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Multidrug transporters play a dual role in haematopoietic cells, mediating the efflux of xenobiotics and regulating cell migration. For several reasons including the lack of specific antibodies, reports of multidrug transporter distribution on lymphocytes conflict. Murine B cells have been reported to completely lack transporter activity. Through analysis of parental and 'knockout' mice we show that, contrary to previous studies, murine B and T lymphocytes possess at least three active multidrug transporters and also a hitherto unrecognised drug-specific import activity. Surprisingly, the drug specificity of P-glycoprotein appears cell type dependent. The data indicate that a range of developmentally regulated, multidrug transporters can impose a barrier to treatment of immune disorders. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) is a drug transporter which pumps toxic hydrophobic compounds out of cells, conferring mutidrug resistance. P-gp is predicted to consist of 12 transmembrane α- helices and there is a strong body of experimental support for this model. However, a number of studies, including those on Pgp expressed in E. coli, have reported topologies with fewer than 12 transmembrane α-helices, leading to the hypothesis that the transmembrane topology of the protein changes during function. It is well established that P-gp undergoes conformational changes during its transport cycle and it has been recently shown that these changes are large in magnitude and could, potentially, reflect a changing transmembrane topology. One therefore, reassessed the transmembrane topology of P-gp expressed in E. coli and compared it directly with the topology ofthe protein express ed in mammalian cells. It was clear that the transmembrane topology of the protein was different in the different cell types and that the misfolding of P-gp in E. coli was due to the misrecognition of multiple P-gp sequences as topogenic signals. Thus, the alternative transmembrane topologies reported for P-gp in E. coli are artefacts of the heterologous expression system used, and models based on such data in which the transmembrane topology changes during drug transport are unlikely to be correct. Instead, the large conformational changes observed during the transportcycle are more likely due to changes in α-helix packing. [ABSTRACT FROM AUTHOR]

Examines the effects of the vinblastine binding site during a transport cycle of P-glycoprotein (P-gp). Determination of the alteration of drug binding site parameters; Exhibition of low-affinity substrate binding by the posthydrolytic form of P-gp; Outcome of the release of phosphate post-adenosine triphosphate hydrolysis.

In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted “stability” to this perturbation of bilayer organization by P-gp and moreover this led ... [ABSTRACT FROM AUTHOR]

Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a memeber of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapuetic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathways through the membrane through which substrates are transpoerted, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonellla tyhimuruim and the cystic fibrosis trransbrane conductance regulator, are accesible from the extracelular surface of the cell, spanning the membrane directly or potenially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBss of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters,the data provided into the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed. [ABSTRACT FROM AUTHOR]

Presents research which expressed a protein in Lactococcus lactis, LmrA, in human lung fibroblast cells. Antibiotic-resistance mechanisms in bacteria; Role of LmrA; Human multidrug-resistance P-glycoprotein; Pharmacological characteristics of LmrA and P-glycoprotein expressing lung fibroblasts very similar; Kinetic analysis of drug dissociation from LmrA; Implications; LmrA and P-glycoprotein interchangeable.

Applies complementary research techniques in the form of experimental cysteine-directed chemical cross-linking and computational homology modeling to produce an atomic scale model for the human multidrug transporter P-glycoprotein (P-gp). Domains of P-gp; Cross-linking analysis of P-gp mutant isoforms; Reconciliation of cross-linking data with a P-gp model.

The article reflects on the 20th anniversary of the identification of adenosine-triphosphate-binding cassette (ABC) transporters. This superfamily of primary active proteins, which play diverse physiological roles and are found in all cell in all kingdom was identified in 1986. The year 2006 also marks the 30th anniversary of the molecular identification of P-glycoprotein which is the first mammalian example of the ABC transporter family.

The proteasome is a multi-protein complex that degrades cellular proteins as well as foreign proteins destined for antigen presentation. The latter function involves the immunoproteasome, in which several proteasome subunits are exchanged for γ-interferon-induced subunits. The transporter associated with antigen processing (TAP) transports proteasome-generated peptides across the membrane of the endoplasmic reticulum (ER) prior to presentation on the plasma membrane. We demonstrate interactions between the cytoplasmic domains of TAP subunits and subunits of both the proteasome and the immunoproteasome, suggesting direct targeting of antigenic peptides to the ER via a TAP–proteasome association. We also show interaction between one of the cytoplasmic domains of P-glycoprotein and a proteasome subunit, but not the corresponding immunoproteasome subunit, suggesting a possible role for P-glycoprotein in the transport of proteasome-derived peptides. [Copyright &y& Elsevier]

Shows that in K562 leukemic cells the levels of MDR1 messenger RNA (mRNA) increase in a dose- and time-dependent manner upon transient exposure to a variety of cytotoxic drugs. Stabilization of mRNA as cause, not transcriptional activation; Possibilities for treatment regimes to circumvent multidrug resistance in leukemia.

Examines the role of three-dimensional structures of the mammalian multidrug resistance P-glycoprotein in major conformational changes in the transmembrane domains upon nucleotide binding. Use of electron crystallography of negatively stained crystals; Reorganization of the transmembrane domains throughout the entire depth of the membrane; Importance of the reorganization of the transmembrane domains.