title: Basis of a high-throughput method for nuclear receptor ligands.
abstract: Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.
authors.affiliation: Laboratory of Environmental Biochemistry, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Kanayama
authors.forename: Satoru
authors.initials: S
authors.lastname: Mamiya
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nishihara
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Nishikawa
mesh_terms: D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism*; D001681:Biological Assay; D003065:Coenzyme A / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008297:Male; D051958:Nuclear Receptor Coactivator 2; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015262:Xenobiotics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008024:Ligands; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015262:Xenobiotics; D005982:Glutathione Transferase; D000469:Alkaline Phosphatase; D003065:Coenzyme A