Characterization of nuclear import of the glucocorticoid receptor mediated by the nuclear localizaton signal in the ligand binding domain.

Abstract

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors. Before exposure to hormone, GR is situated in the cytoplasm in a heterocomplex consisting of heat shock proteins and immunophilins. Upon addition of hormone, GR dissociates from this complex and translocates to the nucleus. After hormone withdrawal, GR returns to the cytoplasm where it is found once again associated in a heterocomplex. GR contains two nuclear localization signals (NLS) in its primary sequence. Nuclear localization signal 1 (NL1), a hormone-independent NLS, is located in the hinge region from amino acid residue 486 to 524 in rat GR. Nuclear localization signal 2 (NL2), a hormone-dependent NLS, is found in the ligand binding domain but its sequence has not been delimited further. To determine the contribution by NL2 to the nucleocytoplasmic trafficking of the full length GR, a GR mutant (mutGR) with no NL1 activity was generated by mutation of the three lysine (K) residues at amino acid residues 513 to 515 to asparagine (N). Characteristics of nuclear uptake and occupancy of the full length wildtype receptor (wtGR) and mutGR were determined by a semi-quantitative indirect immunofluorescence (IIF) assay. (Abstract shortened by UMI.)