ABCMdb

ABCB1 p.Phe983Ala

Predicted by SNAP2:	A: D (75%), C: D (66%), D: D (91%), E: D (91%), G: D (91%), H: D (85%), I: D (63%), K: D (91%), L: D (63%), M: D (59%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (75%), T: D (63%), V: D (75%), W: D (85%), Y: N (57%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: 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: N,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[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.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] A single amino acid residue contributes to distinc... Biochemistry. 1999 May 18;38(20):6630-9. Dey S, Hafkemeyer P, Pastan I, Gottesman MM
A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.
Biochemistry. 1999 May 18;38(20):6630-9., 1999-05-18 [PMID:10350482]

Abstract [show]
Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
37 In this study we demonstrate that substitution of a single phenylalanine residue with alanine at position 983 in TM 12 of Pgp affects inhibition of Pgp-mediated drug transport by both cis(Z)- and trans(E)-flupentixol, and significantly alters their stereospecific effect on ATP hydrolysis and substrate recognition by Pgp, suggesting a common site of functional interaction for both isomers of flupentixol. Login to comment
126 RESULTS Substitution of Phenylalanine Residue 983 with Alanine SelectiVely Affects Inhibition of Drug Transport by both cis(Z)- and trans(E)-Flupentixol. Login to comment
127 In a recent study, seven amino acid residues, L975, V981, F983, M986, V988, Q990, and V991, in the putative TM 12 of human Pgp were substituted individually by alanine (L975A, V981A, F983A, M986A, V988A, Q990A, and V991A). Login to comment
133 Consistent with the previous report, all the mutant Pgp`s (L975A, V981A, F983A, M986A, V988A, Q990A, and V991A) were expressed on the cell surface at a comparable level to that of wild-type (WT) Pgp (Figure 1A, upper panel). Login to comment
141 Human osteosarcoma (HOS) cells infected with vTF7-3 were transfected with either pTM1 (control) (s), pTM1-MDR1 (wild type) (-), pTM1-MDR1-L975A (L975A) (‚‚‚), pTM1-MDR1-V981A (V981A) (- - -), pTM1-MDR1-F983A (F983A) (- -), pTM1-MDR1-M986A (M986A) (thick dashes), pTM1-MDR1-V988A (V988A) (-‚‚-), pTM1-MDR1-Q990A (Q990A) (-‚-), or pTM1-MDR1-V991A (V991A) (-‚‚‚-) plasmid DNA. Login to comment
143 (A, upper panel) Cells were subjected to FACS analysis after staining with human Pgp external epitope-specific monoclonal antibody MRK-16 (14), as described under Experimental Procedures. (A, lower panel) Total cell lysates were prepared from each cell type, and immunoblot analysis with Pgp-specific monoclonal antibody C219 was performed as described under Experimental Procedures. (B) Similar to section A; cells were infected with vTF7-3, and transfected with either pTM1 (control) (s), pTM1-MDR1 (wild type) (-), pTM1-MDR1-L975A (L975A) (‚‚‚), pTM1-MDR1-V981A (V981A) (- - -), pTM1-MDR1-F983A (F983A) (- -), pTM1-MDR1-M986A (M986A) (thick dashes) pTM1-MDR1-V988A (V988A) (-‚‚-), pTM1-MDR1-Q990A (Q990A) (-‚-), or pTM1-MDR1-V991A (V991A) (-‚‚‚-) plasmid DNAs. Login to comment
157 However, the steady-state accumulation of the verapamil derivative in cells expressing F983A was minimally altered in the presence of 10 µM cis(Z)- or 5 µM trans(E)-flupentixol (Figure 2A,B), suggesting an impaired ability of this mutant to respond to inhibition of drug transport by both isomers of flupentixol. Login to comment
160 Since substitution of F983 had the most profound effect on the inhibitory potential of flupentixol, the mutant F983A was our obvious choice for further characterization. Login to comment
161 Stimulation of ATP Hydrolysis by cis(Z)-Flupentixol Is Considerably Altered in F983A. Login to comment
163 Since drug transport by F983A was considerably less sensitive to inhibition by cis(Z)- flupentixol, we studied the ability of cis(Z)-flupentixol to modulate the rate of ATP hydrolysis by this mutant. Login to comment
165 Steady-state accumulation of Bodipy FL-verapamil was measured by FACS in the presence (- - -) and absence (s) of 10 µM cis(Z)-flupentixol (A) or 5 µM trans(E)-flupentixol (B) in HOS cells infected with vTF7-3 and transfected with either pTM1 (pTM1), pTM1-MDR1 (WT), pTM1-MDR1-L975A (L975A), pTM1-MDR1-V981A (V981A), or pTM1-MDR1-F983A (F983A). Login to comment
166 For details, see Experimental Procedures. the wild-type Pgp or F983A, and Pgp-ATPase activity was measured in the presence of varying concentrations of either verapamil or cis(Z)-flupentixol. Login to comment
167 Consistent with their ability to transport Bodipy FL-verapamil, ATP hydrolysis by both wild-type Pgp and F983A in isolated membranes was stimulated by verapamil in a concentration-dependent manner (Figure 3A). Login to comment
168 Although the rate of ATP hydrolysis by F983A in the absence of verapamil was about 70% of that of the wild-type Pgp, the verapamil-stimulated activity was about the same in both Pgp`s. Login to comment
169 A maximum rate of ATP hydrolysis between 25 and 28 nmol mg-1 min-1 was achieved by both wild-type and F983A at a verapamil concentration of 25 µM. Login to comment
173 In contrast, stimulation by cis(Z)-flupentixol of ATP hydrolysis by F983A was significantly impaired, with a maximum of only 12-14 nmol mg-1 min-1 of ATP hydrolyzed at cis(Z)- flupentixol concentrations of 50 µM (Figure 3B). Login to comment
174 Due to a low basal rate of ATP hydrolysis by F983A, the fold stimulation by cis(Z)-flupentixol (2-fold) was about the same for both wild-type Pgp and F983A; however, in F983A this was achieved at a considerably higher concentration (Figure 3B). Login to comment
175 The Ability of trans(E)-Flupentixol To Inhibit Verapamil-Stimulated ATP Hydrolysis Is Markedly Reduced in F983A. Login to comment
176 Since Bodipy FL-verapamil transport by F983A was less sensitive to inhibition by trans(E)-flupentixol, we studied its effect on verapamil-stimulated ATP hydrolysis by F983A. Isolated cell membranes from HeLa cells, expressing either wild-type Pgp or F983A, were preincubated with varying concentrations of trans(E)-flupentixol prior to the measurement of vanadate-sensitive ATP hydrolysis in the presence of 25 µM verapamil. Login to comment
177 Consistent with the previous experiment, in the absence of trans(E)-flupentixol the rate of verapamil-stimulated ATP hydrolysis by the wild-type and the mutant (F983A) was around 26 nmol mg-1 min-1. Login to comment
179 In contrast, the ability of trans(E)- flupentixol to inhibit verapamil-stimulated ATP hydrolysis by F983A was significantly reduced (Figure 4A). Login to comment
181 This indicated that residue F983A also plays a crucial role in the inhibition of ATP hydrolysis by trans(E)-flupentixol. Login to comment
182 To determine whether the loss of sensitivity to trans(E)- flupentixol in F983A reflected a general defect of the mutant FIGURE 3: Effect of verapamil and cis(Z)-flupentixol on ATP hydrolysis by the wild-type and F983A Pgp`s. Login to comment
183 Membranes were isolated from HeLa cells infected with vTF7-3 and transfected with either pTM1, pTM1-MDR1 (WT), or pTM1-MDR1-F983A (F983A) plasmid DNAs. Login to comment
184 HeLa cell membranes (20 µg of protein) containing wild-type Pgp (0) or F983A (b) were incubated either with the indicated concentrations of verapamil (A) or with cis(Z)-flupentixol (B) prior to measuring the rate of ATP hydrolysis. Login to comment
188 FIGURE 4: Effect of trans(E)-flupentixol and cyclosporin A on verapamil-stimulated ATP hydrolysis by the wild-type and F983A Pgp`s. Login to comment
189 Vanadate-sensitive ATP hydrolysis by wild-type Pgp (0) and F983A (b) (20 µg of membrane protein) in HeLa cell membranes was measured as described in Figure 3, in the presence of 5 mM ATP, 10 mM MgCl2, 25 µM verapamil, and the indicated concentrations of either trans(E)-flupentixol (A) or cyclosporin A (B). Login to comment
192 in its ability to respond to inhibitors of Pgp-ATPase activity, we studied the effect of cyclosporin A on verapamil-stimulated ATP hydrolysis by wild-type Pgp and F983A. Login to comment
193 Cyclosporin A inhibited verapamil-stimulated ATPase activity with equal potency in both wild type and F983A (Figure 4B). Login to comment
194 The concentrations for half-maximal inhibition were about 0.4 µM for both wild type and F983A. Login to comment
196 Stimulation by cis(Z)-Flupentixol of Photoaffinity Labeling with the Substrate Analogue [125 I]IAAP Is Considerably Affected in F983A. Login to comment
198 Since cis(Z)-flupentixol was unable to reverse drug transport by F983A, we examined the effect of cis(Z)-flupentixol on [125 I]IAAP labeling of F983A. Isolated membranes from HeLa cells expressing wild-type Pgp or F983A were photoaffinity labeled with 4 nM [125 I]IAAP in the presence of varying (0-100 µM) concentrations of cis(Z)-flupentixol. Login to comment
200 In contrast, stimulation of [125I]IAAP labeling was significantly reduced in F983A (Figure 5B) with a maximal stimulation of only 2-fold (Figure 5C). Login to comment
201 However, stimulation reached its maximum at the same concentration of cis(Z)- flupentixol (10 µM) for both wild-type Pgp and F983A (Figure 5C). Login to comment
205 Photoaffinity Labeling of F983A with [125I]IAAP Is RelatiVely Resistant to Inhibition by trans(E)-Flupentixol. Login to comment
209 Inhibition of [125I]IAAP labeling by trans(E)-flupentixol was considerably altered in F983A, in which case the concentration of trans(E)-flupentixol required for half-maximal inhibition was about 4-fold higher (about 40 µM) than that for the wild-type Pgp. Login to comment
210 Photoaffinity labeling of the wild-type Pgp and F983A with [125 I]IAAP was also carried out in the presence of varying concentrations of cyclosporin A. Login to comment
212 Concentrations of cyclosporin A required for half-maximal and maximal inhibitions were around 0.035 µM for both wild-type Pgp and F983A. Login to comment
213 This excluded the possibility of a general loss of sensitivity to inhibitors of substrate recognition in F983A. Login to comment
218 In this study we demonstrate that a single substitution of alanine for phenylalanine at position 983 in the putative TM 12 of Pgp markedly alters the ability of both cis and trans isomers of flupentixol to modulate ATPase activity and [125 I]IAAP labeling of Pgp, and also FIGURE 5: Effect of cis(Z)-flupentixol on [125I]IAAP labeling of the wild-type Pgp and F983A. Login to comment
219 Membranes were isolated from HeLa cells infected with vTF7-3 and transfected with either (A) pTM1-MDR1 (WT) or (B) pTM1-MDR1-F983A (F983A) plasmid DNAs. Login to comment
221 (C) Quantification of radioactivity associated with the wild-type (0) and F983A (b) Pgp`s bands was done by a STORM 860 phosphorimaging system as described under Experimental Procedures. Login to comment
239 Although drug transport was inhibited by both cis(Z)- and trans(E)-flupentixol in V981A and M986A (data not shown), inhibition was not complete, FIGURE 6: Effect of trans(E)-flupentixol and cyclosporin A on [125I]IAAP labeling of the wild-type Pgp and F983A. Login to comment
240 HeLa membranes (25 µg of protein) containing either wild-type Pgp (top) or F983A (bottom) were preincubated at room temperature in the presence of the indicated concentrations of either trans(E)-flupentixol (A) or cyclosporin A (B) prior to incubation with 4 nM [125I]IAAP, for 10 min, as described under Experimental Procedures. Following incubation, membranes were photo-cross-linked, and 10 µg of membrane proteins was analyzed by SDS-PAGE and subjected to autoradiography. Login to comment
243 Radioactivity associated with the wild-type (0) and F983A (b) Pgp bands was expressed as percent control using the same method as in Figure 5. Login to comment
246 It is to be noted that in all three mutants (F983A, V981A, and M986A) the inhibitory effect of both isomers (cis and trans) of flupentixol was affected. Login to comment
255 In F983A the inhibitory effect of cis(Z)- and trans(E)-flupentixol on Pgp-mediated drug transport was considerably reduced without much alteration in the ability of the protein to transport substrates (Figures 1B and 2A,B). Login to comment
257 Therefore, this dissociation of susceptibility to inhibition by flupentixol from drug transport in F983A strongly indicates the allosteric nature of the flupentixol interaction site with Pgp. Login to comment
258 Consistent with this, in F983A the modulatory potency of cis(Z)- and trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered with only a minimal effect on the basal level of ATPase activity and [125 I]IAAP labeling. Login to comment
266 Consistent with the idea of a single site of interaction, both cis(Z)-flupentixol-mediated stimulation (Figure 5C) and trans(E)-flupentixol-mediated inhibition (Figure 6A) of [125I]IAAP labeling were also significantly reduced in F983A. Login to comment
269 In F983A, stimulation of [125I]IAAP labeling was significantly altered with a maximal stimulation of only 2-fold compared to 9-fold stimulation in the wild-type Pgp (Figure 5A-C). Login to comment
270 However, the decrease in stimulation of [125I]IAAP labeling was evident at the same concentration of cis(Z)-flupentixol for both F983A and wild-type Pgp. Login to comment

