Gly 338 Ala, Ala 339 Pro Col J, Vbl J, Act-D + Devine et al., 199242 Pro 866 Ala Col J, Vbl 4, Act-D J Loo and Clarke, 199341 Ser 941 Phe Col J, Vbl +,Dox J Gros et d.,199143 FIGURE4. Login to comment

[hide] A strong association between mefloquine and halofa... Am J Trop Med Hyg. 1994 Nov;51(5):648-58. Peel SA, Bright P, Yount B, Handy J, Baric RS
A strong association between mefloquine and halofantrine resistance and amplification, overexpression, and mutation in the P-glycoprotein gene homolog (pfmdr) of Plasmodium falciparum in vitro.
Am J Trop Med Hyg. 1994 Nov;51(5):648-58., [PMID:7985758]

Abstract [show]
Stepwise selection for increased mefloquine resistance in a line of Plasmodium falciparum in vitro resulted in increased resistance to halofantrine and quinine, increased sensitivity to chloroquine, and amplification and overexpression of the P-glycoprotein gene homolog (pfmdr1). A point mutation (tyrosine to phenylalanine) noted at amino acid 86 in pfmdr1 in the mefloquine-resistant line W2mef was amplified in more resistant lines derived from it by in vitro selection pressure with mefloquine. Conversely, lines selected for increased chloroquine resistance exhibited a revertant phenotype that was sensitive to mefloquine and halofantrine. These lines also demonstrated increased sensitivity to quinine, loss of amplification of pfmdr1, loss of the mefloquine/halofantrine phenylalanine-86 mutation, and selection for a tyrosine-86 mutation previously associated with chloroquine resistance. These findings provide strong evidence for pfmdr1 mediating cross-resistance to halofantrine and mefloquine in P. falciparum in vitro.

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130 Cross-resistance in human KB carci noma cells was found to result from a cluster of point mutations in the MDRI gene that resulted in a Gly-185 to Val-l85 substitution in the P glycoprotein.38 The mutations were identified in a cell line where the wild-type MDR1 was am plified, yet selection for the next level of resis tance resulted in all amplified copies of the gene carrying the same mutations.39 While the IC@ of chloroquine has decreased in Mef 2.4, the par asite remains resistant to chloroquine, suggest ing that either 86-Tyr is not critical for resis tance, or that the 86-Phe change remains a competent allele for chloroquine resistance. Login to comment

[hide] Altered drug-stimulated ATPase activity in mutants... J Biol Chem. 1996 Jan 26;271(4):1877-83. Muller M, Bakos E, Welker E, Varadi A, Germann UA, Gottesman MM, Morse BS, Roninson IB, Sarkadi B
Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.
J Biol Chem. 1996 Jan 26;271(4):1877-83., 1996-01-26 [PMID:8567633]

Abstract [show]
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.

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5 In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. Login to 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
119 As shown above, in the case of the Gly185 to Val mutant we could not see a significant difference in the maximum level of verapamil-stimulated ATPase activity or in its MgATP concentration dependence. Login to comment
192 A single point mutation in human Pgp, a spontanous exchange of Gly to Val at position 185 (Choi et al., 1988), was reported to result in an increased relative resistance to colchicine and etoposide, while unchanged or slightly reduced resistance toward vinblastine and vincristine (Choi et al., 1988; Currier et al., 1992; Cardarelli et al., 1995). Login to comment
193 In this study we have reproduced the Gly185 to Val point mutation in the baculovirus-Sf9 expression system for MDR1. Login to comment
194 Our experiments showed no significant alteration in the maximum activity of the MDR1-ATPase or in its activation kinetics by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. Login to comment
196 Moreover, the data indicating that the Gly185 to Val exchange, while increasing colchicine extrusion and colchicine stimulation of the MDR1-ATPase activity, reduces the binding of this drug to the MDR1 protein (Safa et al., 1990), may suggest that in the molecular mechanism of drug extrusion, ATP splitting is required for the dissociation of the drug from the transporter protein. Login to comment
198 While the present paper was under revision, a publication by U. S. Rao (1995) reported the expression and partial characterization of the Gly185 to Val MDR1 mutants in Sf9 cells. Login to comment

[hide] P-glycoprotein, multidrug resistance and protein k... Stem Cells. 1996 Jan;14(1):47-55. Fine RL, Chambers TC, Sachs CW
P-glycoprotein, multidrug resistance and protein kinase C.
Stem Cells. 1996 Jan;14(1):47-55., [PMID:8820951]

Abstract [show]
The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-alpha isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.

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142 The human MDR1 P-glycoprotein, with a mutation of amino acid glycine 185 to valine, was also expressed in Sf9 cells and used as membrane vesicles. Login to comment

[hide] Genetic analysis of the multidrug transporter. Annu Rev Genet. 1995;29:607-49. Gottesman MM, Hrycyna CA, Schoenlein PV, Germann UA, Pastan I
Genetic analysis of the multidrug transporter.
Annu Rev Genet. 1995;29:607-49., [PMID:8825488]

Abstract [show]
The analysis of how human cancers evade chemotherapy has revealed a rich variety of cell-based genetic changes resulting in drug resistance. One of the best studied of these genetic alterations is increased expression of an ATP-dependent plasma membrane transport system, known as P-glycoprotein, or the multidrug transporter. This transporter actively effluxes a large number of natural product, hydrophobic, cytotoxic drugs, including many important anticancer agents. This review focuses on the genetic and molecular genetic analysis of the human multidrug transporter, including structure-function analysis, pre- and posttranslational regulation of expression, the role of gene amplification in increased expression, and the properties of transgenic and "knock-out" mice. One important feature of the MDR gene is its potential for the development of new selectable vectors for human gene therapy.

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177 One of the best-characterized mutations, of Gly to Val at position 185, results in increased resistance to colchicine and etoposide but decreased resistance to actinomycin D, vinblastine, doxorubicin, vincristine, and taxol (52, 63,129,197). Login to comment
1088 Mutation of glycine 185 to valine alters the ATPase function of the human P-glycoprotein expressed in Sf9 cells. Login to comment
1087 Mutation of glycine 185 to valine alters the ATPase function of the human P-glycoprotein expressed in Sf9 cells. 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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143 We have demonstrated previously that for the G185V mutation, which conferred increased resistance to colchicine, the MDR1 ATPase activity measurements in Sf9 cell membranes correlated well with the drug resistance profile [16]. Login to comment

[hide] P-glycoprotein function involves conformational tr... Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12908-13. Mechetner EB, Schott B, Morse BS, Stein WD, Druley T, Davis KA, Tsuruo T, Roninson IB
P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity.
Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12908-13., 1997-11-25 [PMID:9371774]

Abstract [show]
The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.

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185 On the other hand, these three compounds also differ from the other Pgp-transported drugs in some sterical aspect of their interaction with Pgp, because the ability of Pgp to transport these three drugs is selectively increased by a mutation (G185V) which, at the same time, decreases the Pgp activity toward those drugs that were found in the present study to increase UIC2 reactivity (36). Login to comment

[hide] Protein kinase C phosphorylation disengages human ... J Physiol. 1998 Apr 15;508 ( Pt 2):333-40. Bond TD, Valverde MA, Higgins CF
Protein kinase C phosphorylation disengages human and mouse-1a P-glycoproteins from influencing the rate of activation of swelling-activated chloride currents.
J Physiol. 1998 Apr 15;508 ( Pt 2):333-40., 1998-04-15 [PMID:9508799]

