ABCMdb

ABCB1 p.Gly185Val

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PMID: 15670731 [PubMed] Ozvegy-Laczka C et al: "Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition."

No. Sentence Comment

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

PMID: 16259577 [PubMed] Sakurai A et al: "Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications."

No. Sentence Comment

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

PMID: 16399366 [PubMed] Ishikawa T et al: "High-speed screening of human ATP-binding cassette transporter function and genetic polymorphisms: new strategies in pharmacogenomics."

No. Sentence Comment

150 G185V is an acquired mutation. Login to comment

PMID: 17015488 [PubMed] Sarkadi B et al: "Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system."

No. Sentence Comment

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

PMID: 17323126 [PubMed] Huang Y et al: "Pharmacogenetics/genomics of membrane transporters in cancer chemotherapy."

No. Sentence Comment

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

PMID: 18855611 [PubMed] Zhou SF et al: "Clinical pharmacogenetics and potential application in personalized medicine."

No. Sentence Comment

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

PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."

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

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

No. Sentence Comment

47 Table 1 List of mutations in human, mouse, and hamster P-glycoproteins that affect substrate specificitya aa mutation Region Sourceb Reference H61R, F, K, M, W, Y TM 1 Human MDR1 149, 150 ABC20c G64R TM 1 Human MDR1 150 L65R TM 1 Human MDR1 150 aa78-97 EC 1 Human MDR1 151 Q128Hd TM 2 Mouse mdr3 152 R138H IC 1 Mouse mdr3 152 Q139H, R IC 1 Mouse mdr3 152 Q141V IC 1 Human MDR1 15319, Q145H IC 1 Mouse mdr3 152 E155G, K IC 1 Mouse mdr3 152 F159I IC 1 Mouse mdr3 152 D174G IC 1 Mouse mdr3 152 S176G, P IC 1 Mouse mdr3 152 K177I IC 1 Mouse mdr3 152 N179S IC 1 Mouse mdr3 152 N183S/G185V IC 1 Human MDR1 154 G183D IC 1 Mouse mdr3 152 G185V IC 1 Human MDR1 155-157 G187V IC 1 Human MDR1 153 A192T TM 3 Mouse mdr3 152 F204S EC 2 Mouse mdr3 152 W208G EC 2 Mouse mdr3 152 K209E EC 2 Mouse mdr3 152 L210I TM 4 Mouse mdr3 152 T211P TM 4 Mouse mdr3 152 I214T TM 4 Mouse mdr3 152 P223A TM 4 Human MDR1 158 G288V IC 2 Human MDR1 153 I299M, T319S, L322I, TM 5, EC3, Human MDR1 159 G324K, S351N IC 3 F335A TM 6 Human MDR1 19 F335 TM 6 Human MDR1 160 V338A TM 6 Human MDR1 161 G338A, A339P TM 6 Hamster PGY1 162, 163 A339P TM 6 Hamster PGY1 163 G341V TM 6 Human MDR1 161 K536R, Q N-NBD Human MDR1 164 ERGA → DKGT N-NBD Mouse mdr3 165 aa 522-525 T578C N-NBD Mouse mdr3 165 (Continued) G830V IC 4 Human MDR1 P866A TM 10 Human MDR1 158 F934A TM 11 Mouse mdr3 166 G935A TM 11 Mouse mdr3 166 I936A TM 11 Mouse mdr3 166 F938A TM 11 Mouse mdr3 166 S939A TM 11 Mouse mdr3 166 S939F TM 11 Mouse mdr3 167, 168 S941F TM 11 Mouse mdr1 167, 168 T941A TM 11 Mouse mdr3 166 Q942A TM 11 Mouse mdr3 166 A943G TM 11 Mouse mdr3 166 Y946A TM 11 Mouse mdr3 166 S948A TM 11 Mouse mdr3 166 Y949A TM 11 Mouse mdr3 166 C952A TM 11 Mouse mdr3 166 F953A TM 11 Mouse mdr3 166 F983A TM 12 Human MDR1 169 L975A, V981A, F983A TM 12 Human MDR1 169 M986A, V988A, Q990A, TM 12 Human MDR1 169 V991A V981A, F983A TM 12 Human MDR1 169 L975A, F983A TM 12 Human MDR1 169 L975A, V981A TM 12 Human MDR1 169 F978A TM 12 Human MDR1 19 a aa,amino acid; EC, extracellular loop; IC, intracellular loop; TM,transmembrane domain; NBD, nucleotide binding/utilization domain. Login to comment
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

PMID: 10388000 [PubMed] Fine RL et al: "P-Glycoprotein, Multidrug Resistance and Protein Kinase C."

No. Sentence Comment

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

PMID: 10400654 [PubMed] Vo QD et al: "Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport."

No. Sentence Comment

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

PMID: 10585407 [PubMed] Loo TW et al: "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."

No. Sentence Comment

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

PMID: 10694816 [PubMed] Licht T et al: "Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice."

No. Sentence Comment

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

PMID: 10698967 [PubMed] Lee CG et al: "Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter."