[hide] Chemoprotection of hematopoietic cells by a mutant... Hum Gene Ther. 2000 Mar 1;11(4):555-65. Hafkemeyer P, Licht T, Pastan I, Gottesman MM
Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers.
Hum Gene Ther. 2000 Mar 1;11(4):555-65., 2000-03-01 [PMID:10724034]

Abstract [show]
Cancers are frequently chemoresistant because of overexpression of P-glycoprotein. Two different approaches to improve cancer treatment are currently being investigated in clinical trials: inhibition of P-glycoprotein function by reversing agents, and alleviation of leukocytopenia by MDR1 gene transfer to normal bone marrow of patients. We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein. Transfer of either mutant or wild-type MDR1 to K562 erythroleukemia cells or primary murine bone marrow resulted in reduced accumulation of daunomycin and vinblastine because of increased drug efflux.trans-(E)-Flupentixol at concentrations up to 10 microM failed to reverse drug efflux mediated by the product of the mutant MDR1 while wild-type P-glycoprotein was inhibited. In the presence of 2 microM trans-(E)-flupentixol chemoresistance to daunomycin was circumvented only in K562 cells transduced with wild-type, but not with mutant, MDR1. Moreover, drug resistance of KB-8-5 epidermoid cancer cells, which express the wild-type MDR1 gene at levels comparable to clinical specimens from multidrug-resistant cancers, was fully overcome in the presence of trans-(E)-flupentixol. Vectors expressing mutant P-glycoprotein may help improve chemotherapy by allowing safe dose intensification under conditions in which multidrug-resistant cancers are rendered drug sensitive by reversing agents.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
3 We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein. Login to comment
42 We have introduced a point mutation that leads to a substitution of phenylalanine by alanine in position 983. Login to comment
45 In the present work, we have constructed retroviral vectors containing this mutant MDR1-F983A cDNA. Login to comment
46 With the exception of the inserted point mutation, pHaMA-F983A is identical to a previously described vector, pHaMA (Pastan et al., 1988; Metz et al., 1995), with long-term inal repeats (LTRs) from Harvey sarcoma virus. Login to comment
48 The MDR1-F983A vectors were characterized in KB-3-1 epidermoid cancer cells, and hematopoietic cells from the K562 erythroleukemia line or primary mouse bone marrow. Login to comment
51 To construct vector pHaMA-F983A, the NsiI-XhoI fragment of previously described plasmid pTM1MDR1 (Hafkemeyer et al., 1998) containing the F983A-mutant fragment was cloned into the corresponding sites of pHaMA. Login to comment
53 The vectors LZRS- pBMN-MDR1 and LZRS-pBMN-MDR1-F983A, respectively, were obtained after excision of lacZ by EcoRI digest of LZRS- pBMN-lacZ, and blunt ending of the vector backbone with T4 polymerase and Klenow enzyme. Login to comment
54 pHaMA and pHaMA-F983A were then blunt ended after SacII-XhoI digestion. Login to comment
55 The thereby obtained MDR1 and MDR1-F983A fragments, respectively, were ligated into the EcoRI-digested and blunt-ended backbone of LZRS-pBMN-lacZ to yield LZRS-pBMN-MDR1 and LZRS- pBMN-MDR1-F983A. Login to comment
61 Cell transfections and transductions KB-3-1 cells were transfected by calcium phosphate coprecipitation with 10 m g of plasmid DNA (pHaMA or pHaMA-F983A) per 1 3 105 cells as described (Germann et al., 1989). Login to comment
64 The same method was used to generate ecotropic retroviral producer cells from GP1 E86 packaging cells (ATCC) with plasmid pHaMA-F983A. Login to comment
67 Wild-type and F983A-mutant MDR1 producer lines were obtained by transient transfection with 10 m g of either LZRS- pBMN-MDR1 or LZRS-pBMN-MDR1-F983A, respectively. Login to comment
81 To assess the chemosensitivity of K562 parental cells, and of K562 cells transduced with either the LZRS-pBMN-MDR1 or LZRS- pBMN-MDR1-F983A construct, 200 cells/ml were mixed with daunomycin (Calbiochem , San Diego, CA) and trans-(E)-flupentixol (RBI, Natick, MA) at various concentrations in MethoCult 4330 medium containing 0.8% methylcellulose (Stem Cell Technologies, Vancouver, BC, Canada). Login to comment
85 RESULTS Reversal of the functional activity of wild-type P-gp in KB cancer cells We first analyzed the reversing potential of trans-(E)-flupentixol in multidrug-resist ant cells expressing either the wild-type or the F983A-mutant P-gp. Login to comment
90 We also analyzed KB-3-1 cells stably transfected with MDR1-containing plasmids, pHaMA or pHaMA-F983A, and selected with vincristine; clones G2 (wild-type) and 7A (mutant) expressed similar levels of P-gp (Fig. 1a). Login to comment
97 At 2 m M trans-(E)-flupentixol, 7A cells expressing F983A-mutant P-gp accumulated three- to fivefold less [3H]daunomycin than G2 cells (Fig. 1b). Login to comment
104 (a)DetectionofP-gponthecellsurfaceofparentalKB-3-1cellsandmul- tidrug-resistantKB-8-5cells,andKB-3-1cellsstablytransfectedwithpHaMA(wild-typeMDR1,cloneG2)orpHaMA-F983A(mutantMDR1-F983A,clone7A).Cellswerestained with5mgofmonoclonalantibodyMRK16(specificforhumanP-gp)conjugatedtoFITC,oranequalconcentrationofanisotype-identifiedcontrolantibody.Afterwashing,cells wereanalyzedbyflowcytometry. Login to comment
106 A, another P-gp inhibitor, was comparable in all investigated KB cells, regardless of the presence of the F983A point mutation (data not shown). Login to comment
107 Development of retroviruses containing mutant MDR1-F983A for transduction of hematopoietic cells Ecotropic retroviral producer cells were generated by stable transfection of GP1 E86 packaging cells with plasmid pHaMA-F983A (Fig. 4a). Login to comment
112 Transduction levels for the EBV-retroviral vectors containing mutant or wild-type MDR1 cDNAs were 80% (LZRS-pBMN-MDR1-F983A) and 68% (LZRS-pBMN-MDR1) over background. Login to comment
114 EBV-hybrid vectors with titers of 5-7 3 105 CFU/ml were generated by transient transfection of amphotropic 293T (Phoenix) cells with plasmid LZRS-pBMN-MDR1 (wild-type) or LZRS-pBMN-MDR1-F983A (mutant). Login to comment
118 Modulation of drug transport by trans-(E)-flupentixol in hematopoietic cells transduced with F983A-mutant or wild-type MDR1 vectors Transfer of either the F983A-mutant or the wild-type MDR1 cDNA to K562 cells caused them to accumulate far less [3H]daunomycin and [3H]vinblastine than the non-MDR1 control (Fig. 5b). Login to comment
123 Parental K562 cells formed only a few colonies in the presence of daunomycin (10 ng/ml) but transfer of the wild-type or the F983A-mutant MDR1 cDNA rendered them resistant to this concentration. Login to comment
129 retroviruses containing F983A-mutant MDR1 but reduced the number of resistant colonies from cells expressing wild-type P-gp. Login to comment
130 Moreover, daunomycin at 30 ng/ml, combined with 2 m M trans-(E)-flupentixol, killed all cells transduced with wild-type MDR1 while 10% of the respective cells transduced with the F983A-mutant vector were protected. Login to comment
131 Thus, K562 cells expressing wild-type MDR1 were slightly more chemoresistant than those transduced with the mutant vector, but this resistance was not reversible in cells expressing F983A-MDR1. Login to comment
138 Drug extrusion from K562 cells transduced with the mutant MDR1-F983A vector was not overcome by trans-(E)-flupentixol at concentrations that reverse P-gp function in KB-8-5 cells. Login to comment
139 Correspondingly , daunomycin resistance was reversible only in K562 cells that had been transduced with wild-type but not with the F983A-mutant MDR1 cDNA. Login to comment
141 Although P-gp expression levels and the total amount of drug efflux were lower than in the K562 cell population because no selection was applied, trans-(E)-flupentixol failed to reverse drug efflux from BMCs transduced with the mutant MDR1-F983A vector. Login to comment
153 (a)MapsoftheproviralstructuresofpHaMAandpHaMA-F983Acon- tainingfull-lengthwild-typeormutantMDR1cDNAs,respectively.TheEBV-derivedretroviralhybridvectorsareLZRS-pBMN-MDR1,containingawild-typeMDR1cDNA,and LZRS-pBMN-MDR1-F983A,containingtherespectivemutantMDR1-F983AcDNA. Login to comment