Abstract [show]
1. Whole-cell, swelling-activated Cl- currents, ICl(swell), were characterized in Chinese hamster ovary (CHO) cells and found to exhibit time-dependent inactivation at depolarizing potentials, tamoxifen and dideoxyforskolin sensitivity, and an anion permeability sequence: SCN- > I- > Br- > Cl- > F- > gluconate-. 2. CHO cells permanently transfected with either the human MDR1 or mouse mdr1a cDNAs demonstrated an increased rate of activation of ICl(swell) compared with parental cells or those permanently transfected with the mouse mdr1b cDNA. However, no differences in the magnitude of the currents were observed at steady state. 3. Pretreatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect ICl(swell) in MDR1 or mdr1a permanently transfected CHO cells. In contrast, pretreatment with TPA reduced ICl(swell) in MDR1(G185V)-expressing transfected NIH3T3 fibroblasts. Subsequently, the CHO cell lines were shown to contain significantly reduced levels of protein kinase C (PKC), suggesting that PKC concentrations might be limiting in these cell lines, at least under whole-cell patch clamp conditions. 4. Addition of purified PKC to the pipette solution, followed by a pretreatment with TPA, reduced the rate of ICl(swell) activation in human Pgp- and mouse Pgp1a-expressing CHO cells to the levels observed in parental and mouse Pgp1b-expressing cells. This confirms that PKC is limiting in these cells under whole-cell, patch clamp conditions. Furthermore, these results suggest that PKC-mediated phosphorylation of human Pgp and mouse Pgp1a disengages the influence which these Pgps have on ICl(swell). 5. These studies also demonstrate a functional distinction between the two mouse homologues, Pgp1a and Pgp1b. Although both can function as drug efflux pumps, only Pgp1a can act like human Pgp to influence ICl(swell).

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13 In contrast, pretreatment with TPA reduced ICl(swell) in MDR1(G185V)-expressing transfected NIH3T3 fibroblasts. Login to comment
34 NIH3T3-MDR1(G185V) cells are NIH3T3 fibroblasts permanently transfected with human MDR1 cDNA containing the G185V mutation which increases the relative resistance to colchicine (Pastan, Gottesman, Ueda, Lovelace, Rutherford & Willingham, 1988). Login to comment
59 In simultaneous experiments, TPA reduced ICl(swell) in NIH3T3-MDR1(G185V) cells (P = 0·007; Fig. 2A), as reported previously. Login to comment
60 It should be noted that the G185V mutation in MDR1 does not influence its ability to modulate channel activation. Login to comment

[hide] Mutational analysis of the P-glycoprotein first in... Biochemistry. 1998 Mar 10;37(10):3337-50. Kwan T, Gros P
Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains.
Biochemistry. 1998 Mar 10;37(10):3337-50., 1998-03-10 [PMID:9521654]

Abstract [show]
The role of individual intracellular (IC) loops linking transmembrane (TM) domains in P-glycoprotein (P-gp) function remains largely unknown. The high degree of sequence conservation of these regions in the P-gp family and other ABC transporters suggests an important role in a common mechanism of action of these proteins. To gain insight into this problem, we have randomly mutagenized a portion of TM2, the entire IC1 loop, TM3, the entire extracellular loop (EC2), and part of TM4, and analyzed the effect of such mutations on P-gp function. Random mutagenesis was carried out using Taq DNA polymerase and dITP under conditions of low polymerase fidelity, and the mutagenized segments were reintroduced in the full length mdr3 cDNA by homologous recombination in the yeast Saccharomyces cerevisiae strain JPY201. The biological activity of mutant P-gp variants was analyzed in yeast by their ability to confer cellular resistance to the antifungal drug FK506 and the peptide ionophore valinomycin, and by their ability to complement the yeast Ste6 gene and restore mating in a yeast strain bearing a null mutation [Raymond, M., et al. (1992) Science 256, 232-4] at this locus. The analysis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in the mutagenized segment resulting in an altered P-gp function. The mutants could be phenotypically classified into two major groups, those that resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity. Several of the mutants affecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positions 169-172) with such a phenotype. On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC2 segment and in the adjacent portion of the neighboring TM4 domain. Expression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associated with loss of drug-induced ATPase activity of P-gp. Therefore, it appears that TM domains, IC and EC loops, are structurally and functionally tightly coupled in the process of drug stimulatable ATPase characteristic of P-gp.

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34 An important role for the predicted IC1 in this process has been suggested by the analysis of the G185V mutant of human MDR1 which emerged during in vitro selection for colchicine resistance (39). Login to comment
35 G185V maps in IC1, alters substrate specificity (increased colchicine resistance, reduced vinblastine resistance) by modulating drug binding to the protein (40), but also affects drug-induced ATPase activity of the protein (41, 43). Login to comment

[hide] Mutations in the nucleotide-binding sites of P-gly... Biochemistry. 1998 Jun 23;37(25):9073-82. Beaudet L, Urbatsch IL, Gros P
Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity.
Biochemistry. 1998 Jun 23;37(25):9073-82., 1998-06-23 [PMID:9636053]

Abstract [show]
The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.

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243 It is interesting to note that the altered drug resistance profile of the G185V mutation in the human MDR1 (23, 55) has also been found associated with alterations in the pattern of drug-stimulatable ATPase activity (56, 57). Login to comment

[hide] Structural flexibility of the linker region of hum... Biochemistry. 1998 Sep 29;37(39):13660-73. Hrycyna CA, Airan LE, Germann UA, Ambudkar SV, Pastan I, Gottesman MM
Structural flexibility of the linker region of human P-glycoprotein permits ATP hydrolysis and drug transport.
Biochemistry. 1998 Sep 29;37(39):13660-73., 1998-09-29 [PMID:9753453]

Abstract [show]
P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two homologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this connecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections of Pgp mutants in mammalian cell lines and a vaccinia virus transient expression system. A 17 aa insertion of predicted flexible structure between amino acids 681 and 682 resulted in a functional Pgp molecule that was capable of conferring drug resistance. In contrast, an 18 aa peptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the linker region (Delta653-686) resulted in a protein expressed at the cell surface in amounts comparable to that of wild-type Pgp but unable to confer drug resistance. No apparent differences in drug or [alpha-32P]-8-azido-ATP photoaffinity labeling were observed. However, both ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion disconnected substrate binding from ATP hydrolysis and transport. This mutant also failed to exhibit an ATP hydrolysis-dependent enhancement of binding of a conformation-sensitive monoclonal antibody, UIC2. Upon replacement with a 17 aa linker peptide having a predicted flexible secondary structure, but bearing no homology to the deleted 34 aa segment, normal Pgp transport and basal and drug-stimulated ATPase activities were restored along with increased UIC2 binding in the presence of substrate, suggesting a dramatic conformational change between the nonfunctional and functional molecules. Taken together, these data suggest a flexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifically required for the proper interaction of the two ATP binding sites.

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83 pSX-MDR1/A is a pGEM3-based vector in which the multiple cloning site has been eliminated and replaced with a SacII-XhoI site to introduce a cDNA encoding the G185V MDR1 variant. Login to comment
111 Transfections followed by colchicine selection with the modified pSK4ADA-MDR1 (G185V) DNA (flexible linker experiments) and modified pHaMDR1/A (G185V) DNA (R-helical experiments) were performed similarly but using 100 mm dishes throughout. Login to comment
188 Table 1 demonstrates that similar numbers of drug-resistant colonies were obtained with unmodified MDR1 and MDR1-(flexible insertion), indicating that the modified transporter was approximately as effective as the G185V variant Pgp in conferring resistance to colchicine. Login to comment
191 The experiments described in Table 1 were performed with the G185V variant of Pgp, a well-characterized mutant that has been shown to change the substrate specificity of Pgp compared to that of the wild type, increasing its ability to confer resistance to colchicine (23). Login to comment
192 We found that the insertion mutant in Table 1 does not have an altered phenotype compared to the G185V variant, suggesting that the insertion of these additional amino acid residues did not affect the ability of the transporter to confer resistance to colchicine. Login to comment
200 To further examine the failure of the R-helical peptide insertion to confer drug resistance, parental G185V Pgp and the R-helical peptide insertion construct were transfected into NIH3T3 cells and the resultant transiently expressing populations were analyzed by FACS. Login to comment

