Members of the ligand-induced nuclear receptor superfamily are medically important regulators of cellular activity. Hormones initiate a receptor activation process leading to receptor redistribution and binding to specific DNA recognition sites in the promoter regions of their target genes. Using microscopy and fluorescent protein chimeras of nuclear receptors, we pioneered studies establishing that ligand binding regulates the subcellular targeting of glucocorticoid (GR), vitamin D (VDR), and retinoid X receptors (RXR). Our studies demonstrated that ligand binding induces formation of multiple nuclear foci of GFP-GR, GFP-VDR, YFP-RXR, and GFP-ER. Mutational analysis demonstrated a correlation between hormone-dependent nuclear foci formation and DNA binding for both the GFP-VDR and the GFP-GR. This notion was further supported by our co-localization and fluorescence energy transfer experiments (FRET). After calcitriol treatment, GFP-VDR and RXR-BFP co-localized in the nuclear foci and FRET demonstrated formation of VDR/RXR dimers at these foci. Dimerization incompetent GFP-VDR and RXR-BFP failed to co-localize and failed to generate FRET signal at the foci. Because VDR and RXR bind to DNA as heterodimer, this finding suggested a correlation between focal receptor accumulation and DNA-binding. Furthermore, dynamic microscopy experiments such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) revealed that both VDR and RXR move rapidly within the nucleus and that hormone binding slows this intranuclear movement. Our ongoing studies are aimed to identify the protein responsible for keeping nuclear receptors in motion within the nucleus and to identify the spatial and temporal relationships between type II nuclear receptors and corepressors. The second aim of our studies is to elucidate the roles of receptor trafficking between the cytoplasm and the nucleus in the regulation of hormone actions. Our dynamic microscopy and cell permeabilization experiments demonstrated that liganded and unliganded YFP RXR and GFP-VDR both shuttle rapidly between the cytoplasm and the nucleus. Using mutational analysis, we identified two nuclear localization signals (NLSs) in the DNA-binding region of the RXR and clarified the contribution of several putative NLSs in the nucleocytoplasmic shuttling of VDR. We also identified regions of VDR and RXR significant for export. In addition, export of the unliganded GFP-VDR was sensitive to treatment with leptomycin B (LMB), an inhibitor of cargo protein binding to the Crm-1 export receptor. Experiments with NLS and NES mutants of VDR and RXR demonstrated that redirection of VDR and RXR signaling with either nuclear exclusion or retention results in the development of abnormal hormone-induced transcriptional regulation. Our results indicated that both VDR and RXR import contributes to calcitriol induced transcriptional activities. Moreover, our results provided the first demonstration of the importance of export for transcriptional activity of a nuclear receptor. Our studies have changed the paradigm of VDR and RXR localization within the cell and introduced a dynamic model for VDR activation. Ongoing studies are aimed to understand the roles of direct and indirect receptor export in the mechanism of hormone actions and expand our mutational analysis to other members of the type II nuclear receptor family.
NIH ExPorter
NIH ExPorter