Multidrug resistance protein (MRP) 1 is a member of the ABCC branch of the ATP binding cassette (ABC) transporter superfamily that can confer resistance to natural product chemotherapeutic drugs and transport a variety of conjugated organic anions, as well as some unconjugated compounds in a glutathione- (GSH-) dependent manner. In addition to the two tandemly repeated polytopic membrane-spanning domains (MSDs) typical of ABC transporters, MRP1 and its homologues MRP2, -3, -6, and -7 contain a third NH(2)-terminal MSD. The cytoplasmic loop (CL3) connecting this MSD, but apparently not the MSD itself, is required for MRP1 leukotriene C(4) (LTC(4)) transport activity, substrate binding and appropriate trafficking of the protein to the basolateral membrane. We have used a baculovirus dual-expression system to produce various functionally complementing fragments of MRP1 in insect Sf21 cells to precisely define the region in CL3 that is required for activity and substrate binding. Using a parallel approach in polarized MDCK-I cells, we have also defined the region of CL3 that is required for basolateral trafficking. The CL3 NH(2)- and COOH-proximal functional boundaries have been identified as Cys(208) and Asn(260), respectively. Cys(208) also corresponds to the NH(2)-proximal boundary of the region required for basolateral trafficking in MDCK-I cells. However, additional residues downstream of the CL3 COOH-proximal functional boundary extending to Lys(270) were found to be important for basolateral localization. Finally, we show that regions in CL3 necessary for LTC(4) binding and transport are also required for binding of the photoactivatable GSH derivative azidophenacyl-GSH.
In mammals, some of the effects of interferon (IFN) on gene transcription are known to be mediated by a family of IFN-inducible DNA-binding proteins, the IFN regulatory factor (IRF) family, which includes both activators and repressors of transcription. Although IFN activities have been described in many vertebrates, little is known about regulation of IFN- or IFN-stimulated genes in species other than human and mouse. Here, we report the cloning of a chicken cDNA, cIRF-3, encoding a protein with a DNA-binding domain similar to that found in the mammalian IRF family of proteins. Similarity between cIRF-3 and the mammalian IRFs is comparable with that between known members of the family. It is most similar to the IRF proteins ICSBP and ISGF3 gamma but is equally divergent from both. Gel mobility shift assays indicate that cIRF-3 is capable of binding a known IFN-stimulated response element that is conserved between the mammalian and chicken Mx genes. Expression of the cIRF-3 gene can be induced to high levels by poly(I).poly(C). Induction is rapid and transient with no requirement for protein synthesis. Co-treatment of cells with cycloheximide results in superinduction of cIRF-3 mRNA. The structural and regulatory characteristics of cIRF-3 indicate that it is the first example of a non-mammalian IRF protein.
Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [ 3 H]25-hydroxycholesterol with an apparent K m of 0.7 M. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase mRNAs. Comparison of wild-type and ABCA1 Ϫ/Ϫ murine fibroblasts indicates that 25-hydroxycholesterol is effluxed ϳ5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1 Ϫ/Ϫ fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor. active transport; cholesterol homeostasis THE ATP BINDING CASSETTE (ABC) protein, ABCA1, was identified during a search for novel ABC proteins expressed in macrophages and at the time of its discovery, its function was unknown (34). Defects in the ABCA1 gene were subsequently shown to be the cause of Tangier disease, which is characterized by a lack, or abnormally low level, of high-density lipoprotein (HDL) and a markedly increased risk of coronary artery disease (5,7,11,56). Studies (2, 6, 14) using knockout mice have confirmed that ABCA1 plays a pivotal role in reverse cholesterol transport and in vitro, increased expression of the protein in several cell types results in elevated net efflux of cellular cholesterol and phospholipids. This efflux is dependent on the presence of an acceptor such as lipid-poor HDL or apolipoprotein A (apoA)-I (19, 70). Other proteins can also act as acceptors although their physiological relevance is presently unclear (46).The mechanism by which ABCA1 mediates efflux of cholesterol and phospholipids remains poorly defined. ABC transporters typically use the energy of ATP binding and hydrolysis to drive the transport of substrate across cellular membranes (26). In the case of hydrophobic compounds, transport is thought to involve ...
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