[hide] Analysis of random recombination between human MDR... Mol Pharmacol. 1998 Oct;54(4):623-30. Shoshani T, Zhang S, Dey S, Pastan I, Gottesman MM
Analysis of random recombination between human MDR1 and mouse mdr1a cDNA in a pHaMDR-dihydrofolate reductase bicistronic expression system.
Mol Pharmacol. 1998 Oct;54(4):623-30., [PMID:9765504]

Abstract [show]
Human P-glycoprotein (Pgp) confers multidrug resistance (MDR) to otherwise sensitive cells. The homologous mouse Pgps, which are encoded by mouse mdr1a (also known as mdr3) and mdr1b (also known as mdr1), confer different degrees of resistance to the same MDR drugs and inhibitors. To create recombinants for the study of sequences responsible for these differences in drug-resistance, chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling. To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a was used. The chimeric proteins were expressed in human KB-3-1 cells. One recombinant Pgp (clone 3-4) with a novel phenotype was analyzed in detail. Inhibitors of Pgp, including verapamil and cyclosporin A, were less effective in reversing resistance of the chimeric Pgp compared with wild-type Pgp, for certain drugs. However, [125I]iodoarylazidoprazosin photoaffinity labeling of the chimeric Pgp and its binding competition with cyclosporin A, showed that cyclosporin A competed for the photoaffinity labeling. The chimeric Pgp cells stained less well with human-specific anti-Pgp mAb MRK16 than wild-type Pgp, despite having the described epitopes for MRK16. Staining with human-specific mAb UIC2 was increased when the chimeric protein was compared with wild-type Pgp. These results suggest an alteration in exposure of human Pgp specific epitopes in this chimeric Pgp, as well as a change in the interaction of reversing agents with the chimeric protein.

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168 It has been previously shown that inhibitors of MDR1 show differential effects on reversing resistance of different drugs depending on whether the Pgp mutant G185V or wild-type transporters are analyzed (Cardarelli et al., 1995). Login to comment

[hide] Interaction of transported drugs with the lipid bi... Biophys J. 2006 Jun 1;90(11):4046-59. Epub 2006 Mar 24. Omote H, Al-Shawi MK
Interaction of transported drugs with the lipid bilayer and P-glycoprotein through a solvation exchange mechanism.
Biophys J. 2006 Jun 1;90(11):4046-59. Epub 2006 Mar 24., [PMID:16565061]

Abstract [show]
Broad substrate specificity of human P-glycoprotein (ABCB1) is an essential feature of multidrug resistance. Transport substrates of P-glycoprotein are mostly hydrophobic and many of them have net positive charge. These compounds partition into the membrane. Utilizing the energy of ATP hydrolysis, P-glycoprotein is thought to take up substrates from the cytoplasmic leaflet of the plasma membrane and to transport them to the outside of the cell. We examined this model by molecular dynamics simulation of the lipid bilayer, in the presence of transport substrates together with an atomic resolution structural model of P-glycoprotein. Taken together with previous electron paramagnetic resonance studies, the results suggest that most transported drugs are concentrated near the surface zone of the inner leaflet of the plasma membrane. Here the drugs can easily diffuse laterally into the drug-binding site of P-glycoprotein through an open cleft. It was concluded that the initial high-affinity drug-binding site was located in the interfacial surface area of P-glycoprotein in contact with the membrane interface. Based on these results and our recent kinetic studies, a "solvation exchange" drug transport mechanism of P-glycoprotein is discussed. A molecular basis for the improved colchicine transport efficiency by the much-studied colchicine-resistance G185V mutant human P-glycoprotein is also provided.

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9 A molecular basis for the improved colchicine transport efficiency by the much-studied colchicine-resistance G185V mutant human P-glycoprotein is also provided. Login to comment
221 The G185V mutation suppresses this effect by increasing the intrinsic rate of transport to the expected one (Fig. 9). Login to comment
222 It should be emphasized that G185V P-glycoprotein does not change the intrinsic drug transport rate or drug binding characteristics of all drugs but only a limited subset such as colchicine and etoposide (33). Login to comment
224 Thus, the G185V mutation distorts the binding site for colchicine while leaving binding sites of other drugs intact. Login to comment
225 Binding of colchicine to WT and G185V P-glycoproteins were characterized by van`t Hoff analysis by methods previously described (52). Login to comment
228 The corresponding values for colchicine binding to G185V P-glycoprotein were ÿ191.4, ÿ176.9, and ÿ14.5 kJ molÿ1 , respectively. Login to comment
229 Here the net driving force for colchicine binding to G185V P-glycoprotein was through favorable noncovalent interactions (H-bonds) compensated somewhat by a large unfavorable entropy term. Login to comment
246 The G185V mutation modified the colchicine binding site in such a fashion as to reduce the contribution of nonpolar interactions. Login to comment
248 These factors improve the colchicine transport rate and coupling efficiency of the G185V mutation as previously described (33). Login to comment
315 In Results and Fig. 9 we demonstrate that G185V P-glycoprotein interacts with colchicine in a manner that removes an overabundance of nonpolar van der Waals interactions while satisfying the available H-bond acceptors. Login to comment
375 This was the case for the improved transport of colchicine by the G185V mutation (33) (Fig. 9). Login to comment

[hide] The P-glycoprotein multidrug transporter. Gen Pharmacol. 1996 Dec;27(8):1283-91. Fardel O, Lecureur V, Guillouzo A
The P-glycoprotein multidrug transporter.
Gen Pharmacol. 1996 Dec;27(8):1283-91., [PMID:9304397]

Abstract [show]
1. P-glycoprotein (P-gp) is a transmembrane protein involved in ATP-dependent efflux of various structurally unrelated anticancer drugs. Its overexpression in cancer cells decreases intracellular drug concentrations and, thus, confers a multidrug resistance phenotype. 2. P-gp is encoded by MDR genes, which constitute a small gene family comprising two genes in humans and three genes in rodents. Only the MDR1 gene in humans and mdr1 and mdr3 genes in rodents have been demonstrated to be involved in drug resistance. 3. P-gp encoded by the human MDR1 gene is a phosphorylated and glycosylated protein 1289 amino acids long, and consists of 2 halves that share a high degree of similarity. 4. A wide variety of cancers have been shown to express P-gp, including solid tumors and hematological malignancies. This P-gp positivity can be evidenced at the time of diagnosis prior to chemotherapy or at relapse after treatment, and has been correlated with treatment failure and poor prognosis in several types of cancer. In addition, P-gp is also expressed by some normal tissues, such as liver and kidney. 5. P-gp expression is regulated by various factors, including xenobiotics and hormones. 6. P-gp-mediated multidrug resistance can be reversed by various unrelated compounds called chemosensitizers or reversing agents. These drugs act through inhibition of P-gp function and have entered clinical trials.

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85 Indeed a change from Gly to Val at position 185 led to reduced vinblastine transport and transport of colchicine was improved (Currier et al., 1992). Login to comment
84 Indeed a change from Gly to Val at position 185 led to reduced vinblastine transport and transport of colchicine was improved (Currier et al., 1992). Login to comment

[hide] A synonymous polymorphism in a common MDR1 (ABCB1)... Biochim Biophys Acta. 2009 May;1794(5):860-71. Epub 2009 Mar 11. Fung KL, Gottesman MM
A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function.
Biochim Biophys Acta. 2009 May;1794(5):860-71. Epub 2009 Mar 11., [PMID:19285158]

Abstract [show]
The MDR1 (ABCB1) gene encodes a membrane-bound transporter that actively effluxes a wide range of compounds from cells. The overexpression of MDR1 by multidrug-resistant cancer cells is a serious impediment to chemotherapy. MDR1 is expressed in various tissues to protect them from the adverse effect of toxins. The pharmacokinetics of drugs that are also MDR1 substrates also influence disease outcome and treatment efficacy. Although MDR1 is a well-conserved gene, there is increasing evidence that its polymorphisms affect substrate specificity. Three single nucleotide polymorphisms (SNPs) occur frequently and have strong linkage, creating a common haplotype at positions 1236C>T (G412G), 2677G>T (A893S) and 3435C>T (I1145I). The frequency of the synonymous 3435C>T polymorphism has been shown to vary significantly according to ethnicity. Existing literature suggests that the haplotype plays a role in response to drugs and disease susceptibility. This review summarizes recent findings on the 3435C>T polymorphism of MDR1 and the haplotype to which it belongs. A possible molecular mechanism of action by ribosome stalling that can change protein structure and function by altering protein folding is discussed.

