Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity.

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

Stabilization and cytoskeletal-association of LDL receptor mRNA are mediated by distinct domains in its 3' untranslated region.

The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.

cIRF-3, a new member of the interferon regulatory factor (IRF) family that is rapidly and transiently induced by dsRNA.

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.

Developmental regulation of specific protein interactions with an enhancerlike binding site far upstream from the avian very-low-density apolipoprotein II gene.

Expression of the avian very-low-density apolipoprotein II (apoVLDLII) gene is completely dependent on estrogen and restricted to the liver. We have identified binding sites for nonhistone nuclear proteins located between -1.96 and -2.61 kilobases. One of these sites, located at -2.6 kilobases (designated site 1), was found to span an MspI site that becomes demethylated between days 7 and 9 of embryogenesis, the stage of development at which competence to express the apoVLDLII gene begins to be acquired. Levels of the factor(s) involved were high at day 7 of embryogenesis, decreased two- to threefold by days 9 to 11, and continued to decline more slowly until hatching. Furthermore, the mobility of the complex formed underwent a well-defined shift between days 11 to 13 embryogenesis. Methylation interference studies showed that modification of the outer guanosines of the MspI site resulted in marked inhibition of the formation of the protein-DNA complex. Competition studies, fractionation of nuclear extracts, and tissue distribution indicated that the factor was not the avian homolog of hepatocyte nuclear factor 1, nuclear factor 1, or CCAAT/enhancer-binding protein (C/EBP). However, site 1 could complete for binding to an oligonucleotide, previously shown to be recognized by C/EBP, in a nonreciprocal fashion. These studies demonstrate that the sequence recognized by the protein includes a C/EBP consensus sequence but that elements in addition to the core enhancer motif are essential for binding.