Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.
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PMID:Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. 889 56

Most antigenic peptides presented to CD8+ T cells are generated from cytosolic precursors and are translocated by TAP into the endoplasmic reticulum, where they associate with MHC class I molecules. TAP-deficient cells exhibit a limited capacity to deliver peptides from cytosolic proteins to class I molecules. One candidate for an alternative peptide transporter is P-glycoprotein, which transports numerous substances, including peptides, across membranes. Elevation of P-glycoprotein expression is partially responsible for the resistance developed by neoplasias to chemotherapeutic drugs. Overexpression of P-glycoprotein has been reported to enhance the expression of class I molecules. Here, we investigated the role of P-glycoprotein in the generation of peptide-MHC complexes. We were unable to detect P-glycoprotein-mediated transport of synthetic peptides into the endoplasmic reticulum of either T2 cells (TAP-deficient) infected with a recombinant vaccinia virus (rVV) expressing P-glycoprotein or drug-resistant cells in which TAP is inactivated by a peptide from the herpes simplex virus ICP47 protein. Expression of rVV-encoded P-glycoprotein in T2 cells was unable to enhance cell surface expression of any of three MHC class I allomorphs tested. rVV-mediated expression of P-glycoprotein enabled T2 cells to produce limited amounts of class I-peptide complexes from cytosolic antigens, but this was not blocked by a drug that inhibits its transporter function, and a similar degree of presentation was mediated by functionally inactive mutated forms of P-glycoprotein. Thus, this was a nonspecific effect that we attributed to diminished membrane integrity resulting from P-glycoprotein overexpression. Taken together, our findings cast serious doubts that P-glycoprotein is a biologically significant transporter of cytosolic peptides.
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PMID:P-glycoprotein plays an insignificant role in the presentation of antigenic peptides to CD8+ T cells. 978 23

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.
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PMID:Contribution to substrate specificity and transport of nonconserved residues in transmembrane domain 12 of human P-glycoprotein. 981 32

Fexofenadine, a nonsedating antihistamine, does not undergo significant metabolic biotransformation. Accordingly, it was hypothesized that uptake and efflux transporters could be importantly involved in the drug's disposition. Utilizing a recombinant vaccinia expression system, members of the organic anion transporting polypeptide family, such as the human organic anion transporting polypeptide (OATP) and rat organic anion transporting polypeptides 1 and 2 (Oatp1 and Oatp2), were found to mediate [(14)C]fexofenadine cellular uptake. On the other hand, the bile acid transporter human sodium taurocholate cotransporting polypeptide (NTCP) and the rat organic cation transporter rOCT1 did not exhibit such activity. P-glycoprotein (P-gp) was identified as a fexofenadine efflux transporter, using the LLC-PK1 cell, a polarized epithelial cell line lacking P-gp, and the derivative cell line (L-MDR1), which overexpresses P-gp. In addition, oral and i.v. administration of [(14)C]fexofenadine to mice lacking mdr1a-encoded P-gp resulted in 5- and 9-fold increases in the drug's plasma and brain levels, respectively, compared with wild-type mice. Also, a number of drug inhibitors of P-gp were found to be effective inhibitors of OATP. Because OATP transporters and P-gp colocalize in organs of importance to drug disposition such as the liver, their activity provides an explanation for the heretofore unknown mechanism(s) responsible for fexofenadine's disposition and suggests potentially similar roles in the disposition of other xenobiotics.
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PMID:OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. 1042 12

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly(-)) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly(-) P-gp (91, 94, and 99N-->Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly(-) P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly(-) D (91, 94, 99N-->D) and Gly(-) Delta (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly(-) D and Gly(-) Delta Pgps were also expressed to the same level as the Gly(-) mutant protein. (35)S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of (35)S-methionine/cysteine in full length Gly(-) P-gp compared to wild-type protein, but the half-life ( approximately 3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of (35)S-methionine/cysteine in Gly(-) P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.
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PMID:Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system. 1066 16

The human MDR1-encoded transporter is a 170-kDa plasma membrane glycoprotein [P-glycoprotein (P-gp)] capable of binding and energy-dependent extrusion of structurally diverse organic compounds and drugs. P-gp seems to play a significant role in uptake, distribution, and excretion of many different drugs. To determine whether common polymorphic forms of P-gp are likely to alter function of P-gp, we characterized five known MDR1 coding polymorphisms (N21D, F103L, S400N, A893S, and A998T) using a vaccinia virus-based transient expression system. Cell surface expression of wild-type P-gp was time-dependent over a time course of 5.5 to 34.5 h; highest expression was obtained by 22 to 26.5 h after infection/transfection, indicating that a semiquantitative assay for P-gp expression levels was possible. HeLa cells stained with the P-gp specific monoclonal antibodies MRK-16 and Western blots probed with C219 revealed similar cell surface expression for the polymorphisms and for wild-type protein. Time-dependent P-gp pump function maximal at 22 h after infection/transfection was demonstrated for the following MDR1 fluorescence substrates: 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester (bodipy-FL)-verapamil, bodipy-FL-vinblastine, calcein-AM, bodipy-FL-prazosin, bisantrene, and bodipy-FL-forskolin, but not for daunorubicin. Transport studies of all tested substrates indicated that the substrate specificity of the pump was not substantially affected by any of the tested polymorphisms. Cell surface expression and function of double mutants including the more common polymorphisms (N21D-S400N, N21D-A893S, and S400N-A893S) showed no differences from wild-type. These results demonstrate that the common MDR1 coding polymorphisms result in P-gps with a cell surface distribution and function similar to wild-type P-gp.
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PMID:Functional characterization of coding polymorphisms in the human MDR1 gene using a vaccinia virus expression system. 1206 48

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.
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PMID:Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1). 1243 56

The organic anion-transporting polypeptides represent an important family of drug uptake transporters that mediate the cellular uptake of a broad range of substrates including numerous drugs. Doxorubicin is a highly efficacious and well-established anthracycline chemotherapeutic agent commonly used in the treatment of a wide range of cancers. Although doxorubicin is a known substrate for efflux transporters such as P-glycoprotein (P-gp; MDR1, ABCB1), significantly less is known regarding its interactions with drug uptake transporters. Here, we investigated the role of organic anion transporting polypeptide (OATP) transporters to the disposition of doxorubicin. A recombinant vaccinia-based method for expressing uptake transporters in HeLa cells revealed that OATP1A2, but not OATP1B1 or OATP1B3, and the rat ortholog Oatp1a4 were capable of significant doxorubicin uptake. Interestingly, transwell assays using Madin-Darby canine kidney II cell line cells stably expressing specific uptake and/or efflux transporters revealed that OATP1B1, OATP1B3, and OATP1A2, either alone or in combination with MDR1, significantly transported doxorubicin. An assessment of polymorphisms in SLCO1A2 revealed that four variants were associated with significantly impaired doxorubicin transport in vitro. In vivo doxorubicin disposition studies revealed that doxorubicin plasma area under the curve was significantly higher (1.7-fold) in Slco1a/1b^-/- versus wild-type mice. The liver-to-plasma ratio of doxorubicin was significantly decreased (2.3-fold) in Slco1a/1b2^-/- mice and clearance was reduced by 40% compared with wild-type mice, suggesting Oatp1b transporters are important for doxorubicin hepatic uptake. In conclusion, we demonstrate important roles for OATP1A/1B in transporter-mediated uptake and disposition of doxorubicin.
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PMID:Contribution of Organic Anion-Transporting Polypeptides 1A/1B to Doxorubicin Uptake and Clearance. 2777 71

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