title: Glucocorticoid receptor concentration and the ability to dimerize influence nuclear translocation and distribution.
abstract: Glucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10(-6) M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10(-10)-10(-9) M). Loss of dimerization results in a slower observed rate of nuclear import (2.5- to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18-27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4- to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5- to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.
authors.affiliation: Department of Biochemistry, University of Stellenbosch, Stellenbosch 7602, South Africa.
authors.forename: Steven
authors.initials: S
authors.lastname: Robertson
authors.forename: Janet P
authors.initials: JP
authors.lastname: Hapgood
authors.forename: Ann
authors.initials: A
authors.lastname: Louw
mesh_terms: D000085:Acetates / Q000494:pharmacology; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003907:Dexamethasone / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014439:Tyramine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C104564:2-(4-acetoxyphenyl)-2-chloro-N-methylethylamine; D000085:Acetates; D011965:Receptors, Glucocorticoid; D049452:Green Fluorescent Proteins; D003907:Dexamethasone; D014439:Tyramine