Evidence for the existence of an oligomeric, non-DNA-binding complex of the progesterone receptor in the cytoplasm

Abstract

Steroid receptors are found as a hetero-oligomeric complex in cell extracts. Due to the dynamic interaction between receptor-associated proteins and receptors, it is difficult to study the oligomeric complex in living cells. Here this was attempted in cells in which the interaction was stabilized by introducing molybdate into the cells or by incubating the cells at low temperature. The complex was studied with an antibody (αD) recognizing only the dissociated form of the chicken progesterone receptor (PR) and with antibodies (PR22, PR6). Recognizing also oligomeric forms of the receptor. When wild-type chicken PR was transfected, all antibodies showed nuclear staining. Molybdate or cold treatment of cells resulted in cytoplasmic accumulation of the PR as detected with PR22/PR6. αD, however, stained predominantly the nuclear PR in treated cells. These findings suggest that when the oligomeric complex of the PR is stabilized in intact cells in vivo and then crosslinked with paraformaldehyde, a portion of the cytoplasmic receptor is seen as an oligomeric complex, whereas, in the nucleus, most, if not all receptor molecules are in dissociated form. Steroid receptors belong to a large family of nuclear receptors functioning as ligand-regulated transcription factors. Receptor function is mediated by receptor-interacting proteins; co-activators interact with agonist-occupied receptors and mediate transcription activation while co-repressors interact with non-liganded or antagonist-occupied and mediate transcription repression. Ligand binding induces a considerable change in the structure of the ligandbinding domain (LBD), affecting the surface exposition of the receptor which dictates the interaction of the cofactors [Moras and Gronemeyer 1998, Renaud et al. 1995, Wurtz et al. 1996. The earliest known receptor-interacting protein is Hsp90 [Dougherty et al. 1984]. It is known to interact in vitro with the LBD of the steroid receptor in a ligand-dependent manner: it binds to non-liganded steroid receptor but not to agonist-occupied receptor. Non-liganded steroid receptor LBDs act as intramolecular repressors of the activation functions of steroid receptors as well as of heterologous proteins when covalently linked to them [Picard et al. 1988]. Deletion of the LBD generates a constitutively active steroid receptor which does not form a stable oligomeric complex with Hsp90 in vitro. In view of these data, it has been proposed that the Hsp90 interaction is responsible for the repressor function of non-liganded LBDs [Scherrer et al. 1993]. Binding of Hsp90 interferes with the DNA binding of the receptor, which has been regarded as one possible mechanism by which Hsp90 functions as a corepressor. Hsp90 is also required for proper ligand binding of the glucocorticoid receptor [Bresnick et al. 1989]. It has been proposed that the oligomeric form of steroid receptors represents a poised conformation, inactive but ready to interact with the ligand. Ligand binding is thought to dissociate the complex and allow receptor binding to ...