We have cloned a cDNA coding for a novel steroid receptor co-activator protein termed SRAP from a rat prostate library. Although 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, McKenna, Onate, Albrecht, Wong, Tsai, Tsai and O'Malley (1999) Cell 97, 17—27], the cDNA of SRAP is capable of generating a functional protein. Glutathione S-transferase pull-down assays showed that SRAP associates with the partial androgen receptor (AR) protein composed of a DNA-binding domain and an activation function 2. Luciferase assays demonstrated that SRAP enhances the transactivation activity of the AR, the glucocorticoid receptor and the peroxisome proliferator-activated receptor γ1 in a ligand-dependent manner. Using a green fluorescent protein (GFP) fusion-protein construct, we demonstrated in vivo translation of the GFP-SRAP fusion protein in HeLa cells co-transfected with pSG5AR and reporter gene in the presence of 5α-dihydrotestosterone (DHT). Co-transfection of the GFP-SRAP fusion protein expression plasmid enhanced the transactivation 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 reverse transcriptase PCR assays showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and the human prostate cancer cells line DU-145, co-transfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by the anti-androgen flutamide, but the transactivation activities still remained high even in the presence of 5μM flutamide, suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer.
Abbreviations used: AIB1, a steroid receptor co-activator amplified in ovarian and breast cancer 1; AF, activation function; AR, androgen receptor; ARA, AR-associated protein; CBP, CREB-binding protein; DBD, DNA-binding domain; DHT, 5α-dihydrotestosterone; ER, oestrogen receptor; FHL2, four and a half LIM domains 2; GFP, green fluorescent protein; GR, glucocorticoid receptor; GST, glutathione S-transferase; MMTV, mouse mammary tumour virus; ORF, open reading frame; PPAR, peroxisome proliferator-activated receptor; RT-PCR, reverse transcriptase PCR; SRA, steroid receptor RNA activator; SRAP, steroid receptor co-activator protein; SRC, steroid receptor coactivator; TIF, transcriptional mediator/intermediary factor.
The nucleotide sequence reported in this paper has been submitted to the GenBank Nucleotide Sequence Database under accession number AY026354.