155 (c)ExpressionofP-gpinK562cellstransduced witheitherLZRS-pBMN-MDR1-F983A,LZRS-pBMN-MDR1,orLZRS-pBMN-lacZ(negativecontrol).Aftercoculturewithretroviralproducersfor48hr,transducedK562cells wereselectedwithvincristine(100ng/ml)for18days.Cellswerestainedwith5mgofantibodyMRK16conjugatedtoFITCoranFITC-labeledcontrolantibody.After30min, cellswerewashed,andfluorescenceintensitieswereanalyzedbyflowcytometry.Nonviablecellswereexcludedbypropidiumiodidecounterstaining. Login to comment
162 The advantage of amphotropic, EBV-based hybrid vectors, LZRS-pBMN-MDR1 and LZRS-pBMN-MDR1-F983A, is rapid production of retroviruses after transient transfection (Nolan and Shatzman, 1998). Login to comment
169 (a) Murine BMCs were transduced in cocultures with vectors GP1 E86 MDR1-F983A (d ), GP1 E86 MDR1/A (wild-type) (r ), or packaging cells (h ) that did not confer drug resistance. Login to comment
171 (b) K562 cells, transduced and selected as described in the legend to Fig. 4c, were analyzed under identical conditions (h , negative control; r , LZRS-pBMN-MDR1; d , LZRS-pBMN-MDR1-F983A). Login to comment
183 High concentrations of trans-(E)-flupentixol (20 m M and higher) are not useful because they reduce chemoprotection of hematopoietic cells mediated by the F983A-MDR1 transgene (Fig. 5). Login to comment
185 The mutation F983A in the putative transmembrane domain TM 12 had little effect on drug binding to P-gp but almost abolished cis-(Z)-flupentixol-stim ulated ATPase activity of the transporter (Dey et al., 1999). 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.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Allosteric modulation of human P-glycoprotein. Inh... J Biol Chem. 2003 May 16;278(20):18132-9. Epub 2003 Mar 17. Maki N, Hafkemeyer P, Dey S
Allosteric modulation of human P-glycoprotein. Inhibition of transport by preventing substrate translocation and dissociation.
J Biol Chem. 2003 May 16;278(20):18132-9. Epub 2003 Mar 17., 2003-05-16 [PMID:12642584]

Abstract [show]
The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z)-flupentixol, a thioxanthene derivative, occurs through an allosteric mechanism. Unlike competitive inhibitors, such as cyclosporin A and verapamil, cis-(Z)-flupentixol does not interfere with substrate ([(125)I]iodoarylazidoprazosin) recognition by Pgp, instead it prevents substrate translocation and dissociation, resulting in a stable but reversible Pgp-substrate complex. cis-(Z)-Flupentixol-induced complex formation requires involvement of the Pgp substrate site, because agents that either physically compete (cyclosporin A) for or indirectly occlude (vanadate) the substrate-binding site prevent formation of the complex. Allosteric modulation by cis-(Z)-flupentixol involves a conformational change in Pgp detectable by monoclonal antibody UIC2 binding to a conformation-sensitive external epitope of Pgp. The conformational change observed is distinct from that induced by Pgp substrates or competitive inhibitors. A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Taken together, our results describe the molecular mechanism by which the Pgp modulator cis-(Z)-flupentixol allosterically inhibits drug transport.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
6 A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Login to comment
45 In addition, two cell lines NIHMDR1-WT and NIHMDR1-F983A were generated. Login to comment
46 NIHMDR1-WT and NIHMDR1-F983A were created by stepwise selection of NIH3T3 cells transfected with pHaMDR1 and pHaMDR1-F983A plasmids, respectively. Login to comment
48 To construct the vector pHaMDR1-F983A, the NsiI-XhoI fragment of the previously described plasmid pTM1MDR1 containing the F983A mutation (25) was cloned into the corresponding sites of pHaMDR1. Login to comment
49 The region corresponding to the NdeI-PstI fragment within pHaMDR1-F983A including the insertion points and the flanking regions was sequenced in its entirety in both directions by automated sequencing (PRISM Ready Reaction DyeDeoxy Terminator Sequencing Kit, PerkinElmer Life Sciences). Login to comment
50 The plasmids pHaMDR1 and pHaMDR1-F983A were calcium phosphate-transfected into NIH3T3 cells and selected with vincristine. Login to comment
52 A stepwise selection was carried out with increasing concentrations of vincristine to generate NIHMDR1-WT and NIHMDR1-F983A cell lines that were able to grow in the presence of 1 ␮M vincristine. Login to comment
164 A Single Amino Acid Substitution That Alters the Inhibitory Potential of cis-(Z)-Flupentixol Also Affects cis-(Z)-Flupentixol-induced Formation of Pgp-[125 I]IAAP Complex-To determine the functional significance of the Pgp-[125 I]IAAP complex formation and the elevated level of [125 I]IAAP associated with Pgp-expressing cells in the presence of cis-(Z)-flupentixol, both phenomena were tested in the Pgp mutant F983A, which is largely insensitive to modulation by cis-(Z)-flupentixol (22). Login to comment
165 The intracellular accumulation of [125 I]IAAP in cells expressing either the wild-type Pgp (NIHMDR1-WT) or the mutant F983A Pgp (NIHMDR1-F983A) was considerably lower than that of the control NIH3T3 cells, suggesting efficient transport of [125 I]IAAP by both the wild-type and F983A mutant Pgp. Login to comment
166 However, the elevated level of cellular [125 I]IAAP association, in the presence of 5 ␮M cis-(Z)-flupentixol, was selectively abrogated in NIHMDR1-F983A (Fig. 4A). Login to comment
167 Photocross-linking for 5 min at the end of the uptake assay showed no increase in interaction (photocross-linking) between [125 I]IAAP and F983A by cis-(Z)-flupentixol, although under the same conditions increased complex formation was observed between wild-type Pgp and [125 I]IAAP in NIHMDR1-WT cells (Fig. 4B, upper panel). Login to comment
168 In the absence of cis-(Z)-flupentixol, there was no significant difference in the interaction with [125 I]IAAP between the wild-type and the F983A mutant. Login to comment
169 Cyclosporin A (5 ␮M), a competitive inhibitor of Pgp, inhibited both transport and [125 I]IAAP binding by wild-type and F983A with similar efficiency (Fig. 4B, upper panel). Login to comment
170 Similar results were obtained from experiments with membranes isolated from NIHMDR1-WT and NIHMDR1-F983A cells (Fig. 4B, lower panel). Login to comment
176 To investigate the mechanistic significance of this conformational change, UIC2 reactivity was studied in cells expressing the Pgp mutant F983A, which has impaired sensitivity to inhibition by cis-(Z)- flupentixol (Fig. 5B). Login to comment
177 cis-(Z)-Flupentixol was unable to induce any negative effect on UIC2 binding to NIHMDR1-F983A cells FIG. 3. Login to comment
190 Because increased or decreased UIC2 reactivity induced by cyclosporin A or vanadate, respectively, was unaffected in NIHMDR1-F983A (Fig. 5B), compared with that observed in NIHMDR1-WT cells, the possibility of a nonspecific effect could be ruled out. Login to comment
204 NIH3T3, NIHMDR1-WT (wild-type Pgp), and NIHMDR1-F983A (mutant F983A) cells were incubated with 1.5 nM [125 I]IAAP in the presence and absence of 5 ␮M cis-(Z)-flupentixol for 60 min at 37 °C. Login to comment
207 Data points represent average value of three identical experiments. B, interaction of [125 I]IAAP with wild-type (WT) Pgp and F983A, in intact cells (upper panel) and in isolated membranes (lower panel). Login to comment
218 B, UIC2 reactivity of Pgp mutant F983A. Login to comment
219 UIC2 binding to NIHMDR1-WT and NIHMDR1-F983A cells was carried out as mentioned above either in the presence of 1 mM sodium orthovanadate (Vi), 5 ␮M cis-(Z)-flupentixol (Cis(Z)), 5 ␮M cyclosporin A (CsA), or in the absence of any modulators. Login to comment
260 Interestingly, formation of the stable Pgp-[125 I]IAAP complex induced by cis-(Z)-flupentixol was remarkably affected in the Pgp mutant F983A (Fig. 4B), drug transport function by which is not inhibited by cis-(Z)-flupentixol (Fig. 4A). Login to comment
262 Because the mutation F983A did not affect [125 I]IAAP transport or the ability of cyclosporin A to block the substrate site (Fig. 4B) and inhibit transport (Fig. 4A), any global effect of the mutation can be ruled out. Login to comment
270 No such conformational change was induced by cis-(Z)- flupentixol in the Pgp mutant F983A, whereas the ability of the competitive inhibitor cyclosporin A to induce its characteristic change in conformation remained unaltered (Fig. 5B), underscoring the functional distinctness of the allosteric site. Login to comment

