Identification, Expression And Nucleocytoplasmic Shuttling Analysis Of Androgen Receptor In The Rice Field Eel

Androgens are essential for male sexual differentiation, maturation, and maintenance of secondary male sexual characteristics. Biological effects of androgens are mediated by androgen receptors (Evans,1988; Mangelsdorf et al.,1995). Androgen receptor (AR) belongs to a large family of nuclear receptors, which play key roles in regulation of gene transcription in processes of reproduction, development and metabolism. Androgen receptors contain a hypervariable N-terminal transactivation domain (TAD), a highly conserved DNA-binding domain (DBD), a hinge region and a moderately conserved C-terminal ligand-binding domain (LBD).Here we report isolation and characterization of an isoform of AR of the rice field eel, and anlyze AR mRNA levels in various tissues and in different gonads during sex reversal. We reveal a conserved NLS of AR protein of the rice field eel within DBD and Hinge region. Furthermore, a critical ligand binding sequence and a NES sequence were identified in the LBD of the AR. Our studies provide a new insight into mechanism of fish AR in sexual differention.1. Molecular cloning of AR gene in the rice field eelAndrogen receptor cDNA of the rice field eel was cloned by degenerate PCR and RACE techniques. The multiple alignments showed that the TAD and DBD of the AR was highly homologous to other known ARs of vertebrates, and the LBD was lowly homologous to the others. Phylogenetic tree construction revealed that AR protein of the rice field eel was close to sea bass AR with the highest homology.2. Expression pattern of AR gene in the rice field eelBoth semi-quantity RT-PCR (reverse transcription-PCR) and real-time RT-PCR results indicated that AR was expressed ubiquitously in adult tissues, and the expression was higher in liver and much lower in brain. AR expression showed an increasing tendency during the gonadal transformation of the rice field eel from female via intersex to male. To examine the expression sites of AR in gonads, in situ hybridization was performed onto gonadal sections. AR expression was observed in leydig cells and developing germ cells, especially in developing oocytes, spermatogonia and spermatocytes. These results suggest that AR potentially has important roles in gonad differentiation and spermiogenesis during sex reversal of the rice field eel.3. Identification of NLS in the DBD and hinge regionThe unliganded AR of rice field eel was localized predominantly in cytoplasm and partially translocated into nuclei in the presence of DHT. Ligand binding was an outer condition for AR nuclear localization, while NLS sequence localized in AR was an inner condition. Comparison of available DBD and hinge region from different vertebrates including the rice field eel revealed a highly conserved two basic clusters (KK and RKLKK) between amino acids 448 and 464 in eel AR, which corresponded to bipartite NLS (RK and RKLKK) in the DBD and hinge region of human AR. Deletion mutants, which included NLS sequence, could efficiently target the GFP to the nuclei completely. Deletion mutant, which lack of NLS sequence, showed even localization between cytoplasm and nuclear. These results suggested that NLS sequence between 448 and 464 was important for eel AR nuclear localization. Site-direceted mutant revealed that K461, K463, K464 were pivotal for rice field eel AR nuclear localization. The NLS of eel AR is important for its nuclear localization and transactivational function.4. Identification of NES in the LBDGFP-AR translocated into nuclei in the presence of DHT, and back to cytoplasm by DHT withdrwal, indicating the exsitance of a NES in the eel AR. Different deletion mutants were performed to identified a NES sequence within amino acid residues 553-575 in LBD of rice field eel AR. The NES was necessary for rice field eel AR nuclear export and was dominant over the NLS in the DBD and hinge region in the absence of ligand. A Leucine-rich sequence, which was similar to L-x(2,3)-[LIVFM]-x(2,3)-L-x-[LI], was found in the NES, site-direceted mutants revealed that the Leu or Val within this region is not critical to the cytoplasmic localization of eel AR. LMB treatment experiment suggested that the nuclear export of rice field eel AR was CRM1-indepentdent.5. Definition of critical amino acids for ligand binding in the LBDSimulation model of the 3D structure of eel AR LBD exhibiting a ligand-binding pocket (LBP), which composed of eel helix 3,4, and 5, may be critical for DHT recognition and binding. Comparison the transcriptional activity of rice field eel AR and human AR following DHT expoure, we found that the transcriptional activity of human AR was higher than that of eel AR, indicating that DHT binds to eel AR with low efficiency. This conclusion was further supported by comparing the nuclear translocation of mouse AR and eel AR following DHT expoure. Deletion mutant and site-directed mutant in eH5 and Site-directed mutant in eH4 showed restriction in transactivity and nuclear import following DHT exposure. The a-helix 3 was conserved between human and the rice field eel, Seven of eight key point mutations, which leaded to low or no binding capacity to ligands were identical between human and the rice field eel. These results indicated that eH3, eH4 and eH5 were critical for the ligand binding.The results clue to an important role for androgens to regulate sexual differentiation and sexual maturation through AR in rice field eel.