Intracellular localization of RORα is isoform and cell line-dependent

AbstractThe retinoid-related orphan receptor α (RORα) belongs to the nuclear receptor superfamily and comprises four isoforms generated by different promotor usage and alternative splicing. To better understand its function, the subcellular distribution of RORα was investigated. We could show that subcellular distribution of RORα is cell line and isoform-dependent. Isoform specific differences were mediated by the A/B domains which with the exception of RORα1 contain a signal that mediates cytoplasmic localization. The lack of this signal in RORα1 results in a complete nuclear localization and prevents cell membrane association observed for RORα2, 3, and 4. The region responsible for membrane association was identified as the C-terminal α-helix 12. Furthermore, the hinge region/ligand binding domain mediates nuclear localization. Our results show that isoform specific activity of RORα is not only regulated by different expression and DNA binding affinities but also by different subcellular distribution. Different access to the nucleus reveals an important mechanism regulating the activity of this constitutively active nuclear receptor

Overexpression, refolding, and purification of polyhistidine-tagged human retinoic acid related orphan receptor RORα4

RORα4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORα4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6 M guanidinium–HCl and renatured by gradual removal of guanidinium–HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20 mg of pure and native RORα4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORα in mice with high sensitivity and specificity