[hide] Photoaffinity labeling of P-glycoprotein. Mini Rev Med Chem. 2005 Feb;5(2):165-72. Peer M, Csaszar E, Vorlaufer E, Kopp S, Chiba P
Photoaffinity labeling of P-glycoprotein.
Mini Rev Med Chem. 2005 Feb;5(2):165-72., [PMID:15720286]

Abstract [show]
The aim of the present review is to summarize recent progress in identifying substrate binding domains of P-glycoprotein by photoaffinity labeling. Preferred substrate binding regions have been identified using a number of photoaffinity ligands, including anthracyclines, the quinazoline iodoarylazidoprazosine (IAAP), dihydropyridines, taxanes and propafenones. These studies allowed identification of protein regions, which are involved in ligand interaction.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
87 Cell lines expressing wt and mutant proteins were subjected to accumulation studies using Bodipy FL-verapamil in the presence of cis(Z)- or trans(E)- flupentixol. Both flupentixol isomers were shown to loose their inhibitory function in the F983A mutant. Login to comment
88 The large increase in labeling with [125I]IAAP in the presence of cis(Z)-flupentixol was lost in the F983A mutant. Login to comment
90 A general loss of sensitivity to inhibitors was excluded, because labeling of F983A with IAAP stayed sensitive to Cyclosporin A inhibition. Login to comment

[hide] P-glycoprotein--implications of metabolism of neop... Curr Cancer Drug Targets. 2005 Sep;5(6):457-68. Breier A, Barancik M, Sulova Z, Uhrik B
P-glycoprotein--implications of metabolism of neoplastic cells and cancer therapy.
Curr Cancer Drug Targets. 2005 Sep;5(6):457-68., [PMID:16178819]

Abstract [show]
Multidrug resistance (MDR) of neoplastic tissues is a major obstacle in cancer chemotherapy. The predominant cause of MDR is the overexpression and drug transport activity of P-glycoprotein (P-gp, a product of the MDR gene). P-gp is a member of the ATP binding cassette (ABC) transporters family, with broad substrate specificity for several substances including anticancer drugs, linear and cyclic peptides, inhibitors of HIV protease, and several other substances. The development of P-gp-mediated MDR is often associated with several changes in cell structure and metabolism of resistant cells. In the present review are discussed the relations between glucosylceramide synthase activity, Pregnane X receptor and development of P-gp mediated MDR phenotype. Attention is also focused on the changes in protein kinase systems (mitogen-activated protein kinases, protein kinase C, Akt kinase) that are associated with the development of MDR phenotype and to the possible role of these kinase cascades in modulation of P-gp expression and function. The overexpression of P-gp may be associated with changes in metabolism of sugars as well as energy production. Structural and ultrastructural characteristics of multidrug resistant cells expressing P-gp are typical for cells engaged in a metabolically demanding process of protein synthesis and transport. P-gp mediated MDR phenotype is often also associated with alterations in cytoskeletal elements, microtubule and mitochondria distribution, Golgi apparatus, chromatin texture, vacuoles and caveolae formation. The current review also aims at bringing some state-of-the-art information on interactions of P-glycoprotein with various substances. To capture and transport the numerous unrelated substances, P-gp should contain site(s) able to bind compounds with a molecular weight of several hundreds and comprising hydrophobic and/or base regions that are protonated under physiological conditions. Drug binding sites that are able to recognize substances with different chemical structures may have a complex architecture in which different parts are responsible for binding of different drugs. For P-gp substrates and inhibitors, a pharmacophore-based model has been described. The pharmacophores have to contain parts with hydrophobic and aromatic characteristics and functional groups that can act as hydrogen-bond donors and/or acceptors. Several drugs are known to be P-glycoprotein antagonizing agents. They represent a large group of structurally unrelated substances that can act via direct interaction with P-gp and inhibition of its transport activity, or via possible modulation of processes (such as phosphorylation) regulating P-gp transport activity. Effects of MDR reversal agents on the P-gp expression have also been reported. Function and expression of P-gp can be affected indirectly as well, e.g. through cyclooxygenase-2 or carbonic anhydrase-IX expression and effects.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
185 Several other authors described modulation of drug efflux capacity of mutant P-gp after site directed mutagenesis of an amino acid in the transmembrane domains, e.g. histidine 61 in domain 1 [134], single serines replacements by phenylalanines in transmembrane domain 11 [135], leucine 975, valine 981 and phenylalanine 983 by alanine in transmembrane domain 12 [136]. Login to comment

[hide] Modulator-induced interference in functional cross... Biochemistry. 2006 Feb 28;45(8):2739-51. Maki N, Moitra K, Silver C, Ghosh P, Chattopadhyay A, Dey S
Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein.
Biochemistry. 2006 Feb 28;45(8):2739-51., 2006-02-28 [PMID:16489767]

