RESUMO
Several members of the nuclear receptor superfamily including RXR (retinoid X receptor) bind to a specific retinoic acid response element (site A) of the apoAI promoter. However, transcriptional activation of the apoAI gene by different homo- and heterodimeric forms of RXR or RAR (retinoic acid receptors) cannot be evaluated in mammalian cells, which contain endogenous RXR or RAR. In order to circumvent this limitation, we assessed the DNA-binding activities and transcriptional activation of different homo- and heterodimers of these receptors in yeast. Electrophoretic mobility shift assays (EMSA) demonstrated that yeast expressed RARalpha does not bind to site A of the apoAl promoter, whereas binding of RARbeta to site A is ligand-dependent. Both RARalpha and RARbeta form heterodimers with RXRalpha and bind to site A with high affinity. These DNA-binding studies correlate with the transcriptional data, which indicated that RARbeta but not RARalpha activates transcription from site A in response equally well to 9-cis and all-trans retinoic acids. 9-cis RA is a more potent ligand than all-trans RA to activate transcription via RXR/RAR heterodimers. We conclude that this yeast expression system is a useful tool to elucidate the 'transactivation code' for apoAl site A via specific combinations of different homo and heterodimeric versions of RXR and RAR.
Assuntos
Apolipoproteínas A/genética , Receptores do Ácido Retinoico/genética , Fatores de Transcrição/genética , Ativação Transcricional , Sequência de Bases , Técnicas de Transferência de Genes , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Saccharomyces cerevisiae/genéticaRESUMO
An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.
