Abstract
The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.
Highlights
The pharmacological actions of morphine and morphinelike drugs such as heroin are mediated primarily through the opioid receptor
We reported that Oprm1 transcription is regulated by a cis-acting single strand DNA sequence in the mouse Oprm1 promoter through the binding of Poly(C)-binding protein 1 (PCBP1) [17, 19]
Our earlier studies showed that mouse Oprm1 transcription was regulated by a cis-acting single strand DNA sequence that was essential for the activity of the mouse Oprm1 promoter through the binding of ␣CP1 (i.e. PCBP1) [19, 30]
Summary
Plasmid Construction and in Vitro Translation—A luciferase fusion plasmid (pGL450; Ϫ450 to ϩ1 bp, relative to the translation start site (ϩ1) of the mouse Oprm1) was generated by ligating the PCR product (Ϫ450 to ϩ1) into the SacI and HindIII sites of pGL3-basic (Promega, Madison, WI). The end-labeled DNA probes were incubated with in vitro translated proteins in a final volume of 20 l of EMSA buffer (10 mM Tris (pH 7.5), 5% glycerol, 1 mM EDTA (pH 7.1), 50 mM NaCl, 1 mM DTT, and 0.1 mg/ml poly(dI-dC)) at room temperature for 20 min. To eliminate nuclear proteins that might bind nonspecifically, control experiments were performed by mixing 5000 pmol of nonbiotinylated single strand DNA (10ϫ competitor) with 1 mg of nuclear proteins for 15 min on ice. The nuclear extracts containing the 10ϫ competitor were added to the affinity particles and incubated for 1 h at 4 °C. The IPG strips were incubated for 15 min with an equilibration solution (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), and 2% DTT (w/v)) followed by equilibration for another 15 min in the same buffer containing 2.5% iodoacetamide (w/v) inmagnet.
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