Basis of a High-Throughput Method for Nuclear Receptor Ligands

• Published in: Journal of biochemistry (Tokyo) Vol. 133; no. 6; pp. 791 - 797
• Main Authors: Kanayama, Tomohiko, Mamiya, Satoru, Nishihara, Tsutomu, Nishikawa, Jun-ichi
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-06-2003
• Subjects: alkaline phosphatase; Alkaline Phosphatase - drug effects; Alkaline Phosphatase - metabolism; Biological Assay; Coenzyme A - genetics; Coenzyme A - metabolism; Drug Evaluation, Preclinical - methods; Escherichia coli - genetics; estrogen receptor; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; glutathione-S-transferase; GST; hormone receptor; human nuclear receptor; Humans; in vitro screening method; IPTG; isopropyl-β-d-thiogalactopyranoside; Key words: endocrine disruptor; LBD; ligand binding domain; Ligands; Male; nuclear receptor; Nuclear Receptor Coactivator 2; Protein Binding; RAR; Receptors, Cytoplasmic and Nuclear - drug effects; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; retinoic acid receptor; retinoid X receptor; RXR; SDS-PAGE; SDS–polyacrylamide gel electrophoresis; Sensitivity and Specificity; thyroid hormone receptor; TIF2; Transcription Factors - genetics; Transcription Factors - metabolism; transcriptional intermediary factor 2; VDR; vitamin D receptor; Xenobiotics - metabolism; Xenobiotics - pharmacology
• ISSN: 0021-924X
• DOI: 10.1093/jb/mvg101

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 (ERα/β, TRα, RARα/γ, RXRα,and VDR) and confirmed that the method works well.