|Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Glucocorticoid-receptor (GR), a member of the superfamily of ligand-dependent transcription factor, exerts its functions by binding to specific DNA sequences named glucocorticoid-response elements (GREs). Possibly, after binding to the GREs, GR interacts directly or indirectly with chromatin remodeling complexes and coactivators, such as SCR-l/NcoAl, p300/CBP, GRIP-1/TIF-2/NcoA2, Bafl 55, hnRNP U and hBRGl/BAFs that may contribute to the transcriptional activation of organized chromatin template. Furthermore, we found that GR interacts directly with a macromolecular-translocation inhibitor III.This protein may play an important role in the regulation of the gene transcription by glucocorticoid hormone, especially in the "switch off" mechanism. Moreover, we found that GR directly binds to histone. GR has higher affinity for histones H3 and H4 than for histones H2A and H2B, negligible affinity for histone H1. Thus, the interactions of GR with other proteins are important regulatory steps of hormone-dependent activation of gene transcription, but it is not clear how GR interacts with these regulatory molecules, which part of the GR domain structures involves in these protein-protein complex formations, and how these complexes function.
GR protein consists of three distinct functional domains. The N-terminal region of the GR have the major transactivation activity. The central region is the DNA binding domain (DBD) containing two zinc finger motifs, and the C-terminal region is the steroid binding domain. Each domain consists of several subdomains and is suggested to interact with different proteins. To investigate the GR-protein interactions precisely in vitro, it is prerequisite to obtain highly purified GR protein in abundance. Thus, we tried to isolate cDNA coding rat GR by reverse transcriptase-polymerase chain reaction (PCR) amplification, and inserted it into E.coli expression vector and eukaryotic expression vector.