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146 A missense mutation found at position 183 is very close to two amino acids before the G185V mutation site. Login to comment
147 This G185V mutation has been identified in drug-resistant cell lines, but not in humans. Login to comment
145 A missense mutation found at position 183 is very close to two amino acids before the G185V mutation site. Login to comment

[hide] P-Glycoprotein is not present in mitochondrial mem... Exp Cell Res. 2007 Aug 15;313(14):3100-5. Epub 2007 Apr 24. Paterson JK, Gottesman MM
P-Glycoprotein is not present in mitochondrial membranes.
Exp Cell Res. 2007 Aug 15;313(14):3100-5. Epub 2007 Apr 24., [PMID:17512524]

Abstract [show]
Recent reports have indicated the presence of P-glycoprotein in crude mitochondrial membrane fractions, leading to the assumption that P-glycoprotein is present in mitochondrial membranes, and may be involved in transport across these membranes. To determine the validity of this claim, two cell lines overexpressing endogenous P-glycoprotein were investigated. Using various centrifugation steps, mitochondria were purified from these cells and analyzed by Western blot reaction with the anti-P-glycoprotein antibody C219 and organelle-specific antibodies. While P-glycoprotein is present in crude mitochondrial fractions, these fractions are contaminated with plasma membranes. Further purification of the mitochondria to remove plasma membranes revealed that P-glycoprotein is not expressed in mitochondria of the KB-V1 (vinblastine-resistant KB-3-1 cells) or MCF-7(ADR) (adriamycin-resistant MCF-7 cells) cell lines. To further substantiate these findings, we used confocal microscopy and the anti-P-glycoprotein antibody 17F9. This demonstrated that in intact cells, P-glycoprotein is not present in mitochondria and is primarily localized to the plasma membrane. These findings are consistent with the role of P-glycoprotein in conferring multidrug resistance by decreasing cellular drug accumulation. Therefore, contrary to previous speculation, P-glycoprotein does not confer cellular protection by residing in mitochondrial membranes.

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153 [21] M. Ramachandra, S.V. Ambudkar, M.M. Gottesman, I. Pastan, C.A. Hrycyna, Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system, Mol. Biol. Cell 7 (1996) 1485-1498. Login to comment
151 [21] M. Ramachandra, S.V. Ambudkar, M.M. Gottesman, I. Pastan, C.A. Hrycyna, Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system, Mol. Biol. Cell 7 (1996) 1485-1498. Login to comment

[hide] P-glycoprotein enhances TRAIL-triggered apoptosis ... Biochem Pharmacol. 2006 Jul 28;72(3):293-307. Epub 2006 May 4. Park SJ, Wu CH, Choi MR, Najafi F, Emami A, Safa AR
P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5.
Biochem Pharmacol. 2006 Jul 28;72(3):293-307. Epub 2006 May 4., [PMID:16753135]

Abstract [show]
The death-inducing cytokine TRAIL is a promising agent for anticancer therapy since it preferentially kills cancer versus normal cells; however, some cancer cells are TRAIL-resistant. We initially explored whether overexpression of the MDR1 gene product P-glycoprotein (P-gp), which causes multidrug resistance (MDR) in cancer cells, also contributes to TRAIL-resistance. Surprisingly, our results revealed that P-gp-overexpression enhances TRAIL-induced apoptosis not only in neoplastic cells transfected with the MDR1 gene but also in MDR variants selected with cytotoxic anticancer agents. Mechanistic analysis of TRAIL-induced apoptosis in the MDR1-transfected MCF-7 breast cancer cell line BC-19 revealed that TRAIL-triggered significantly more apoptosis in these cells compared with parental MCF-7 cells by binding to the TRAIL receptor DR5. DR5 but not DR4 engagement by TRAIL attenuated cellular ATP levels by robustly stimulating P-gp ATPase activity, and thus triggered P-gp-dependent apoptosis by depletion of the cellular ATP pool. In addition to hyperactive P-gp-mediated ATP hydrolysis, TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspases-6, -7, -8, and -9; Bid cleavage; and mitochondrial depolarization. P-gp interacted with the TRAIL receptors DR4, DR5, and DcR1 in plasma membranes and enhanced TRAIL binding to DR5. Interestingly, the decreased level of the decoy TRAIL receptor, DcR1, in BC-19 cells further sensitized these cells to TRAIL. Therefore, both extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment preferentially causes apoptosis in P-gp-overexpressing MDR cells, and suggests significant clinical implications for the use of TRAIL in treating neoplasms that have failed chemotherapy.

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139 It is therefore intriguing that TRAIL induced much more apoptosis in the KB-3-1 transfectant KB-VSV1, which expresses the mutant G185V P-gp, than in the wt P-gp transfectant KB-GSV2 (Table 1) (see Section 4). Login to comment
297 The immunoblot was probed for the presence of (A) DR5, (B) DR4, (C) DcR1, and P-gp as described in Section 2. in cells transfected with the MDR1 and overexpressing P-gp; (2) treating P-gp-expressing BC-19 cells with the anti-P-gp monoclonal antibody MRK-16 decreased TRAIL-induced apoptosis to similar levels induced in their drug-sensitive counterparts; (3) TRAIL induced preferential apoptosis in P-gp- overexpressing cells by depleting ATP and increasing the ATPase activity of P-gp; (4) KB-VSV1 transfectants with P-gp mutated at position 185 (glycine-185 to valine, G185V) underwent much more TRAIL-induced apoptosis than the transfectants expressing wild-type P-gp. Login to comment
298 The superior TRAIL sensitization elicited by G185V P-gp strengthens the linkage between P-gp transporter function and enhancement of TRAIL-induced apoptosis, because thermodynamic analysis has revealed that G185V P-gp is more efficient in the ATP-dependent transport of substrates than wt P-gp, owing to a lower Arrhenius activation energy for the rate-limiting step of ATP hydrolysis-coupled substrate transport [31]. Login to comment
138 It is therefore intriguing that TRAIL induced much more apoptosis in the KB-3-1 transfectant KB-VSV1, which expresses the mutant G185V P-gp, than in the wt P-gp transfectant KB-GSV2 (Table 1) (see Section 4). Login to comment
296 The immunoblot was probed for the presence of (A) DR5, (B) DR4, (C) DcR1, and P-gp as described in Section 2. in cells transfected with the MDR1 and overexpressing P-gp; (2) treating P-gp-expressing BC-19 cells with the anti-P-gp monoclonal antibody MRK-16 decreased TRAIL-induced apoptosis to similar levels induced in their drug-sensitive counterparts; (3) TRAIL induced preferential apoptosis in P-gp-overexpressing cells by depleting ATP and increasing the ATPase activity of P-gp; (4) KB-VSV1 transfectants with P-gp mutated at position 185 (glycine-185 to valine, G185V) underwent much more TRAIL-induced apoptosis than the transfectants expressing wild-type P-gp. Login to comment

[hide] New light on multidrug binding by an ATP-binding-c... Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20. Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW
New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20., [PMID:16545467]

Abstract [show]
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

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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

[hide] Implications of genetic polymorphisms in drug tran... Cancer Lett. 2006 Mar 8;234(1):4-33. Epub 2006 Feb 28. Kerb R
Implications of genetic polymorphisms in drug transporters for pharmacotherapy.
Cancer Lett. 2006 Mar 8;234(1):4-33. Epub 2006 Feb 28., [PMID:16504381]

