title: Overexpressed glucocorticoid receptor negatively regulates gene expression under conditions that favour accumulation of non-hormone-binding forms of the receptor.
abstract: Previous reports have suggested that the native hormone-responsive glucocorticoid receptor is a heterocomplex with hsp90 and that the receptor constantly cycles between the hormone-responsive and an inactive state, with complex assembly and turnover being driven by hsp70 and hsp90, respectively. Since hsp70 appears to be titrated in cells that transiently overexpress the receptor, assembly intermediates may accumulate when more receptor is produced than can be assembled to hormone-responsive complex. Comparison of receptor protein and hormone-binding levels in extracts from transiently transfected COS-7 cells revealed the presence of non-hormone-binding receptor forms in addition to the native heterocomplex. The receptor was predominantly nuclear in the majority of the transfected cells even in the absence of hormone, with the DNA-binding domain (DBD) being necessary for nuclear localisation. Moreover, the unliganded receptor exhibited constitutive DNA-binding activity and reactivity towards antibodies against the hinge region where NLS1 is known to reside. By comparing fluorography to immunoblotting of two-dimensional SDS-PAGE of cross-linked [3H]dexamethasone-mesylate-labelled receptor, we detected non-hormone-binding receptor species capable of binding DNA in vitro. In addition, using a constitutively active receptor mutant, we found that the overexpressed wild-type receptor was capable of repressing mutant-activated transcription of transiently and stably transfected reporter genes alike in a DBD-dependent manner.
authors.affiliation: Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, 11635 Athens, Greece.
authors.forename: Despina
authors.initials: D
authors.lastname: Siriani
authors.forename: Dimitra J
authors.initials: DJ
authors.lastname: Mitsiou
authors.forename: Michael N
authors.initials: MN
authors.lastname: Alexis
mesh_terms: D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008024:Ligands; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D003432:Cross-Linking Reagents; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D011965:Receptors, Glucocorticoid; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase