Identification and characterization of physically interacting partners of retinoic acid receptor alpha in Sertoli cells

Vitamin A is essential for male reproduction as demonstrated by the sterility of the rats fed with vitamin A-deficient diet. The vitamin A signal is mediated by two families of retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Genetic studies have shown that RARA is critical for spermatogenesis, as indicated by the sterility of the Rara-null mice. RARA mediates the transcription of target genes, but it can be regulated by many factors such as posttranslational modifications and coregulators. The studies presented herein were to understand mechanisms by which modulators affect the functions of RARA. We performed a yeast two-hybrid screening to identify RARA-interacting proteins with RARA as the bait and proteins from a Sertoli cell cDNA library as the prey, and obtained 15 candidates, of which small ubiquitin-related modifier-2 (SUMO-2) and glucose-regulated protein 58 (GRp58) were further investigated.;We identified SUMO-2 modification of RARA as a novel posttranslational modification for RARA. The atRA ligand and the amount of SUMO-2 are two key components regulating sumoylation of RARA. SUMO-2 modification of RARA affects its stability, nuclear localization and transcriptional activity. Furthermore, we also characterized the subcellular localization and protein expression of SUMO-2/3, 95% similar to each other, in testes. We found that different RA signaling pathways modulated the expression and subcellular localization of SUMO-2/3 in testes. These studies demonstrate that the relationship among RA, RARA and SUMO-2 is finely tuned in testes, and manipulation of SUMO-2 could be an alternative strategy to control the functions of RARA.;We also demonstrated that GRp58 is a novel molecular chaperone required for nuclear localization of RARA. The atRA was able to mobilize both GRp58 and RARA into the nucleus, and then GRp58 accompanied RARA to the ER for degradation, before it could de-couple from RARA and recycle back to the cytoplasm. Studies with sulfhydryl modifying agents allowed us to speculate that the thiol-oxidoreductase activity of GRp58 on cysteine residues in RARA might be important for ligand binding to RARA and nuclear localization of RARA. These studies indicate manipulation of GRp58 could be an alternative mechanism to modulate the functions of RARA.