No. Sentence Comment

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

PMID: 10951193 [PubMed] Westerhoff HV et al: "Relating multidrug resistance phenotypes to the kinetic properties of their drug-efflux pumps."

No. Sentence Comment

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

PMID: 11455011 [PubMed] Song J et al: "Transmembrane domain (TM) 9 represents a novel site in P-glycoprotein that affects drug resistance and cooperates with TM6 to mediate [125I]iodoarylazidoprazosin labeling."

No. Sentence Comment

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

PMID: 12172212 [PubMed] Tang K et al: "Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations."

No. Sentence Comment

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

PMID: 12419946 [PubMed] Sakaeda T et al: "MDR1 genotype-related pharmacokinetics and pharmacodynamics."

No. Sentence Comment

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

PMID: 12831320 [PubMed] Sakaeda T et al: "Pharmacogenetics of MDR1 and its impact on the pharmacokinetics and pharmacodynamics of drugs."

No. Sentence Comment

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

PMID: 1347044 [PubMed] Sarkadi B et al: "Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase."

No. Sentence Comment

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

PMID: 14513768 [PubMed] Nakamura T et al: "[MDR1 genotypes related to pharmacokinetics and MDR1 expression]."

No. Sentence Comment

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

PMID: 14551217 [PubMed] Al-Shawi MK et al: "Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein."

No. Sentence Comment

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

PMID: 14576852 [PubMed] Ambudkar SV et al: "P-glycoprotein: from genomics to mechanism."

No. Sentence Comment

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

PMID: 15049699 [PubMed] Omote H et al: "Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation."

No. Sentence Comment

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

PMID: 15212152 [PubMed] Pauli-Magnus C et al: "Functional implications of genetic polymorphisms in the multidrug resistance gene MDR1 (ABCB1)."

No. Sentence Comment

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

PMID: 15256718 [PubMed] Ishikawa T et al: "Pharmacogenomics of drug transporters: a new approach to functional analysis of the genetic polymorphisms of ABCB1 (P-glycoprotein/MDR1)."

No. Sentence Comment

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

PMID: 15379652 [PubMed] Sakaeda T et al: "Pharmacogenetics of drug transporters and its impact on the pharmacotherapy."

No. Sentence Comment

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

PMID: 15499164 [PubMed] Ishikawa T et al: "Functional evaluation of ABCB1 (P-glycoprotein) polymorphisms: high-speed screening and structure-activity relationship analyses."

No. Sentence Comment

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

PMID: 16211086 [PubMed] Zaboikin M et al: "Gene therapy with drug resistance genes."

No. Sentence Comment

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

PMID: 16257832 [PubMed] Mizutani T et al: "New horizon of MDR1 (P-glycoprotein) study."

No. Sentence Comment

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

PMID: 16370938 [PubMed] Dey S et al: "Single nucleotide polymorphisms in human P-glycoprotein: its impact on drug delivery and disposition."

No. Sentence Comment

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

PMID: 16415525 [PubMed] Sakaeda T et al: "MDR1 genotype-related pharmacokinetics: fact or fiction?"

No. Sentence Comment

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

PMID: 16686372 [PubMed] Xia CQ et al: "Comparison of species differences of P-glycoproteins in beagle dog, rhesus monkey, and human using Atpase activity assays."

No. Sentence Comment

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

PMID: 16691488 [PubMed] Al-Shawi MK et al: "The remarkable transport mechanism of P-glycoprotein: a multidrug transporter."

No. Sentence Comment

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

PMID: 17352537 [PubMed] Jeong H et al: "Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay."

No. Sentence Comment

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

PMID: 17608770 [PubMed] Kimura Y et al: "Mechanism of multidrug recognition by MDR1/ABCB1."

No. Sentence Comment

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

PMID: 21389634 [PubMed] Ueda K et al: "ABC proteins protect the human body and maintain optimal health."

No. Sentence Comment

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

PMID: 21625253 [PubMed] Wolf SJ et al: "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."

No. Sentence Comment

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

PMID: 7568044 [PubMed] Volkman SK et al: "Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum."

No. Sentence Comment

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

PMID: 7605985 [PubMed] Sorrentino BP et al: "Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 7731035 [PubMed] Leveille-Webster CR et al: "The biology of the P-glycoproteins."

No. Sentence Comment

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

PMID: 7866993 [PubMed] Cardarelli CO et al: "Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters."

No. Sentence Comment

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

PMID: 7910372 [PubMed] Stein WD et al: "Kinetic evidence suggesting that the multidrug transporter differentially handles influx and efflux of its substrates."

No. Sentence Comment

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

PMID: 7912913 [PubMed] Gottesman MM et al: "Gene transfer of drug resistance genes. Implications for cancer therapy."

No. Sentence Comment

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

PMID: 7985758 [PubMed] Peel SA et al: "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."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 8820951 [PubMed] Fine RL et al: "P-glycoprotein, multidrug resistance and protein kinase C."

No. Sentence Comment

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

PMID: 8825488 [PubMed] Gottesman MM et al: "Genetic analysis of the multidrug transporter."