Abstract [show]
Drug transporters are increasingly recognized as a key determinant of drug disposition and response. It is now widely appreciated that expression of the ATP-dependent efflux transporter, MDR1 (ABCB1, P-glycoprotein), in organs such as the gastrointestinal tract, liver and kidney significantly alters the extent of drug absorption and excretion. Moreover, expression of MDR1 at the level of the blood-brain barrier limits the entry of many drugs into the central nervous system. Given such an important role of MDR1 in the drug disposition process, it is not surprising to see increasing focus on the role of single nucleotide polymorphisms (SNPs) in this transporter as a potential determinant of interindividual variability in drug disposition and pharmacological response. However, drug transport is often the result of the concerted action of efflux and uptake pumps located both in the basolateral and apical membranes of epithelial cells. A growing list of membrane-spanning proteins involved in the in- or outward transport of a large variety of drugs has been recognized and characterized over the past few years in almost all tissues, including organic anion and cation transporters (OAT, OCT, solute carrier family SLC22A), organic anion transport proteins (OATP, solute carrier family SLCO, formerly SLC21A), and MRPs (ABCCs), other members of the ATP-binding cassette family. We are just beginning to appreciate their role for drug delivery and disposition and the contribution of genetic polymorphisms in these transport proteins to interindividual variability in the efficacy and safety for pharmacotherapy. This review summarizes the consequences of inherited differences in drug transport for pharmacotherapy. With the main focus on ABCB1, an update of recent advances is given and clinically relevant examples are used to illustrate how heritable differential drug transport can help to explain individual variability in drug response. The pharmacogenetics of other transporters is briefly introduced.

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64 R. Kerb / Cancer Letters 234 (2006) 4-338 naturally-occurring polymorphisms in the human ABCB1 gene reported was the amino acid substitution Gly185Val [89], and more recently Ala893Ser and Met986Val [90]. Login to comment
63 naturally-occurring polymorphisms in the human ABCB1 gene reported was the amino acid substitution Gly185Val [89], and more recently Ala893Ser and Met986Val [90]. Login to comment

[hide] Molecular genetic analysis and biochemical charact... Semin Cell Dev Biol. 2001 Jun;12(3):247-56. Hrycyna CA
Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance.
Semin Cell Dev Biol. 2001 Jun;12(3):247-56., [PMID:11428917]

Abstract [show]
A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional drug therapies. This phenomenon is due in large part to the overexpression of a 170 kDa plasma membrane ATP-dependent pump known as the multidrug resistance transporter or P-glycoprotein. P-glycoprotein is a member of the large ATP binding cassette (ABC) superfamily of membrane transporters. This review focuses on the use of structure-function analyses to elucidate further the mechanism of action of mammalian P-glycoproteins. Ultimately, a complete understanding of the mechanism is important for the development of novel strategies for the treatment of many human cancers.

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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

[hide] Coordinate changes in drug resistance and drug-ind... Biochemistry. 2001 Apr 10;40(14):4332-9. Ruth A, Stein WD, Rose E, Roninson IB
Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein.
Biochemistry. 2001 Apr 10;40(14):4332-9., [PMID:11284689]

Abstract [show]
The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.

