In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the “chemoimmunity” network, which has a number of features reminiscent of classical immunology.
ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.
Currently 30 human ABC proteins are represented by full sequences in various databases, and this paper provides a brief overview of these proteins. ABC proteins are composed of transmembrane domains (TMDs), and nucleotide binding domains (NBDs, or ATP-binding cassettes, ABSs). The arrangement of these domains, together with available membrane topology models of the family members, are presented. Based on their sequence similarity scores, the members of the human ABC protein family can be grouped into eight subfamilies. At present the MDR/TAP, the ALD, the MRP/CFTR, the ABC1, the White, the RNAseL inhibitor, the ANSA, and the GCN20 subfamilies are identified. Mutations of many human ABC proteins are known to be causative in inherited diseases, and a short description of the molecular pathology of these ABC gene-related genetic diseases is also provided.
The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD 0 ) connected to the core by a cytoplasmic linker (L 0 ). We have studied truncated MRP1 versions containing either the MDRlike core alone or the core plus linker L 0 , produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Fulllength MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C 4 and N-ethylmaleimide glutathione. In addition, leukotriene C 4 -stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L 0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD 0 region but still containing L 0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD 0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD 0 L 0 and the MRP1 minus TMD 0 L 0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD 0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane. MDR1 P-glycoprotein (MDR1 Pgp)1 and MRP1 (multidrug resistance protein 1) are members of the ATP binding cassette (ABC) transporter family that can cause multiple drug resistance in tumor cells. MDR1 Pgp is an ATP-dependent drug extrusion pump and confers resistance to a wide variety of hydrophobic toxic agents (1). MRP1 has been shown to be a high affinity primary active transporter for the glutathioneconjugated eicosanoid, leukotriene C 4 (LTC 4 ) (2, 3) and to transport various other compounds that are conjugated to glutathione, sulfate, or glucuronide (2, 4 -6). The physiological functions of MRP1 range from the mediation of an inflammatory response to the elimination of certain xenobiotics (7-11), and this protein may play a role in the chemotherapy resistance of several types of cancer (12).Vanadate inhibits ATP-dependent drug transport both by MDR1 and MRP1 (1, 7), and in the presence of vanadate, the trapping of an adenine nucleotide in these proteins has been demonstrated (13,14). Transported compounds specifically increase the rate of vanadate-dependent nucleotide occlusion in MRP1 (14), similarly to what has been shown for hydrophobic drugs in the case of MDR1 Pgp (15). Vanadate-dependent, drug-stimulated nucleotide trapping reflects a partial reaction of the multidrug transporters and thus can be used to examine their functional characteristics.MDR1 Pgp and MRP1 share a similar core structure, consisting of a tandem repeat of transmembrane domains (TMDs) and cytoplasmic ABC-containing regions. However,...
The human multidrug resistance protein MRP1 and its homolog, MRP2, are both suggested as being involved in cancer drug resistance and the transport of organic anions. We expressed MRP1 and MRP2 in Spodoptera frugiperda ovarian cells and compared their ATP-dependent transport properties and vanadate-sensitive ATPase activities in isolated membrane vesicles. Both MRP1 and MRP2 actively transported leukotriene C 4 and N-ethylmaleimide glutathione (NEM-GS), although the relative affinity of MRP2 for these substrates was found to be significantly lower than that of MRP1. Methotrexate was actively transported by both proteins, although more efficiently by MRP2. ATP-dependent NEM-GS transport by MRP1 and MRP2 was variably modulated by organic anions. Probenecid and furosemide inhibited, whereas under certain conditions sulfinpyrazone, penicillin G, and indomethacin greatly stimulated, MRP2-mediated NEM-GS uptake. Vanadate-sensitive ATPase activity in isolated membranes containing MRP1 or MRP2 was significantly stimulated by NEM-GS and reduced GS, although these compounds acted only at higher concentrations in MRP2. ATP hydrolysis by MRP2 was also effectively stimulated by methotrexate. Probenecid, sulfinpyrazone, indomethacin, furosemide, and penicillin G all significantly increased MRP2-ATPase activity, whereas these compounds acted more as ATPase inhibitors on MRP1. These results indicate that MRP1 is a more efficient transporter of glutathione conjugates and free glutathione than MRP2, whereas several anions are preferred substrates for MRP2. Our data suggest that MRP2 may be responsible for the active secretion of pharmacologically relevant organic anions, such as diuretics and antibiotics, and indicate different modulation possibilities for MRP1 or MRP2 in drug-resistant tumor cells.
An important issue for in vivo gene therapy for cystic fibrosis (CF) is the percentage of cells within the CF airway that will require correction. In this study, we mixed populations of a CF airway cell line expressing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA (corrected cells) or a reporter gene in defined percentages. As few as 6-10% corrected cells within an epithelial sheet generated C1-transport properties similar to sheets comprised of 100% corrected cells. Cell-cell coupling may serve as the mechanism for amplification of the functional effects of corrected cells. These data suggest that in vivo correction of all CF airway cells may not be mandatory.
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