Biol. Pharm. Bull. 30(6) 1056—1064 (2007)

gand-inducible transcription factors. They include the receptor proteins for steroids, thyroid hormone, vitamin D, and retinoids. Steroid hormone receptors are structurally related and composed of six major functional domains. Domain A/B in the NH2-terminal region of the protein is the constitutive activation 1 (AF-1). Domain C, the DNA-binding domain, is arranged in two zinc-stabilized DNA-binding finger motifs. Region D contains a nuclear localization signal. The ligandbinding and ligand-dependent transcriptional activation function 2 (AF-2) is located in domain E/F of the COOH-terminal region of the protein. For the activation of transcription, steroid hormone receptors recruit coactivators such as SRC-1/p160, p300/CBP, ARA70, Tip60, and RIP140, through binding to AF-1 and AF-2 of the receptors. This kind of coactivator recruits histone acetyltransferase (HAT) or bear HAT activity on itself. Recruitment of HAT allows the local decondensation of chromatin. In the next step, the DRIP/TRAP complex binds to the AF-2 of the receptor and mediates transcriptional activation. This kind of coactivator does not recruit HAT. Thus, these coactivators facilitate the transcription process. The thyroid hormone receptor (TR) and retinoid X receptor (RXR) in the absence of ligand bind to the corepressors, such as N-CoR and SMRT, respectively, suppressing transcriptional activity. The corepressors recruit histone deacetylase (HDAC). The chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes regulate chromatin structure and transcription so that they might be coordinated to create a DNA template that is accessible to the general transcription apparatus. We employed a yeast two-hybrid system for cloning the cDNA of the protein that interacted with domains C and D of the rat estrogen receptora (rERaC/D) from the rat ovary cDNA library and cloned the cDNA of a novel steroid receptor-binding (SRB) protein bearing the regulator of the G-protein signaling (RGS) domain at the COOH-terminal, designated as SRB-RGS. RGS proteins are GTPase accelerating proteins, which interact with the Ga-subunits that are linked to seven transmembrane G-protein coupled receptor. SRBRGS bears a PDZ domain that is a multi-functional protein– protein interaction module that plays important roles in organizing signal transduction complexes, clustering membrane proteins, and maintaining cell polarity at the NH2-terminal, as well. rERaC/D interacted in vitro with partial SRB-RGS1-495 amino acid (a.a.). SRB-RGS suppressed the transcriptional activities of ERa and the other promoters. We could show that the full-length SRB-RGS interacted with either the full-length ERa or ERb by a coimmunoprecipitation assay. The full-length SRB-RGS and full length ERa interacted in COS-7 cell by a mammalian two-hybrid system. The interaction between intrinsic SRB-RGS and ERs in the nuclear ER extract from the rat uteri was suggested by the gel-shift assay. The human (h) ERa-, hERb-, hERa hERb-mediated transcriptional activities were suppressed by overexpression of SRB-RGS. Enhanced fluorescence protein (EGFP)-tagged SRB-RGS was localized in the nucleus and the cytoplasm of the HeLa cells and the COS-7 cells. Similar results were shown by immunostaining of the HeLa cells. Intrinsic SRB-RGS was identified by the immunostaining and Western blotting. Overexpression of SRB-RGS induced the cell death in the HeLa cells. SRB-RGS could interact with ERs bound DNA in the nucleus and suppressed the ERs-mediated transcriptional activities. 1056 Vol. 30, No. 6