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1 The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. Login to comment
2 MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. Login to comment
3 This selection yielded the I186N substitution, adjacent to G185V. Login to comment
4 Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. Login to comment
5 In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. Login to comment
21 The first altered-function mutation to be identified in ABC transporters was the substitution of glycine to valine at position 185 (G185V), adjacent to TM3 of the human MDR1 Pgp (11). Login to comment
31 At the same time, G185V decreased cellular resistance to vinblastine and Taxol, compared to that of the wild-type Pgp (11, 12), and altered the sensitivity of Pgp to different inhibitors (13, 14). Login to comment
34 Biochemical assays showed that G185V enhances the ability of colchicine and inhibits the ability of vinblastine to stimulate the ATPase activity of Pgp (17, 18). Login to comment
35 On the other hand, G185V was found to decrease the level of Pgp binding of a photoactive analogue of colchicine and to increase the level of binding of a vinblastine analogue, suggesting that this mutation changes the transport efficiency by affecting the release of the substrates from Pgp to the outside of the cell (12). Login to comment
38 The first functional mutation identified by this selection turned out to affect amino acid 186, which is immediately adjacent to the previously identified G185V substitution. Login to comment
50 The procedures for the propagation of retroviral plasmid vector pLMDR1 (23), carrying MDR1 cDNA with a G185V mutation, in the mutD strain of Escherichia coli, transfection of BOSC 23 ecotropic packaging cells (24), retroviral transduction of NIH 3T3 cells, etoposide selection, and PCR-based recovery and recloning of the integrated MDR1 cDNA have been previously described (19). Login to comment
76 To isolate Pgp mutants that would further increase the resistance to etoposide conferred by the previously described G185V mutant, we have used the strategy of function-based selection of mutagenized retroviral vectors (19). Login to comment
77 A retroviral plasmid vector carrying the coding sequence of human MDR1 cDNA with the G185V mutation was propagated in a mutator strain of E. coli, and the resulting randomly mutagenized retrovirus was transduced into murine NIH 3T3 cells. Login to comment
84 One of the selected mutants produced elevated etoposide resistance relative to that of the parental G185V MDR1 cDNA. Login to comment
85 This mutant was found to carry a single ATT f AAT mutation of codon 186, which resulted in a change of isoleucine to asparagine at the corresponding position (I186N), adjacent to the original G185V substitution. Login to comment
86 Effects of G185V and I186N Substitutions on Drug Resistance Profiles. Login to comment
87 To confirm that the I186N mutation was indeed responsible for elevated etoposide resistance and to investigate how this mutation interacts with G185V, we used site-directed mutagenesis to introduce the I186N substitution into both G185V and the wild-type versions of the MDR1 cDNA. Login to comment
88 Retroviral vectors carrying the wild-type MDR1, MDR1 carrying the individual G185V or I186N mutation, and MDR1 with both G185V and I186N mutations (G185V/ I186N) were individually transduced into NIH 3T3 cells. Login to comment
93 The etoposide assays (Figure 2A) showed that the G185V (O) substitution increased etoposide resistance relative to cells transduced with the wild-type MDR1 (b), in agreement with our previous report (12). Login to comment
94 The etoposide resistance of G185V was further increased by combining it with I186N [G185V/I186N (4)]. Login to comment
97 The overlapping FACS profiles represent four different NIH 3T3 populations expressing either the wild-type MDR1 or MDR1 mutants G185V, I186N, or G185V/I186N (double mutant). Login to comment
100 NIH 3T3 cells were transduced with insert-free LXSN vector (9), with the wild-type MDR1 (b), with G185V (O), with I186N (1), or with G185V/I186N (3). Login to comment
103 G185V (Figure 2A). Login to comment
105 Colchicine assays (Figure 2C) showed that both I186N and G185V increased colchicine resistance relative to the wild-type MDR1, but G185V (a mutation that was originally found in colchicine-selected cells) had a stronger effect. Login to comment
106 Colchicine resistance conferred by G185V/I186N was indistinguishable from the effect of G185V alone. Login to comment
107 A major functional difference between G185V and I186N mutations was observed, however, in the vinblastine (Figure 2D) and Taxol (Figure 2E) assays. Login to comment
108 In agreement with our previous reports (11, 12), G185V decreased the resistance to both of these drugs relative to the wild-type MDR1. Login to comment
110 These opposite effects of the two mutations were balanced out in the G185V/I186N double mutant, which produced vinblastine and Taxol resistances that were indistinguishable from that of the wild type (Figure 2D,E). Login to comment
111 These results indicate that I186N increases the ability of MDR1 to confer resistance to all the tested drugs and augments the effect of G185V in providing a high level of etoposide resistance. Login to comment
112 Analysis of the Effects of the G185V and I186N Mutations on Drug-Induced Conformational Transitions of Pgp Using UIC2 ImmunoreactiVity. Login to comment
114 We have used changes in UIC2 reactivity to investigate how G185V and I186N mutations change the effects of transported drugs on the conformation of Pgp. Login to comment
119 NIH 3T3 cells were transduced with wild-type MDR1 (b), with G185V (O), with I186N (1), or with G185V/I186N (3). Login to comment
123 Measurement of the level of cellular ATP in oligomycin-treated and control untreated samples showed that oligomycin decreased cellular ATP levels to 3.2, 3.3, 3.4, and 2.9% of the control in the wild type, G185V, I186N, and G185V/ I186N, respectively. Login to comment
131 The G185V mutation had no effect on UIC2 reactivity in the absence of drugs, but it raised the reactivity in the presence of all the drugs or oligomycin to a level similar to that of MRK16 (Figure 4). Login to comment
132 Unlike G185V, I186N produced different effects on the fold increase in UIC2 reactivity by different drugs. Login to comment
135 The G185V/I186N double mutant showed the same increase in UIC2 reactivity with all three drugs and with oligomycin as I186N alone (Figure 4). Login to comment
151 As shown in many previous studies, including those that analyzed the effects of G185V on the accumulation (12) and transport (15) of different drugs, changes in Pgp-mediated drug resistance reflect the corresponding changes in the outward drug pumping. Login to comment
152 We also investigated the effects of I186N, together with a previously identified specificity-altering mutation of the adjacent amino acid, G185V, on substrate-induced conformational transitions of the transporter. Login to comment
153 This analysis revealed that mutations that affect the ability of Pgp to transport Table 1: Effects of G185V and I186N Mutations on the Relative Resistance and Conformational Effects of Vinblastine, Colchicine, and Etoposidea changes in UIC2 reactivity MDR1 mutant relative resistance fold increase Km (µM) Hill number vinblastine wild type 7.8 2.2 0.24 ( 0.01 2.1 ( 0.2 G185V 5.7 3.9 0.10 ( 0.01 1.2 ( 0.2 I186N 10.8 3.8 0.44 ( 0.02 1.9 ( 0.3 G185V/I186N 8.1 4.2 0.24 ( 0.04 0.9 ( 0.1 colchicine wild type 4.5 2.3 1700 ( 400 0.7 ( 0.1 G185V 15.8 3.6 2400 ( 200 2.0 ( 0.4 I186N 8.6 1.8 5700 ( 1200 1.8 ( 0.2 G185V/I186N 14.6 1.9 4800 ( 500 3.7 ( 1.1 etoposide wild type 4.3 1.7 220 ( 10 2.1 ( 0.2 G185V 9.3 3.8 480 ( 30 2.9 ( 0.6 I186N 9.3 2.5 600 ( 70 3.3 ( 0.2 G185V/I186N 13.1 2.4 730 ( 20 4.2 ( 0.3 a Relative resistance is the ratio of the LD50 of NIH 3T3 cells transduced with the corresponding form of MDR1 to the LD50 of cells transduced with the control vector LXSN. Login to comment
159 This residue abuts TM3 and is immediately adjacent to the G185V mutation, which was present in the original MDR1 cDNA used for mutagenesis and which was already known to increase etoposide resistance. Login to comment
160 This coincidence raised the possibility that I186N could act by augmenting the effect of G185V and would only be functional in combination with the latter mutation. Login to comment
161 Testing the effects of G185V and I186N mutations individually and in combination showed that this is not the case. Login to comment
162 I186N alone increased etoposide resistance to the same extent as G185V (~2-fold), while combining these two mutations enhanced the ability of Pgp to confer etoposide resistance approximately 3-fold relative to that of the wild-type protein. Login to comment
163 Another possibility suggested by the proximity of the two mutations was that I186N would affect Pgp function in the same way as G185V. Login to comment
165 While G185V increased the resistance to some Pgp-transported drugs, it also decreased the activity of Pgp toward other substrates, whereas I186N increased the Pgp activity toward all five of the tested drugs. Login to comment
168 G185V and I186N Mutations Change the Parameters of Drug-Induced Conformational Transitions of Pgp. Login to comment
169 The mechanistic effects of the G185V and I186N mutations were approached by analyzing the effects of drugs on the conformation of Pgp, as measured by increased UIC2 reactivity in the presence of drugs. Login to comment
173 This limit of UIC2 reactivity in NIH 3T3 cells was overcome by the mutation G185V, which increased the highest levels of UIC2 reactivity in the presence of all three tested transport substrates and oligomycin to the levels obtained with MRK16. Login to comment
174 Unlike the G185V mutation, the I186N mutation allowed the maximal level of UIC2 reactivity in the presence of vinblastine, but not in the presence of etoposide, colchicine, or oligomycin. Login to comment
175 The G185V/I186N double mutant was essentially indistinguishable in this respect from I186N alone, indicating that the restrictions on the UIC2 reactivity shift imposed by I186N are dominant over the "releasing" effect of G185V. Login to comment
177 Meaningful correlations with drug resistance were observed, however, for the effects of G185V and I186N mutations on the Km and Hill number values for this drug-induced increase in UIC2 reactivity. Login to comment
179 This assumption is confirmed by a comparison between the effects of G185V on the Km values for UIC2 reactivity changes and the previously reported effects of this mutation on drug binding to Pgp. Login to comment
180 In the study presented here, we observed that G185V increases the Km for colchicine (i.e., decreases its apparent affinity) and decreases the Km for vinblastine (i.e., increases the apparent affinity of this drug). Login to comment
181 These findings are in complete agreement with the earlier work (12), where we found that G185V decreases the level of binding of a photoactive colchicine analogue and increases the level of binding of a vinblastine analogue to Pgp. Login to comment
183 Since changes in drug binding conferred by G185V were inversely associated with changes in the transport of the corresponding drug, we had suggested that it was debinding at the off site that was affected by this mutation (12). Login to comment
185 Despite the results with G185V, we cannot tell a priori whether a higher Km found in other cases should always point to a higher rate of debinding of the substrate at the off site, or whether it may also reflect drug binding at the on site. Login to comment
194 G185V, which decreases vinblastine resistance, also decreases the Km value in the UIC2 shift assay. Login to comment
195 As discussed above, this observation, together with the previous results (12), suggests that G185V decreases the level of vinblastine debinding at the off site. Login to comment
196 Strikingly, G185V also causes an apparent loss of one vinblastine-binding site, as it decreases the Hill number for vinblastine from 2 to 1. Login to comment
197 The loss of the binding site could conceivably provide a mechanism for the decreased level of transport of vinblastine by G185V. Login to comment
198 In contrast to G185V, the I186N mutation increases the vinblastine resistance, and this effect is associated with an increase in the Km value. Login to comment
201 The Hill number for vinblastine is 1 in the G185V/I186N double mutant, indicating that I186N cannot reverse the loss of a drug-binding site caused by G185V. Login to comment
202 The double mutant shows an intermediate Km between those of G185V and I186N, which happens to match the Km for the wild-type Pgp, and G185V/I186N shows the same level of vinblastine resistance as wild-type Pgp. Login to comment
205 The G185V mutation, which originally arose in a colchicine-selected cell line and which strongly increases colchicine resistance, produces a moderate increase in the Km value for colchicine, in agreement with the previous study (12). Login to comment
207 I186N, like G185V, increases resistance to colchicine, and this is accompanied by a higher Km and an increase in the Hill number from 1 to 2. Login to comment
208 At the quantitative level, however, I186N provides a stronger increase in the Km value, but G185V confers a larger increase in resistance. Login to comment
209 This contradiction raises the question of whether the effect of G185V on colchicine resistance may be due not only to increased debinding but also to some additional factors, or whether an increased colchicine Km (at least for I186N) may reflect changes in both drug binding and debinding. Login to comment
210 G185V/ I186N produces a higher Hill number than either mutation alone, suggesting an additive effect of the two mutations on the apparent number of drug-binding sites. Login to comment
211 The effects of the mutations on the Km value and on drug resistance, however, do not appear to be additive, since the G185V/ I186N double mutant has about the same Km as the I186N mutant but produces the same level of colchicine resistance as the G185V mutant. Login to comment
212 In contrast to the complicated colchicine situation, etoposide, the drug that was originally used to select the G185V/ I186N double mutant, provides the most straightforward correlation between drug resistance, on one hand, and the Km and Hill number changes, on the other hand. Login to comment
213 Both G185V and I186N increase the apparent number of etoposide-binding sites from 2 to 3, while combining these mutations brings this number up to 4. Login to comment
214 Similarly, the Km values are increased by either G185V or I186N, and the double mutant exhibits the highest Km value, i.e., the lowest apparent affinity. Login to comment
215 The changes in both of these parameters agree with the effects of the mutations on etoposide resistance, which is higher in the G185V or I186N mutant than in the wild type and becomes the highest in the double mutant. Login to comment
216 In summary, the observed effects of the G185V and I186N mutations on the transport of individual drugs can be interpreted through the effects of these mutations on the ability of the drugs to alter Pgp conformation, as reflected by the process of the change in UIC2 reactivity. Login to comment

