| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2002: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
We have isolated a cDNA coding for a novel steroid receptor coactivator protein termed SRAP from a rat prostate library. The nucleotide sequence of the SRAP has 78.2 % identity to that of the human steroid receptor RNA activator (SRA), a novel RNA molecule which was reported to act as an RNA transcript without being translated into protein [Lanz, R. B., McKenna, N. J., Onate, S. A., Albrecht, U., Wong, J., Tsai, S. Y., Tsai, M. -J. and O'Malley, B. W. (1999) Cell 97, 17-27]. However, the cDNA of SRAP is capable of generating a functional protein, Glutathione S-transferase (GST) pull-down assays showed that SRAP interacts with the partial androgen receptor (AR) protein composed of a DNA binding domain (DBD) and an activation function 2 (AF-2). Luciferase assays demonstrated that SRAP enhances the transactivation activity of the androgen receptor (AR), the glucocorticoid receptor (GR) and the peroxisome proliferator-activated receptor γ_1 (PPARγ_1) a ligand-dependent manner. Using a GFP (green fluorescent protein) fusion protein construct, we showed in vivo translation of the GFP-SRAP fusion protein in HeLa cells cotransfected with pSG5AR and reporter gene in the presence of 5α-dihydrotestosterone (DHT). Cotransfection of the GFP-SRAP fusion protein expression plasmid enhanced the activity of AR whereas incorporation of mutations in SRAP of the fusion protein resulted in loss of enhancement of the transactivation activity. Northern blot analysis and RT-PCR showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and human prostate cancer cells, DU-145, cotransfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by an antiandrogen flutamide, but the transactivation activities remained high even in the presence of 5 μM flutamide suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer.
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