P-84: Characterization of Androgen Receptor Structure and Nucleocytoplasmic Shuttling of the Rice Field Eel

Background: Androgen receptor (AR) plays a critical role in prostate cancer and male sexual differentiation.Mechanisms by which AR acts and regulations of AR nucleocytoplasmic shuttling are not understood well. Materials and Methods: Degenerate PCR and RACE Cloning of AR Gene; Phylogenetic Analysis and Molecular Modeling;Real-time Fluorescent Quantitative RT-PCR; Northern Blot Hybridization;In Situ Hybridization Analysis;Cell Preparation, Transfection, and DHT/ leptomycin B (LMB) Treatments; Transcriptional Activity Analysis; GST Pull-down; DHT Binding Assay. Results: We have identified AR from a primitive vertebrate with a sex reversal characteristic, the rice field eel. AR of this species (eAR) is distinct from human AR, especially in the ligand binding domain (LBD), and its expression in gonads shows an increasing tendency during gonadal transformation from ovary via ovotestis to testis. eAR has a restricted androgen-dependent transactivation function after a nuclear translocation upon dihydrotestosterone exposure. A functional nuclear localization signal was further identified in the DNA binding domain and hinge region. Although nuclear export is CRM1-independent, eAR has a novel nuclear export signal, which is negatively charged, indicating that a nuclear export pathway may be mediated by electrostatic interaction. Further, our studies have identified critical sequences for ligand binding in the C terminus. A structure of three -helices in the LBD has been conserved from eels to humans during vertebrate evolution, despite a distinct amino acid sequence. Mutation analysis confirmed that the LBD is essential for dihydrotestosteroneinduced nuclear import of eAR and following transactivation function in the nucleus. In addition, eAR interacts with both Sox9a1 and Sox9a2, and their interaction regulates transactivation of eAR. Conclusion: Our data suggest that the primitive species conserves and especially acquires key novel domains, the nuclear export signal and LBD, for the eAR function in spite of a rapid sequence evolution.