Analysis of the subcellular trafficking of the glucocorticoid receptor and properties of the ligand binding domain

The glucocorticoid receptor (GR) is a ligand dependent transcription factor and member of the nuclear receptor superfamily. Nuclear import and export of transcription factors is accomplished through nuclear localization signals (NLS) and nuclear export signals (NES), respectively. We have determined that L687 and L690 of rat GR are necessary for the characteristically slow nuclear export of GR and may be included in the signal sequence responsible for directing post-agonist withdrawn GR from the nucleus to the cytoplasm. We also suggest that L687 and L689 of rat GR are required for efficient NL2-mediated nuclear translocation. Substitutions L687A and L689A mildly affect steroid binding and steroid off-rate, yet significantly increase the concentration of steroid required for inducing nuclear import of naive GR. When introduced into GRNL1-, these substitutions compromise the receptor's ability to transfer to the nucleus, suggesting they partially abrogate NL2 activity. We have also observed that NL1-dependent transfer does not begin until 10 -7M steroid, demonstrating that NL2 is the primary physiological mediator of GR nuclear import