Abstract [show]
The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
7 In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Login to comment
24 1 Abbreviations: Pgp, P-glycoprotein; [125 I]IAAP, [125 I]iodoarylazidoprazosin; [R-32 P]-8N3ATP, [R-32 P]-8-azido-ATP; ATP, adenosine triphosphate; ADP, adenosine diphosphate; TM, transmembrane; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Cis(Z), cis-(Z)-flupentixol; CsA, cyclosporin A; Vi, vanadate; F983A, Pgp F983A mutant; EGTA, ethylene glycol bis( -aminoethyl)-N,N,N',N'- tetraacetic acid. Login to comment
58 The recombinant baculovirus containing the Pgp mutant F983A [BV-MDR1-(H6)-F983A] was constructed in the same way as BV-MDR1-(H6) (25) using recombinant plasmid pBac- PakMDR1-(H6)-F983A. Login to comment
59 The NdeI-XhoI fragment of pTM1-MDR1-(H6)-F983A (containing the codon for amino acid phenylalanine 983 changed to that of alanine) (47) was cloned into the wild-type MDR1 coding region of plasmid pBacPak9-MDR1-(H6) to generate pBacPakMDR1-(H6)- F983A. Login to comment
60 The recombinant baculovirus BV-MDR1(H6)-F983A was generated according to ref 25. Login to comment
61 For expression of the wild-type Pgp and the mutant F983A, cells were propagated to 80% confluency at 27 °C in the monolayer and infected with the recombinant baculoviruses with a multiplicity of infection of 10 and harvested after 72 h of infection. Login to comment
159 When dissociation of bound [125 I]IAAP in response to vanadate trapping was studied in the Pgp F983A mutant that is impaired in modulation by cis-(Z)-flupentixol (38, 39), no such delay in dissociation was observed even in the presence of 25 µM cis-(Z)-flupentixol (k ) 1.86 ( 0.25/ min, t1/2 ) 0.37 min) (R2 ) 0.99) (Figure 2C,D), suggesting that the inhibitory effect on substrate dissociation is dependent on a functional interaction of cis-(Z)-flupentixol with Pgp. Login to comment
171 Consistent with that, stimulation of ATP hydrolysis by 50 µM prazosin in the Pgp F983A mutant was minimally affected by cis-(Z)- flupentixol (25 µM) (Figure 3B, right), further supporting that the inhibitory effect on substrate-stimulated ATP hydrolysis is mediated through a specific interaction of cis- (Z)-flupentixol with the allosteric site of Pgp. Login to comment
187 Vanadate trapping of wild-type (top and middle panels) and F983A (bottom panel) Pgp was carried out in the presence (middle and lower panels) and absence (top panel) of 25 µM cis-(Z)-flupentixol, similar to the above experiment, except that nonradioactive 8-azido-ADP (1.25 mM) was used for trapping, and trapped samples were incubated and photo-cross-linked with 5 nM [125I]IAAP, a Pgp substrate. Login to comment
190 Key: no modulator, O; 25 µM cis-(Z)-flupentixol, b; 25 µM cis-(Z)-flupentixol in F983A, 0. Login to comment
207 The rate of ATP hydrolysis by the wild-type (left and middle histograms) and F983A (right histogram) Pgp was measured as mentioned in the presence of 50 µM prazosin and varying concentrations (0-50 µM) of the allosteric modulator cis-(Z)- flupentixol (left and right panels) or the competitive modulator verapamil (middle panel). Login to comment
221 Since the stimulatory effect on both substrate interaction and ATP hydrolysis is abrogated in the Pgp F983A mutant (38), which is impaired in modulation by cis-(Z)-flupentixol (38, 39), it suggests that both phenomena are mediated through modulator interaction at the allosteric site. Login to comment
238 The reduced effectiveness of cis-(Z)-flupentixol to inhibit the prazosin-stimulated ATP hydrolysis (Figure 3B, right) in the Pgp F983A mutant further confirms that the inhibitory action of the modulator is mediated through its interaction at the allosteric site of Pgp and not by its nonproductive (nonstimulating) association with the substrate site, blocking access to prazosin. Login to comment
245 The dissociation of bound [125 I]IAAP coupled to vanadate trapping was minimally affected by cis-(Z)-flupentixol in the Pgp F983A mutant (Figure 2C,D), further emphasizing the involvement of the allosteric modulator site in the phenomenon. Login to comment
252 In the Pgp F983A mutant, the stimulatory signal (for ATP hydrolysis) originating from the substrate site is minimally affected in the presence of cis-(Z)-flupentixol (Figure 3B, right); this suggests that the modulator-induced interference in cross talk between the FIGURE 5: Effect of cis-(Z)-flupentixol on dissociation of trapped [R-32P]-8-azido-ADP. Login to comment

[hide] Allosteric modulation bypasses the requirement for... J Biol Chem. 2006 Apr 21;281(16):10769-77. Epub 2006 Feb 27. Maki N, Moitra K, Ghosh P, Dey S
Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein.
J Biol Chem. 2006 Apr 21;281(16):10769-77. Epub 2006 Feb 27., 2006-04-21 [PMID:16505485]

Abstract [show]
ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
50 Baculovirus Expression of Human Pgp-Recombinant baculoviruses BV-MDR1-(His6) (19) and BV-MDR1-(His6)-F983A (32) harboring the wild type and the Pgp F983A mutant, respectively, were used to infect High Five insect cells grown in Excell 400 medium as described (34). Login to comment
86 No modulator-induced regeneration of the [125 I]IAAP binding was observed in the Pgp F983A mutant that is impaired in interaction with cis-(Z)-flupentixol (Fig. 1, A (bottom) and B). Login to comment
93 A, isolated membranes (0.1 mg/ml protein) from High Five insect cells, infected eitherwithrecombinantbaculovirusBV-MDR1(His6) or with BV-MDR1(His6)-F983A, were vanadate-trapped with 1.25 mM 8-azido-ATP (8-N3-ATP) and 0.25mM sodiumorthovanadate.Vanadate-trapped Pgp, either the wild type (top three panels) or the Pgp F983A mutant (bottom panel), in isolated membranes was pelleted, resuspended, and incubated in a vanadate- and nucleotide-free medium for the indicated time periods in the absence (second panel from top) and presence of either 1.25 mM 8-azido-ATP (top) or 25 ␮M cis-(Z)-flupentixol (third panel from top and bottom panel), prior to photocross-linking with 5 nM [125 I]IAAP. Login to comment
97 Solid bar, none; open bar, 1.25 mM 8-azido-ATP; hatched bar, 25 ␮M cis-(Z)-flupentixol plus 0.25 mM EDTA; gray bar, F983A/25 ␮M cis-(Z)-flupentixol plus 0.25 mM EDTA. Login to comment

[hide] Phenothiazines and structurally related compounds ... Curr Drug Targets. 2006 Sep;7(9):1123-34. Tsakovska I, Pajeva I
Phenothiazines and structurally related compounds as modulators of cancer multidrug resistance.
Curr Drug Targets. 2006 Sep;7(9):1123-34., [PMID:17017890]

Abstract [show]
Phenothiazines and structurally related compounds alongside their other biological activities are able to modulate multidrug resistance (MDR) in tumor cells. The extensive investigations on their MDR modulation effects consist part of the efforts to overcome MDR - the major obstacle in cancer chemotherapy. In this article we try to systematize the results collected in the last two decades in two main aspects. The first one comprises the mechanism of modulation by phenothiazine-type MDR modulators. Two main possible mechanisms of MDR reversal are reviewed: (i) direct interaction with Pgp; (ii) interactions with membrane phospholipids. The second aspect relates to the structural properties of phenothiazines and related compounds responsible for their MDR reversing effect. The structural alerts and physicochemical properties influencing anti-MDR activity are considered as identified by structure--activity (SAR) or quantitative structure--activity relationship (QSAR) studies. Results discussed in the article point to MDR modulation by phenothiazines and related compounds as a complex process in which more than one mechanism are certainly involved. Further investigations in this direction should contribute to elucidation of the possible mechanisms of MDR modulation by these compounds. On the basis of the studies discussed the potential use of phenothiazine-type MDR modulators as a model system in the further investigations of the MDR phenomenon is outlined.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
76 It was demonstrated that a single substitution of alanine for phenylalanine at position 983 in the putative TM12 of Pgp markedly altered the ability of the stereoizomers to modulate ATP hydrolysis and [125 I] IAAP labeling of Pgp and also affected inhibition of Pgp-mediated transport by both isomers of flupentixol. Login to comment

[hide] Schisandrol A from Schisandra chinensis reverses P... Planta Med. 2007 Mar;73(3):212-20. Epub 2007 Feb 22. Fong WF, Wan CK, Zhu GY, Chattopadhyay A, Dey S, Zhao Z, Shen XL
Schisandrol A from Schisandra chinensis reverses P-glycoprotein-mediated multidrug resistance by affecting Pgp-substrate complexes.
Planta Med. 2007 Mar;73(3):212-20. Epub 2007 Feb 22., [PMID:17318783]

Abstract [show]
Recent studies have shown that dibenzocyclooctadiene lignans may reverse P-glycoprotein-mediated multidrug resistance (Pgp-MDR) in cancer cells; however, the mechanism of action remains unknown. Through screening of herbs, we found that schisandrol A (SCH) isolated from Fructus Schisandrae (the dried fruit of Schisandra chinensis (Turcz.) Baill.) sensitized Pgp-MDR HepG2-DR cells by interfering with the function of Pgp-substrate complexes. In Pgp-MDR cells, SCH enhanced the cytotoxicity of cancer drugs that are Pgp substrates and restored vinblastine-induced G2/M arrest without lowering Pgp expression. SCH increased cellular retention of Pgp substrates such as rhodamine 123. In Pgp-overexpressing membrane preparations, SCH stimulated basal Pgp-ATPase thus showing some substrate-like function. However, SCH was not a competitive inhibitor for verapamil or progesterone and decreased their Km. In the presence of substrates, SCH decreased the reactivity between Pgp and the monoclonal antibody UIC-2 which is normally increased with active substrate-Pgp complexes. The labeling of active Pgp transport sites by [125I]-iodoarylazidoprazosin was partially blocked by SCH. SCH did not affect the activity of the mutant Pgp F983A suggesting that SCH acted differently than the thioxanthene type of Pgp allosteric inhibitors. Our results suggest that SCH acts by affecting the normal formation and functioning of the Pgp-substrate complexes.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
67 Baculovirus-mediated expression of human Pgp and photoaffinity labeling of Pgp with [125 I]IAAP High Five insect cells were transfected with a recombinant baculovirus harboring either the wild type {BV-MDR1-(H6)} [14] or the F983A mutant {BV-MDR1-F983A-(H6)} [15] human MDR1 cDNA [16]. Login to comment
123 To investigate the effect of SCH on Pgp substrate recognition, we performed photoaffinity labeling studies with [125 I]IAAP in insect cells transfected with either wild type or the F983A mutated Pgp. Login to comment
127 The inhibitory effect of SCH on [125 I]IAAP binding was unaltered in the F983A mutatant Pgp, indicating that this specific allosteric regulatory site was not involved in the inhibitory action of SCH. Login to comment
157 Lastly, SCH showed some inhibitory effects on [125 I]IAAP photo-labelling of Fig. 9 Effect of SCH on [125 I]IAAP binding to wild-type and to the F983A mutant of Pgp. Login to comment
158 Membrane preparations (15 mg of protein) containing wild-type Pgp (A) or the F983A mutant (B) were photoaffinity labeled with 5nM [125 I]IAAP in the presence of varying concentrations (0±100 mM) of SCH. Login to comment