No. Sentence Comment

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

PMID: 9169612 [PubMed] Bakos E et al: "Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 9508799 [PubMed] Bond TD et al: "Protein kinase C phosphorylation disengages human and mouse-1a P-glycoproteins from influencing the rate of activation of swelling-activated chloride currents."

No. Sentence Comment

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

PMID: 9521654 [PubMed] Kwan T et al: "Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains."

No. Sentence Comment

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

PMID: 9636053 [PubMed] Beaudet L et al: "Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity."

No. Sentence Comment

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

PMID: 9753453 [PubMed] Hrycyna CA et al: "Structural flexibility of the linker region of human P-glycoprotein permits ATP hydrolysis and drug transport."

No. Sentence Comment

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

PMID: 9765504 [PubMed] Shoshani T et al: "Analysis of random recombination between human MDR1 and mouse mdr1a cDNA in a pHaMDR-dihydrofolate reductase bicistronic expression system."

No. Sentence Comment

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

PMID: 16565061 [PubMed] Omote H et al: "Interaction of transported drugs with the lipid bilayer and P-glycoprotein through a solvation exchange mechanism."

No. Sentence Comment

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

PMID: 9304397 [PubMed] Fardel O et al: "The P-glycoprotein multidrug transporter."

No. Sentence Comment

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

PMID: 19285158 [PubMed] Fung KL et al: "A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function."

No. Sentence Comment

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

PMID: 17512524 [PubMed] Paterson JK et al: "P-Glycoprotein is not present in mitochondrial membranes."

No. Sentence Comment

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

PMID: 16753135 [PubMed] Park SJ et al: "P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5."

No. Sentence Comment

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

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

No. Sentence Comment

58 Although mutation of only one of these residues (L975A, V981A and F983A) has no effect on the phenotype of the protein [20], double mutations either completely inhibit (V981A/F983A and L975A/V981A) or cause 50% inhibition (L975A/F983A) of Table 1. Login to comment
59 Published mutations in human and murine P-glycoprotein that alter drug transport in cells Location of mutation Mutation Refs Mutation Refs Mutation Refs Transmembrane helices H61A and others [14] I214L [60] L868W [59] G64R [15] P223A [65] I936A [21] L65R [15] S224P [60] F938A [21] Q139[H/P/R] [60] I306R [18] S939[A/C/T/Y/W/D/F] [21,22] G141V [17] F335A [16] T941A [21] G185V [61,62] V338A [66] Q942A [21] I186N [61] G338A [67,68] A943G [21] G187V [17] A339P [67,68] Y946A [21] G187E [60] G341A [66] S948A [21] A192T [60] S344[A/T/C/Y] [66] Y949A [21] F200L [60] N350I [19] C952A [21] F204S [60] P709A [65] F953A [21] R206L [60] G830V [17] L975A [20] W208G [60] I837L [23] F978A [16] K209E [60] N839I [23] V981A [20] L210I [60] I862F [19] F983A [20] T211P [60] L865F [19] F978A [16] V213A [60] P866A [65] N988D [59] Intracellular domain T169I [60] K177I [60] G288V [17] R170L [60] E180G [60] A931T [19] L171P [60] G181R [60] F934A [21] T172P [60] G183D [60] G935A [21] S176P [60] D184N [60] NBD D555N [63] K1076M [69] E1197Q [64] D558N [64] D1093N [64] D1203N [64] D592N [64] E1125Q [64] D1237N [64] E604Q [64] S1173A [70] E1249Q [64] Review TRENDS in Pharmacological Sciences Vol.27 No. Login to comment

PMID: 16504381 [PubMed] Kerb R et al: "Implications of genetic polymorphisms in drug transporters for pharmacotherapy."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 11284689 [PubMed] Ruth A et al: "Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein."

No. Sentence Comment

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

PMID: 10790226 [PubMed] Decleves X et al: "A new polymorphism (N21D) in the exon 2 of the human MDR1 gene encoding the P-glycoprotein."

No. Sentence Comment

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

PMID: 9774148 [PubMed] Spitaler M et al: "PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) P-glycoprotein by bryostatin 1."

No. Sentence Comment

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

PMID: 9711572 [PubMed] Germann UA et al: "Baculovirus-mediated expression of human multidrug resistance cDNA in insect cells and functional analysis of recombinant P-glycoprotein."

No. Sentence Comment

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

PMID: 9711563 [PubMed] Greenberger LM et al: "Identification of drug interaction sites in P-glycoprotein."

No. Sentence Comment

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

PMID: 9441945 [PubMed] Ueda K et al: "How does P-glycoprotein recognize its substrates?"

No. Sentence Comment

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

PMID: 8876632 [PubMed] Bosch I et al: "P-glycoprotein multidrug resistance and cancer."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 18404516 [PubMed] Sabirov RZ et al: "ATP release via anion channels."

No. Sentence Comment

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

PMID: 23658020 [PubMed] Wen PC et al: "On the origin of large flexibility of P-glycoprotein in the inward-facing state."

No. Sentence Comment

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

PMID: 23850894 [PubMed] Kopecka J et al: "Insights in the chemical components of liposomes responsible for P-glycoprotein inhibition."

No. Sentence Comment

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