[hide] A new polymorphism (N21D) in the exon 2 of the hum... Hum Mutat. 2000 May;15(5):486. Decleves X, Chevillard S, Charpentier C, Vielh P, Laplanche JL
A new polymorphism (N21D) in the exon 2 of the human MDR1 gene encoding the P-glycoprotein.
Hum Mutat. 2000 May;15(5):486., [PMID:10790226]

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17 (1988) showed for the first time that the G185V variant of P-gp was responsible for preferential resistance to colchicine in multidrug resistance KB cells. Login to comment

[hide] PKC-independent modulation of multidrug resistance... Biochem Pharmacol. 1998 Oct 1;56(7):861-9. Spitaler M, Utz I, Hilbe W, Hofmann J, Grunicke HH
PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) P-glycoprotein by bryostatin 1.
Biochem Pharmacol. 1998 Oct 1;56(7):861-9., [PMID:9774148]

Abstract [show]
Bryostatin 1 is a new antitumor agent which modulates the enzyme activity of protein kinase C (PKC, phospholipid-Ca2+-dependent ATP:protein transferase, EC 2.7.1.37). Several reports have suggested that the pumping activity of the multidrug resistance gene 1 (MDR1)-encoded multidrug transporter P-glycoprotein (PGP) is enhanced by a PKC-mediated phosphorylation. It was shown here that bryostatin 1 was a potent modulator of multidrug resistance in two cell lines over-expressing a mutant MDR1-encoded PGP, namely KB-C1 cells and HeLa cells transfected with an MDR1-V185 construct (HeLa-MDR1-V185) in which glycine at position 185 (G185) was substituted for valine (V185). Bryostatin 1 is not able to reverse the resistance of cells over-expressing the wild-type form (G185) of PGP, namely CCRF-ADR5000 cells and HeLa cells transfected with a MDR1-G185 construct (HeLa-MDR1-G185). Treatment of HeLa-MDR1-V185 cells with bryostatin 1 was accompanied by an increase in the intracellular accumulation of rhodamine 123, whereas no such effect could be observed in HeLa-MDR1-G185 cells. HeLa-MDR1-V185 cells expressed the PKC isoforms alpha, delta and zeta. Down-modulation of PKC alpha and delta by 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect the drug accumulation by bryostatin 1. Bryostatin 1 depleted PKC alpha completely and PKC delta partially. In HeLa-MDR1-V185 cells, short-term exposure to bryostatin 1, which led to a PKC activation, was as efficient in modulating the pumping activity of PGP as long-term exposure leading to PKC depletion. Bryostatin 1 competed with azidopine for binding to PGP in cells expressing the MDR1-V185 and MDR1-G185 forms of PGP. It is concluded that bryostatin 1: i) interacts with both the mutated MDR1-V185 and the wild-type MDR1-G185; ii) reverses multidrug resistance and inhibits drug efflux only in PGP-V185 mutants; and iii) that this effect is not due to an interference of PKC with PGP. For gene therapy, it is important to reverse the specific resistance of a mutant in the presence of a wild-type transporter and vice versa. Our results show that it is possible to reverse a specific mutant PGP.

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30 0043-512-507-3505; FAX 0043-512-507-2872; E-mail: johann.hofmann@uibk.ac.at § Abbreviations: HeLa-WT, drug-sensitive HeLa wild-type cells; HeLa- MDR1-G185, multidrug-resistant HeLa cells transfected with a wild-type MDR1 gene; HeLa-MDR1-V185, multidrug-resistant HeLa cells transfected with a mutant MDR1 gene (valine instead of glycine in position 185); MDR, multidrug resistance; MDR1, multidrug resistance gene 1; PGP, P-glycoprotein; PKC, protein kinase C; and TPA, 12-O-tetradecanoylphorbol-13-acetate. Login to comment
98 It has been reported that KB-C1 cells harbor a mutated MDR1 gene in which glycine at position 185 is replaced by valine [37]. Login to comment
29 Tel. 0043-512-507-3505; FAX 0043-512-507-2872; E-mail: johann.hofmann@uibk.ac.at &#a7; Abbreviations: HeLa-WT, drug-sensitive HeLa wild-type cells; HeLa-MDR1-G185, multidrug-resistant HeLa cells transfected with a wild-type MDR1 gene; HeLa-MDR1-V185, multidrug-resistant HeLa cells transfected with a mutant MDR1 gene (valine instead of glycine in position 185); MDR, multidrug resistance; MDR1, multidrug resistance gene 1; PGP, P-glycoprotein; PKC, protein kinase C; and TPA, 12-O-tetradecanoylphorbol-13-acetate. Login to comment

[hide] Baculovirus-mediated expression of human multidrug... Methods Enzymol. 1998;292:427-41. Germann UA
Baculovirus-mediated expression of human multidrug resistance cDNA in insect cells and functional analysis of recombinant P-glycoprotein.
Methods Enzymol. 1998;292:427-41., [PMID:9711572]

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109 P-GLYCOPROTEIN EXPRESSION IN INSECT CELLS with a Gly-185 --> Val substitution. Login to comment

[hide] Identification of drug interaction sites in P-glyc... Methods Enzymol. 1998;292:307-17. Greenberger LM
Identification of drug interaction sites in P-glycoprotein.
Methods Enzymol. 1998;292:307-17., [PMID:9711563]

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201 In particular, point mutations in G185V in human P-glycoprotein (immediately before TM3)31 and $941F or $939F in TMll of mouse P-glycoprotein encoded by rndrl and mdr3, respectively, altered drug resistance profiles32and the ability to photolabel the mutant proteins.33'34In summary, these data suggest that different drugs have overlapping but distinct binding sites, and some of the drug-binding sites may exist outside the photolabeling regions. Login to comment

[hide] How does P-glycoprotein recognize its substrates? Semin Cancer Biol. 1997 Jun;8(3):151-9. Ueda K, Taguchi Y, Morishima M
How does P-glycoprotein recognize its substrates?
Semin Cancer Biol. 1997 Jun;8(3):151-9., [PMID:9441945]

Abstract [show]
We review how P-glycoprotein recognizes a wide variety of compounds and how it carries its substrates across membranes. Amino acid substitutions that affect the substrate specificity of P-glycoprotein have been found scattered throughout the molecule. In particular, some amino acid residues in the putative transmembrane domain (TM) 1 together with TM5-6 and TM11-12 may help to govern substrate specificity. The features that substrates for P-glycoprotein share are also discussed. The amphipathy of a substrate may decide whether the substrate can be intercalated into the lipid bilayer of the membrane. In addition, only certain molecular volumes and tertiary structures may make it possible for the substrate to fit into the substrate-binding site(s) of P-glycoprotein.