[hide] Contribution to substrate specificity and transpor... Biochemistry. 1998 Nov 17;37(46):16400-9. Hafkemeyer P, Dey S, Ambudkar SV, Hrycyna CA, Pastan I, Gottesman MM
Contribution to substrate specificity and transport of nonconserved residues in transmembrane domain 12 of human P-glycoprotein.
Biochemistry. 1998 Nov 17;37(46):16400-9., 1998-11-17 [PMID:9819232]

Abstract [show]
P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino acid residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, [125I]iodoarylazidoprazosin (IAAP), were abrogated, with little effect on [alpha-32P]-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative amino-proximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
8 The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. Login to comment
50 Moreover, double mutants based on the triple mutant were cloned (L975A-V981A-F983, L975A-V981-F983A, L975- V981A-F983A). Login to comment
98 Alanine mutants were arranged in such a way that three alanine mutations were putatively located in the outer plasma membrane leaflet (L975A, V981A, and F983A) and four in the inner leaflet (M986A, V988A, Q990A, and V991A), assuming an R-helical structure to TM 12 (Figure 1b). Login to comment
124 Double mutants combining pairwise L975A, V981A, and F983A were constructed in order to determine the critical nonconserved residues that were responsible for mediating drug transport in the amino-proximal half of TM 12. Login to comment
125 Those double mutants were L975A-V981A, L975A-F983A, and V981A-F983A. Login to comment
126 The V981-F983 double mutant was Table 1: Properties of Wild-Type and Mutant P-glycoproteinsa drug transporta cell surface expressionc rhodamine daunomycin bodipy-verapamil calcein-AM bodipy-taxol wild-type ++++ ++++ ++++ ++++ ++++ ++++ L975A ++++ ++++ ++++ ++++ ++++ ++++ V981A ++++ ++++ ++++ ++++ ++++ ++++ F983A ++++ ++++ ++++ ++++ ++++ ++++ M986A ++++ ++++ n.d. ++++ ++++ n.d. V988A ++++ ++++ n.d. ++++ ++++ n.d. Q990A ++++ ++++ n.d. ++++ ++++ n.d. V991A ++++ ++++ n.d. ++++ ++++ n.d. L975A, V981A, F983A ++++ no transport no transport ++ ++ ++ M986A, V988A, Q990A, V991A ++++ ++++ +++ ++ ++++ ++++ V981A, F983A ++++ + no transport ++ ++ +++ L975A, F983A ++++ + ++ ++++ +++ ++++ L975A, V981A ++++ ++ no transport ++++ +++ ++++ a Symbols are noted as follows: ++++, wild-type activity; ++, impaired activity; +, residual activity; and n.d., not determined. b Drug transport was determined by FACS. Login to comment
130 Mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A, - -), the quadruple mutant in the carboxy-terminal half of TM 12 (M986A-V988A-Q990A-V991A, ‚‚‚), the double mutants L975A-V981A (- - -), L975A-F983A (-‚‚-), and V981A-F983A (-‚-). Login to comment
134 The double mutants involving L975A and either V981A and/or F983A were still capable of transporting calcein-AM, bodipy-taxol, and bodipy-verapamil but rhodamine 123 and daunorubicin transport was significantly reduced compared to wild-type Pgp (Figure 4), indicating a significant contribution of L975 to drug specificity. Login to comment
141 Mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A) and the quadruple mutant (M986A-V988A-Q990A-V991A) (s). Login to comment
144 Photoaffinity labeling demonstrated that the triple mutant L975A-V981A-F983A displayed a significant reduction in binding of IAAP compared to wild-type Pgp which is consistent with its reduced drug transport ability (Figure 5b). Login to comment
147 The three double mutants L975A-V981A, L975A-F983A, and V981A-F983A were able to bind IAAP but somewhat less than wild-type Pgp as was also true for the single mutants investigated in this study (Figure 5b,c). Login to comment
153 Mutant MDR1s are double mutants L975A-V981A, L975A-F983A, and V981A-F983A (s). Login to comment
159 ATP binding was equivalent for wild-type MDR1 and the MDR1 mutants L975A-V981A-F983A as well as for M986A-V988A-Q990A-V991A (Figure 7). Login to comment
162 Vanadate-trapped [R-32P]-8-azido-ADP was significantly reduced in the triple mutant L975A-V981A-F983A (Figure 8b,c), consistent with the decrease a b c FIGURE 5: Photoaffinity labeling of wild-type and mutant Pgp`s. Login to comment
174 (a) pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), and mutant Pgp`s are the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A) and the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A). Login to comment
176 Mutant MDR1s are the three double mutants L975A-V981A, L975A-F983A, and V981A-F983A. Login to comment
188 pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A), the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A), WT (wild-type human MDR1) in the presence of 250 µM EDTA and without MgCl2 to assess the specificity of the 170 kDa band representing Pgp as indicated with the arrow. Login to comment
193 (Middle panel) Vanadate-induced [R-32P]-8-azido-ADP labeling was performed at 37 °C. pTM: cells infected with vTF 7-3 virus and transfected with the expression vector containing no MDR1 (pTM1) (negative control), WT (wild-type human MDR1), the triple mutant in the amino-proximal half of TM 12 (L975A-V981A-F983A), and the quadruple mutant in the carboxy-terminal half of TM 12 (M986A- V988A-Q990A-V991A). Login to comment
227 To determine which combinations of these three residues of TM 12 predicted to lie in the outer leaflet were responsible for the loss of transport activity, we constructed double mutants (L975A-V981A, L975A-F983A, and V981A-F983A). Login to comment
228 The double mutants showed reduced drug transport suggesting that a change in any two of these three amino Table 2: Properties of Wild-Type and Mutant P-Glycoproteinsa photoaffinity labelinga ATP bindingb ATPase activityc wild-type ++++ ++++ ++++ L975A +++ n.d. +++ V981A +++ n.d. +++ F983A +++ n.d. +++ M986A n.d. n.d. n.d. V988A n.d. n.d. n.d. Q990A n.d. n.d. n.d. V991A n.d. n.d. n.d. L975A,V981A, F983A + ++++ + M986A, V988A, Q990A, V991A ++ ++++ ++ V981A, F983A +++ n.d. +++ L975A, F983A +++ n.d. +++ L975A, V981A +++ n.d. +++ a Symbols are noted as follows: ++++, wild-type activity; ++, impaired activity; +, residual activity; and n.d. not determined. b Photoaffinity labeling was done in HeLa crude membrane preparations using [125 I]iodoarylazidoprazosin. Login to comment
236 While no single residue is crucial, combinations of residues, especially V981A and F983A, affect binding of specific substrates such as daunomycin and rhodamine 123. Login to comment
248 F983A in combination with the other substitutions was also found to have a major effect on fluorescent drug transport. Login to comment

[hide] Single amino acid substitution of rat MRP2 results... Am J Physiol Gastrointest Liver Physiol. 2001 Oct;281(4):G1034-43. Ito K, Suzuki H, Sugiyama Y
Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate.
Am J Physiol Gastrointest Liver Physiol. 2001 Oct;281(4):G1034-43., [PMID:11557524]

Abstract [show]
Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
199 Dey et al. (5) demonstrated that the transport activity of the F983A mutant of P-glycoprotein was the same as that of wild-type P-glycoprotein, whereas the susceptibility of F983A to the stimulatory and inhibitory effect of cis- and trans-flupentixol, respectively, was markedly reduced compared with that of wild-type P-glycoprotein. Login to comment

[hide] Evidence for modulatory sites at the lipid-protein... Biochemistry. 2012 Apr 3;51(13):2852-66. Epub 2012 Mar 22. Mandal D, Moitra K, Ghosh D, Xia D, Dey S
Evidence for modulatory sites at the lipid-protein interface of the human multidrug transporter P-glycoprotein.
Biochemistry. 2012 Apr 3;51(13):2852-66. Epub 2012 Mar 22., [PMID:22360349]

