Characterization and identification of a steroid receptor-binding protein, SRB-RGS.
We cloned the cDNA of a novel steroid receptor-binding protein, SRB-RGS, which suppressed the estrogen receptor (ER)alpha-mediated and other promoter-driven transcriptional activities. This study revealed the interaction between the full-length SRB-RGS and full-length ERalpha or ERbeta by a coimmunoprecipitation assay. The full-length SRB-RGS and full-length ERalpha 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 observed by the gel-shift assay. These results strongly suggested that SRB-RGS interacts with ERs bound to DNA (estrogen response element) in the nuclei of the cells. SRB-RGS suppressed very efficiently the ERalpha-, ERbeta-, and ERalpha+ERbeta-mediated transcriptional activities. Green fluorescence of enhanced green fluorescence protein (EGFP)-tagged SRB-RGS was localized both in the nucleus and in the cytoplasm. Intrinsic SRB-RGS was immunostained in the nucleus and the cytoplasm of HeLa cells. The putative SRB-RGS deduced from cDNA sequence was identified by the immunostaining and Western blotting by using the anti-SRB-RGS antibody. Overexpression of SRB-RGS induced the cell death in the HeLa cells. The nucleotide sequence of SRB-RGS cDNA that we cloned previously is identical with that of the newly isolated RGS3 cDNA. SRB-RGS could interact with ERs bound DNA in the nuclei of the cells and suppressed the ERs-mediated transcriptional activities.
Ikeda
Mitsunori
M
Department of Biochemistry, Kawasaki Medical School, Kurashiki, Okayama, Japan. ikeda@bcc.kawasaki-m.ac.jp
Inoue
Satoshi
S
Muramatsu
Masami
M
Minatogawa
Yohsuke
Y
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D016923:Cell Death; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D011485:Protein Binding; D020710:RGS Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism*; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D020710:RGS Proteins; D011960:Receptors, Estrogen; D011987:Receptors, Steroid; D012097:Repressor Proteins; C437714:Rgs3 protein, rat; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase