Mechanisms of nucleocytoplasmic trafficking of the glucocorticoid receptor

Abstract

Nuclear hormone receptors (NHRs) are ligand-inducible transcription factors that regulate the activity of a wide variety of target genes. The glucocorticoid receptor (GR) is a NHR that is found predominantly in the cytoplasm prior to ligand binding. Nucleo-cytoplasmic shuttling has been proposed to play an important role in the regulation of NHR function by allowing for continuous communication between the nuclear receptor and cytosolic signalling pathways. To study shuttling in a setting that resembles the cellular environment I developed a fluorescence recovery after photobleaching (FRAP) technique using randomly occurring multinucleated Cos7 cells. We found little if any shuttling of the liganded receptor 4 h after photobleaching, which was consistent with the slow rate of redistribution following ligand withdrawal but not consistent with the previously described rapid export that is believed to underlie nucleocytoplasmic shuttling of GR. This persistent nuclear localisation of the liganded receptor was confirmed using two independent assays. However, experiments performed in cells fused by treatment with polyethylene glycol (4000) confirmed earlier reports that liganded GR shuttles rapidly between nucleus and cytoplasm in a manner that is dependent upon direct binding of GR to calreticulin. In contrast, disruption of calreticulin binding did not abrogate the slow export of GR observed by FRAP. Further, redistribution of the receptor to the cytoplasm following ligand withdrawal was not dependent on calreticulin binding. Our results suggest that cell fusion activates a specialised mechanism of GR export that is dependent on calreticulin, and that under normal cellular conditions NHRs may not be able to respond rapidly to cytoplasmic signalling pathways. Additionally, our findings demonstrate that nucleocytoplasmic shuttling as suggested by cell fusion-based assays may not always reflect trafficking behaviour in a normal cell. Within the course of these studies, we noted that the slow rate of GR nuclear export is accelerated upon mutation of the GR nuclear localisation sequence 1 (NL1). This suggested that in addition to mediating nuclear import of GR by binding with importin alpha, the NL1 sequence might function to retain GR in the nucleus. We determined that the hinge region of GR, which includes the NL1 sequence, was sufficient to efficiently repress export mediated by the CRM1-dependent HIV Rev nuclear export sequence (NES). (Abstract shortened by UMI.)