Abstract [show]
The human multidrug transporter P-glycoprotein (Pgp or ABCB1) sets up pharmacological barriers to many clinically important drugs, a therapeutic remedy for which has yet to be formulated. For the rational design of mechanism-based inhibitors (or modulators), it is necessary to map the potential sites for modulator interaction and understand their modes of communication with the other functional domains of Pgp. In this study, combining directed mutagenesis with homology modeling, we provide evidence of two modulator-specific sites at the lipid protein interface of Pgp. Targeting 21 variant positions in the COOH-terminal transmembrane (TM) regions, we find residues M948 (in TM11) and F983, M986, V988, and Q990 (all four in TM12) critically involved in substrate-site modulation by a thioxanthene-based allosteric modulator cis-(Z)-flupentixol. Interestingly, for ATP-site modulation by the same modulator, only two (M948 and Q990) of those four residues appear indispensable, together with two additional residues, T837 and I864 in TM9 and TM10, respectively, suggesting independent modes of communication linking the allosteric site with the substrate binding and ATPase domains. None of the seven residues identified prove to be critical for modulation of the substrate or ATP sites by Pgp modulators that are transported by the pump, such as cyclosporin A or verapamil, indicating their specificity for cis-(Z)-flupentixol. On the other hand, ATP-site modulation by verapamil proves to be highly sensitive to replacement at positions F716 (in TM7) and I765 (in TM8), and to a more moderate extent at I764 and L772 (both in TM8). Homology modeling based on the known crystal structures of the bacterial multidrug transporter SAV1866 and the mouse Pgp homologue maps the identified residues primarily at the lipid-protein interface of Pgp, in two spatially distinct modulator-specific clusters. The two modulatory sites demonstrate negative synergism in influencing ATP hydrolysis, consolidating their spatial distinctness. Because Pgp is known to recruit drug molecules directly from the lipid bilayer, identification of modulatory sites at the lipid-protein interface and at the same time outside the conventional central drug binding cavity is mechanistically revealing.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
28 The desired nucleotide replacements were confirmed by Big-Dye version 3.1 (BD Sciences) nucleotide sequencing of the MDR1 open reading frame in recombinant plasmids pKM2-MDR1- V715A, -F716A, -I719A, -G723A, -I764A, -I765A, -I768A, -F770A, -L772A, -T837A, -I840A, -I864A, -I867A, -A871F, -T945A, -M948A, -F983A, -M986A, -V988A, -Q990A, and -V991A. Login to comment
102 A comparable level of basal binding of [125 I]IAAP to wild-type Pgp and Pgp mutants M948A, F983A, V988A, M986A, and Q990A (Figure 2B) rules out the possibility that the reduced level of stimulation in the mutants is due to loss of [125 I]IAAP binding and not stimulation per se. To investigate whether M948, Q990, F983, M986, and V988 are part of a general module involved in the regulation of substrate binding or are molecular components specific to cis- (Z)-flupentixol, we studied the effect of a structurally unrelated Pgp modulator, cyclosporin A, on binding of [125 I]IAAP to all 21 mutants. Login to comment
110 Stimulation of ATP Hydrolysis by cis-(Z)-Flupentixol and Verapamil cis-(Z)-flupentixol-mediated stimulation verapamil-mediated stimulation maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark maximal stimulation (x-fold basal) concentration for half-maximal stimulation (μM) remark WT 7.3 ± 0.4 6.8 ± 1.5 5.6 ± 0.19 6.6 ± 1.04 V715A 6.2 ± 0.2 5.0 ± 0.9 6.4 ± 0.37 16.3 ± 3.3 V716A 5.1 ± 0.2 4.7 ± 1.1 NDb NDb a I719A 4.6 ± 0.08 4.7 ± 0.4 5.8 ± 0.26 6.5 ± 1.31 G723A 5.3 ± 0.3 10.93 ± 2.7 5.4 ± 0.1 11.6 ± 0.88 I764A 4.5 ± 0.4 4.1 ± 2 3.2 ± 0.26 27.9 ± 8.4 I765A 4.96 ± 0.2 2.3 ± 0.47 NDb NDb a I768A 4.8 ± 0.2 1.2 ± 0.43 4.8 ± 0.19 8.5 ± 1.4 F770A 6.6 ± 0.6 10.9 ± 3.9 8.6 ± 0.59 13.2 ± 3.2 L772A 3.6 ± 0.2 2.96 ± 0.9 3.2 ± 0.11 9.5 ± 1.6 T837A 1.1 ± 0.06 NDb a 2.6 ± 0.1 3.1 ± 0.9 I840A 4.0 ± 0.3 25.6 ± 6.5 6.9 ± 0.2 13.5 ± 0.5 I864A 0.6 ± 0.05 NDb a 4.1 ± 0.25 0.9 ± 1.04 I867A 10.2 ± 1.3 72.3 ± 18.5 10.2 ± 0.52 30.7 ± 3.1 A871F NDb NDb NDb NDb a T945A 5.0 ± 0.3 13.7 ± 3.1 6.4 ± 0.15 6.9 ± 0.71 M948A NDb NDb a 6.8 ± 1.5 44.4 ± 12.4 F983A 4.95 ± 0.36 19.59 ± 5.0 6.1 ± 0.38 6.6 ± 1.8 M986A 3.8 ± 0.3 2.6 ± 1.5 7.7 ± 0.76 30.0 ± 8.6 V988A 3.0 ± 0.2 21.4 ± 7.6 6.5 ± 0.31 16.9 ± 2.9 Q990A 1.8 ± 0.4 18.9 ± 2.6 a 7.7 ± 0.58 12.9 ± 3.7 V991A 3.9 ± 0.2 21.1 ± 5.0 5.9 ± 0.59 21.9 ± 7.2 a Mutants with <2-fold maximal stimulation (<25% of the wild-type level). Login to comment
224 Substitutions F983A, M986A, and V988A completely abrogate stimulation of substrate binding (Figure 2) with only modest effects on ATP-site stimulation (Figure 4), which indicates that the two modulatory events are not tightly coupled to each other. Login to comment

[hide] Gomisin A alters substrate interaction and reverse... Biochem Pharmacol. 2006 Sep 28;72(7):824-37. Epub 2006 Aug 4. Wan CK, Zhu GY, Shen XL, Chattopadhyay A, Dey S, Fong WF
Gomisin A alters substrate interaction and reverses P-glycoprotein-mediated multidrug resistance in HepG2-DR cells.
Biochem Pharmacol. 2006 Sep 28;72(7):824-37. Epub 2006 Aug 4., [PMID:16889754]

Abstract [show]
Through an extensive herbal drug screening program, we found that gomisin A, a dibenzocyclooctadiene compound isolated from Schisandra chinensis, reversed multidrug resistance (MDR) in Pgp-overexpressing HepG2-DR cells. Gomisin A was relatively non-toxic but without altering Pgp expression, it restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are Pgp substrates but may act by different mechanisms. Several lines of evidence suggest that gomisin A alters Pgp-substrate interaction but itself is neither a Pgp substrate nor competitive inhibitor. (1) First unlike Pgp substrates gomisin A inhibited the basal Pgp-associated ATPase (Pgp-ATPase) activity. (2) The cytotoxicity of gomisin A was not affected by Pgp competitive inhibitors such as verapamil. (3) Gomisin A acted as an uncompetitive inhibitor for Pgp-ATPase activity stimulated by the transport substrates verapamil and progesterone. (4) On the inhibition of rhodamine-123 efflux the effects of gomisin A and the competitive inhibitor verapamil were additive, so were the effects of gomisin A and the ATPase inhibitor vanadate. (5) Binding of transport substrates with Pgp would result in a Pgp conformational change favoring UIC-2 antibody reactivity but gomisin A impeded UIC-2 binding. (6) Photocrosslinking of Pgp with its transport substrate [125I]iodoarylazidoprazosin was inhibited by gomisin A in a concentration-dependent manner. Taken together our results suggest that gomisin A may bind to Pgp simultaneously with substrates and alters Pgp-substrate interaction.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
113 Baculovirus mediated expression of human Pgp and photoaffinity labeling of Pgp with [125 I]IAAP Recombinant baculoviruses harboring either the wild type BV-MDR1-(H6) [24] or the F983A mutant BV-MDR1-F983A- (H6)[25] of human MDR1 cDNA, with 6 Â His-tag at the C-terminal end, were used to infect High Five insect cells grown in serum free Excell 400 medium as described [26]. Login to comment
216 To understand whether F983 has any role in the interaction of gomisin A with Pgp, we studied [125 I]IAAP labeling of Pgp F983A mutant in the presence of varying concentrations of gomisin A. Login to comment
237 b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 8 2 4 - 8 3 7 835 Fig. 10 - Effect of gomisin A on [125 I]IAAP photo-labeling of the wild type and F983A mutant Pgp. Login to comment
238 Wild type (Fig. 10A, top panel) or F983A mutant (Fig. 10B, bottom panel) Pgp in isolated membranes was photoaffinity labeled with 5 nM of [125 I]IAAP in the presence of varying concentrations (0-100 mM) of gomisin A (GOM). Login to comment
246 However, gomisin A`s effect on [125 I]IAAP labeling of Pgp was not altered in the Pgp F983A mutant that is impaired of modulation by cis- (Z)-flupentixol. Login to comment
112 Baculovirus mediated expression of human Pgp and photoaffinity labeling of Pgp with [125 I]IAAP Recombinant baculoviruses harboring either the wild type BV-MDR1-(H6) [24] or the F983A mutant BV-MDR1-F983A- (H6)[25] of human MDR1 cDNA, with 6 His-tag at the C-terminal end, were used to infect High Five insect cells grown in serum free Excell 400 medium as described [26]. Login to comment
217 To understand whether F983 has any role in the interaction of gomisin A with Pgp, we studied [125 I]IAAP labeling of Pgp F983A mutant in the presence of varying concentrations of gomisin A. Login to comment
239 Wild type (Fig. 10A, top panel) or F983A mutant (Fig. 10B, bottom panel) Pgp in isolated membranes was photoaffinity labeled with 5 nM of [125 I]IAAP in the presence of varying concentrations (0-100 mM) of gomisin A (GOM). Login to comment
247 However, gomisin A`s effect on [125 I]IAAP labeling of Pgp was not altered in the Pgp F983A mutant that is impaired of modulation by cis- (Z)-flupentixol. Login to comment

[hide] Biochemical and pharmacological properties of an a... Biochem Pharmacol. 2006 Jul 14;72(2):145-55. Epub 2006 Apr 25. Maki N, Dey S
Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1).
Biochem Pharmacol. 2006 Jul 14;72(2):145-55. Epub 2006 Apr 25., [PMID:16729976]