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80 associated with a Gly-to-Val substitution at position 185, in the predicted cytoplasmic loop between TM2 and TM3. Login to comment
98 The effects of amino acid substitutions on substrate specificity of P-glycoprotein can generally be classified into two groups.46 The first group is of mutations Gly185-to-Val, 51,52 Gly141-to-Val, and Gly187- to-Val,54 all in the first cytoplasmic loop; Gly288-to- Val54 in the second cytoplasmic loop; Phe335-to-Ala 39 and Val338-to-Ala 40 in TM6; Gly812-to-Val and Gly830-to-Val 54 in the fourth cytoplasmic loop. Login to comment

[hide] P-glycoprotein multidrug resistance and cancer. Biochim Biophys Acta. 1996 Oct 9;1288(2):F37-54. Bosch I, Croop J
P-glycoprotein multidrug resistance and cancer.
Biochim Biophys Acta. 1996 Oct 9;1288(2):F37-54., [PMID:8876632]

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92 A glycine to valine mutation at position 185, in the first cytoplasmic loop just proximal to transmembrane domain three was identified in a highly resistant cell line selected in colchicine [73]. Login to comment
126 This is indicated by increased colchicine stimulated ATPase activity in mutant P-glycoprotein with the glycine to valine mutation at position 185 [89]. Login to comment
91 A glycine to valine mutation at position 185, in the first cytoplasmic loop just proximal to transmembrane domain three was identified in a highly resistant cell line selected in colchicine [73]. Login to comment
125 This is indicated by increased colchicine stimulated ATPase activity in mutant P-glycoprotein with the glycine to valine mutation at position 185 [89]. Login to comment

[hide] The catalytic cycle of P-glycoprotein. FEBS Lett. 1995 Dec 27;377(3):285-9. Senior AE, al-Shawi MK, Urbatsch IL
The catalytic cycle of P-glycoprotein.
FEBS Lett. 1995 Dec 27;377(3):285-9., [PMID:8549739]

Abstract [show]
P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro. It contains two nucleotide-binding domains, and its structure places it in the 'ABC transporter' family. We review recent evidence that both nucleotide-sites bind and hydrolyse Mg-ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high-energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

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68 G141V, G185V and G830V caused relative changes in degree of activation of ATPase by vinblastine, verapamil and colchic- inc. Login to comment
69 Rao [35] studied the G185V mutation in human Pgp using the membrane fraction from St9 cells, and obtained similar results. Login to comment

[hide] ATP release via anion channels. Purinergic Signal. 2005 Dec;1(4):311-28. Epub 2005 Dec 3. Sabirov RZ, Okada Y
ATP release via anion channels.
Purinergic Signal. 2005 Dec;1(4):311-28. Epub 2005 Dec 3., [PMID:18404516]

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ATP serves not only as an energy source for all cell types but as an 'extracellular messenger' for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg(2+) and/or H(+) salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP(4-) in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.

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116 The whole-cell currents were inhibited by anti-MDR1 antibodies, but the ATP release was insensitive to the mutation of G185V, which alters the substrate selectivity of MDR1. Login to comment

[hide] On the origin of large flexibility of P-glycoprote... J Biol Chem. 2013 Jun 28;288(26):19211-20. doi: 10.1074/jbc.M113.450114. Epub 2013 May 8. Wen PC, Verhalen B, Wilkens S, Mchaourab HS, Tajkhorshid E
On the origin of large flexibility of P-glycoprotein in the inward-facing state.
J Biol Chem. 2013 Jun 28;288(26):19211-20. doi: 10.1074/jbc.M113.450114. Epub 2013 May 8., [PMID:23658020]

Abstract [show]
P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 A, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.

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136 Interestingly, a G185V mutation in human Pgp (equivalent to Gly-181 of mouse Pgp, a glycine that bends helix TM3, supplemental Fig. S6) is well studied and shown to improve the coupling between ATP hydrolysis and transport (72). Login to comment

[hide] Insights in the chemical components of liposomes r... Nanomedicine. 2014 Jan;10(1):77-87. doi: 10.1016/j.nano.2013.06.013. Epub 2013 Jul 10. Kopecka J, Salzano G, Campia I, Lusa S, Ghigo D, De Rosa G, Riganti C
Insights in the chemical components of liposomes responsible for P-glycoprotein inhibition.
Nanomedicine. 2014 Jan;10(1):77-87. doi: 10.1016/j.nano.2013.06.013. Epub 2013 Jul 10., [PMID:23850894]

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In this work we investigated how the surface charge and the presence of polyethylene glycol (PEG) on liposome carriers affect the delivery of the encapsulated doxorubicin in P-glycoprotein (Pgp)-overexpressing cells. We found that neutral net charge was critical to favour the liposome uptake and decrease the Vmax of doxorubicin efflux. PEG-coating was necessary to increase the Km of doxorubicin for Pgp. In particular the PEGylated phospholipid present in neutral liposomes, i.e. PEGylated distearoyl-phosphatidylethanolamine (DSPE-PEG), was a Pgp allosteric inhibitor, increased doxorubicin Km and inhibited Pgp ATPase activity. Site-directed mutagenesis experiments suggested that the domain centred around glycine 185 of Pgp was necessary for these inhibitory properties of DSPE-PEG and PEGylated neutral liposomes. We conclude that both surface charge and PEGylation must be considered to optimize the doxorubicin delivery within chemoresistant cells. DSPE-PEG-enriched particles may represent promising tools for therapeutic and diagnostic applications in tissues with high levels of Pgp. FROM THE CLINICAL EDITOR: These authors investigated how surface charge and PEGylation of liposome carriers affect the delivery of encapsulated doxorubicin to Pgp-overexpressing cells, concluding that both factors need to be considered in order to optimize doxorubicin delivery to chemoresistant cells.

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153 The G185V-mutated Pgp displays an altered binding of verapamil and colchicine13 and is not sensitive to the effects of liposomal doxorubicin.9 To verify whether the domain containing glycine 185 was the putative binding site of DSPE-PEG, we transfected MCF7 cells with the wild-type and the G185V-mutated Pgp expression vectors. Login to comment
155 Whereas DSPE-PEG strongly increased the doxorubicin retention in wild-type Pgp + MCF7-dx cells, it did not in G185V Pgp + MCF7-dx cells (Figure 6, B). Login to comment
156 G185V Pgp had the same affinity for verapamil (Figure 6, C) and a higher affinity for colchicine (Figure 6, D) than wild-type Pgp (Figure 5, A-B). Login to comment
157 No displacement of [3 H]-verapamil (Figure 6, C) or [3 H]-colchicine (Figure 6, D) was achieved by DSPE-PEG on G185V Pgp. Login to comment
158 In addition, DSPE-PEG did not reduce the ATPase activity of G185V Pgp, either after stimulation with verapamil (Figure 6, E) or in basal condition (Figure 6, F). Login to comment
159 Also the whole PEGylated neutral liposomal particles were devoid of any inhibitory effect on G185V Pgp (Figure 6, E-F). Login to comment
184 (A) MCF7 cells, MCF7-dx cells, transfected with the pCDNA3 vector containing wild-type (Pgp wt) or mutated (Pgp G185V) mdr1 sequence, were checked for the expression of Pgp/ABCB1 by Western blotting. Login to comment
190 (C-D) The binding assay of [3 H]-verapamil (C) and [3 H]-colchicine (D), in the presence of increasing concentrations of cold verapamil or colchicine, DSPE-PEG, or DSPE-PEG and doxorubicin, was performed on Pgp-rich membrane vesicles, isolated from G185V Pgp + MCF7-dx cells. Experiments were performed in quadruplicate and data are presented as mean &#b1; SD (n = 3). Login to comment
191 (E-F) G185V Pgp + MCF7-dx cells were lysed and the Pgp-rich membrane vesicles were isolated. Login to comment
196 The displacement assays showed that DSPE-PEG directly competed with verapamil and interfered with colchicine for the binding to Pgp; these data indicate that DSPE-PEG likely interacts with the verapamil/ colchicine binding site, a domain centered around glycine 185.13 Indeed in G185V Pgp + MCF7-dx cells DSPE-PEG completely lost its efficacy, in terms of doxorubicin retention, verapamil/ colchicine displacement and Pgp inhibition, suggesting that DSPE-PEG did not interact with this mutant Pgp. Login to comment