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J. Biochem, 2003, Vol. 133, No. 6 791-797
© 2003 Japanese Biochemical Society

BIOTECHNOLOGY

Basis of a High-Throughput Method for Nuclear Receptor Ligands

Tomohiko Kanayama, Satoru Mamiya, Tsutomu Nishihara and Jun-ichi Nishikawa+

Laboratory of Environmental Biochemistry, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871

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 (ER{alpha}/ß, TR{alpha}, RAR{alpha}/{gamma}, RXR{alpha},and VDR) and confirmed that the method works well.

+ To whom correspondence should be addressed. Tel: +81-6-6879-8241, Fax: +81-6-6879-8244, E-mail: nisikawa{at}phs.osaka-u.ac.jp


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