Abstract [show]
The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
47 Replacement of the F983 with alanine (F983A) abrogates inhibition of [125 I]IAAP transport as well as stimulation of Pgp-[125 I]IAAP interaction by cis-(Z)-flupentixol, without any effect on the basal level of [125 I]IAAP binding and transport by Pgp [25]. Login to comment
59 Previously characterized [25] mouse NIHMDR1-WT and NIHMDR1-F983A cells were used for the studies with intact cells. Login to comment
118 Replacement of F983 by an alanine (F983A) abrogates both stimulation of [125 I]IAAP binding to Pgp as well as inhibition of transport by cis-(Z)- flupentixol [25]. Login to comment
120 We studied [125 I]IAAP binding to the wild type Pgp as well as to the Pgp F983A mutant in NIHMDR1-WT and NIHMDR1-F983A cells, respectively, in the presence of several Pgp modulators, both structurally related (Fig. 1A and B) and unrelated (Fig. 1C) to cis-(Z)-flupentixol. Login to comment
121 A detectable level of [125 I]IAAP binding to the wild type and the Pgp F983A mutant was observed (Fig. 2A-C, autoradiogram, none) in the absence of any modulator. Login to comment
122 The relatively lower level of [125 I]IAAP signal in the Pgp F983A mutant was due to a reduced level of the expression of the mutant Pgp in NIHMDR1-F983A cells, as determined by immunoblotting with a Pgp specific antibody PEPG13 (see immunoblots). Login to comment
123 As evident in Fig. 2A, thioxanthene derivatives cis-(Z)-clopentixol, trans-(E)-clopentixol, cis-(Z)- 753, compound 789, cis-(Z)-768, and trans-(E)-768, all stimulated [125 I]IAAP binding to the wild type Pgp (autoradiogram, upper panel), and the stimulatory effect was abrogated in the Pgp F983A mutant (autoradiogram, lower panel). Login to comment
127 The inhibitory potential of these compounds on [125 I]IAAP binding remained unaltered in the Pgp F983A mutant (Fig. 2C, autoradiogram, lower panel), suggesting no involvement of F983 in the modulation of Pgp function by those compounds. Login to comment
159 b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 4 5 - 1 5 5150 Fig. 2 - (A) Effect of thioxanthenes on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Login to comment
161 Following incubation, cells were exposed to UV irradiation for 5 min, lysed, and resolved by SDS-PAGE (80,000 cells per well) as indicated in Section 2; (B) effect of phenothiazines on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done as described for (A), except that the modulators added were 5 mM cyclosporin A (CsA) (lane 2), 10 mM promethazine (lane 3), 10 mM chlorpromazine (lane 4), 10 mM trifluperazine (lane 5), 10 mM thioridazine (lane 6), or 1% DMSO (none) (lane 1); (C) effect of other modulators on Pgp- [125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done exactly the same way as described in (A), except modulators used were 5 mM cyclosporin A (CsA) (lane 2), 5 mM rapamycin (lane 3), 10 mM nicardipine (lane 4), 10 mM niguldipine (lane 5), or in the absence of any modulator (none) (lane 1). Login to comment
209 The stimulatory effect on [125 I]IAAP binding, and its abrogation in the Pgp F983A mutant, provided us with a convenient assay to study the specificity of the allosteric site. Login to comment
119 Replacement of F983 by an alanine (F983A) abrogates both stimulation of [125 I]IAAP binding to Pgp as well as inhibition of transport by cis-(Z)- flupentixol [25]. Login to comment
124 As evident in Fig. 2A, thioxanthene derivatives cis-(Z)-clopentixol, trans-(E)-clopentixol, cis-(Z)- 753, compound 789, cis-(Z)-768, and trans-(E)-768, all stimulated [125 I]IAAP binding to the wild type Pgp (autoradiogram, upper panel), and the stimulatory effect was abrogated in the Pgp F983A mutant (autoradiogram, lower panel). Login to comment
128 The inhibitory potential of these compounds on [125 I]IAAP binding remained unaltered in the Pgp F983A mutant (Fig. 2C, autoradiogram, lower panel), suggesting no involvement of F983 in the modulation of Pgp function by those compounds. Login to comment
160 b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 1 4 5 - 1 5 5 150 Fig. 2 - (A) Effect of thioxanthenes on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Login to comment
162 Following incubation, cells were exposed to UV irradiation for 5 min, lysed, and resolved by SDS-PAGE (80,000 cells per well) as indicated in Section 2; (B) effect of phenothiazines on Pgp-[125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done as described for (A), except that the modulators added were 5 mM cyclosporin A (CsA) (lane 2), 10 mM promethazine (lane 3), 10 mM chlorpromazine (lane 4), 10 mM trifluperazine (lane 5), 10 mM thioridazine (lane 6), or 1% DMSO (none) (lane 1); (C) effect of other modulators on Pgp- [125 I]IAAP interaction in NIHMDR1-WT (wild type) and NIHMDR1-F983A (mutant F983A) cells. Experiment was done exactly the same way as described in (A), except modulators used were 5 mM cyclosporin A (CsA) (lane 2), 5 mM rapamycin (lane 3), 10 mM nicardipine (lane 4), 10 mM niguldipine (lane 5), or in the absence of any modulator (none) (lane 1). Login to comment
210 The stimulatory effect on [125 I]IAAP binding, and its abrogation in the Pgp F983A mutant, provided us with a convenient assay to study the specificity of the allosteric site. Login to comment

[hide] Allosteric modulation of the human P-glycoprotein ... Arch Biochem Biophys. 2006 Jun 1;450(1):100-12. Epub 2006 Mar 15. Ghosh P, Moitra K, Maki N, Dey S
Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates.
Arch Biochem Biophys. 2006 Jun 1;450(1):100-12. Epub 2006 Mar 15., [PMID:16624245]

Abstract [show]
The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
3 The protection by Xupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by Xupentixols, indicating involvement of the allosteric site in generating the conformational change. Login to comment
46 Baculovirus mediated expression of human Pgp A recombinant baculovirus harboring either the wild-type or the F983A mutant human MDR1 cDNA, with 6 £His-tag at the C-terminal end, BV-MDR1-(H6) [34] was used to infect High Five insect cells grown in serum free Excell 400 medium as described [35]. Login to comment
105 SigniWcantly, no detectable protection from trypsin digestion was observed for the Pgp F983A mutant (Fig. 2C) that is impaired in modulation by Xupentixol [21]. Login to comment
112 Protection to limited trypsin digestion is induced by both isomers of Xupentixol, which is aVected in Pgp F983A mutant. Login to comment
116 (C) Similar experiments were carried out with the Pgp F983A mutant in the presence of varying concentrations (0-25 M) of cis-(Z)-Xupentixol and resolved as stated above. Login to comment
117 198 49.8 115 93 +-Trypsin 0 1 2.5 5 10cis-(Z)-Flupentixol (µM) 250 Wild Type 198 49.8 115 93 +-Trypsin 0 1 2.5 5 10trans-(E)-Flupentixol (µM) 250 Wild Type 198 49.8 115 93 +-Trypsin 0 1 2.5 5 10cis-(Z)-Flupentixol (µM) 250 F983A A B C strate site, both isomers of Xupentixol induced similar conformational changes in Pgp. Login to comment
188 Since the Pgp F983A mutant that is impaired in modulation by Xupentixol [21,22] displays no such protection (Fig. 2C) the functional signiWcance of the conformational change in the mechanism of modulation is underscored. Login to comment
223 Consistent with that, in the Pgp F983A mutant, the loss of cis-(Z)-Xupentixol-induced conformational change coincides with impaired modulation of [125 I]IAAP binding, whereas inhibition of [125 I]IAAP binding by cyclosporin A remained unaltered in the mutant [21]. Login to comment
107 SigniWcantly, no detectable protection from trypsin digestion was observed for the Pgp F983A mutant (Fig. 2C) that is impaired in modulation by Xupentixol [21]. Login to comment
114 Protection to limited trypsin digestion is induced by both isomers of Xupentixol, which is aVected in Pgp F983A mutant. Login to comment
118 (C) Similar experiments were carried out with the Pgp F983A mutant in the presence of varying concentrations (0-25 M) of cis-(Z)-Xupentixol and resolved as stated above. Login to comment
119 198 49.8 115 93 + - Trypsin 0 1 2.5 5 10 cis-(Z)-Flupentixol (&#b5;M) 25 0 Wild Type 198 49.8 115 93 + - Trypsin 0 1 2.5 5 10 trans-(E)-Flupentixol (&#b5;M) 25 0 Wild Type 198 49.8 115 93 + - Trypsin 0 1 2.5 5 10 cis-(Z)-Flupentixol (&#b5;M) 25 0 F983A A B C strate site, both isomers of Xupentixol induced similar conformational changes in Pgp. Login to comment
190 Since the Pgp F983A mutant that is impaired in modulation by Xupentixol [21,22] displays no such protection (Fig. 2C) the functional signiWcance of the conformational change in the mechanism of modulation is underscored. Login to comment
225 Consistent with that, in the Pgp F983A mutant, the loss of cis-(Z)-Xupentixol-induced conformational change coincides with impaired modulation of [125 I]IAAP binding, whereas inhibition of [125 I]IAAP binding by cyclosporin A remained unaltered in the mutant [21]. 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.

Comments [show]

None has been submitted yet.

Sentences [show]
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
61 The same mutations have either no effect (L975A/F983A and L975A/V981A) or cause up to 50% inhibition (V981A/ F983A) in the transport of bodipy-verapamil, calcein-AM and bodipy-taxol. Login to comment
57 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

[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.

Comments [show]

None has been submitted yet.

Sentences [show]
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