👤 Kirsten Prüfer

Liver X receptors (LXRs) alpha and beta are nuclear receptors, which form obligate heterodimers with the retinoid X receptor (RXR). The LXRs regulate both redundantly and non-redundantly the transcription of genes controlling cholesterol metabolism and transport as well as lipogenesis. Previously, we showed that mutations in putative N-terminal nuclear localization sequences (NLSs) within both LXRs inhibit nuclear import. Through in vitro studies, we show here that these NLSs bind importin alpha and are both necessary and sufficient for the nuclear import of LXRs. Imaging, transactivation, and electro-mobility shift experiments show that RXR rescues the nuclear import of the LXRalpha NLS mutant yet does not restore its transcriptional activity despite intact DNA binding. In contrast, RXR partially rescues the import of the LXRbeta NLS mutant, but has no effect on its transcriptional activity due to the loss of DNA binding. Experiments with NLS mutant RXR confirmed that RXR may dominate the nuclear import of the RXR/LXRalpha heterodimer, whereas LXRbeta dominates the nuclear import of the RXR/LXRbeta heterodimer. Intriguingly, our data indicate differences between LXRalpha and LXRbeta in their interaction with RXR and in the role their NLSs play in transactivating functions independent of nuclear import. Show less

The liver X receptors (LXR) alpha and beta are ligand-induced transcription factors that regulate the expression of genes important for cholesterol metabolism, lipogenesis, and other metabolic pathways. Despite their high degree of similarity, LXRs have redundant as well as nonredundant functions. The regulation of LXRs' intranuclear mobility most likely plays a major role in the regulation of their transcriptional activities. In order to elucidate how ligand binding, receptor-protein and receptor-DNA interactions affect intranuclear receptor mobility, we expressed transcriptionally active yellow fluorescent protein (YFP)-LXR alpha and YFP-LXR beta in Cos-7 cells. We used the fluorescence recovery after photobleaching (FRAP) technique and confocal laser scanning microscopy as well as Triton X-100 permeabilization experiments and fluorescence microscopy to measure differences in the intranuclear mobility between LXR alpha and LXR beta. The image analyses revealed that after agonist binding, LXR alpha exhibits slower intranuclear trafficking and greater intranuclear immobilization compared with LXR beta. In addition, mutational analysis showed that the integrity of the Activation Function (AF)-2 region of LXR alpha is essential for its immobilization whereas the integrity of the DNA binding domain is not. These findings imply that specific protein interactions with the AF-2 region of LXR alpha play a role in its intranuclear immobilization. Show less