Cytoplasmic-nuclear transport of the glucocorticoid receptor: Implications forhsp56 and the cytoskeleton.

Abstract

When steroid receptors are translated, they are assembled into heterocomplexes that contain hsp90, hsp56, p23, and often some hsp70, a complex which also exists independently of steroid receptors. The glucocorticoid receptor (GR) is a cytoplasmic steroid receptor that binds glucocorticoids and translocates to the nucleus to act as a ligand-dependent transcription factor. This thesis focuses on the role of hsp56 in the GR heterocomplex, and the process by which the GR moves to the nucleus. In Chapter 2, I determine the protein-protein interactions of the GR heterocomplex. Highly purified mouse hsp90, but not hsp70, binds to purified hsp56 in an equilibrium manner. hsp90 does not bind to purified hsp70 unless another factor is present. This is consistent with the model that hsp70 and hsp56 both bind to hsp90 at different sites and do not interact with each other. hsp56 is weakly associated with the GR complex isolated from WCL2 cytosol, and it is stabilized in the complex by the presence of molybdate. Localization of hsp56 might provide a clue to its function. Immunofluorescent localization of hsp56 reveals primarily a nonrandom nuclear localization identical to the nuclear GR of WCL2 cells indicating it may play a role in the nuclear activity of the GR. hsp56 also localizes to microtubules, a pathway along which the GR could potentially move into the nucleus following steroid-binding. A conserved region of hsp56 is electrostatically complementary to nuclear localization signals, and may recognize the GR's nuclear localization signal, which becomes active following steroid binding. Microinjection of antibodies against this region of hsp56 into L(M)tk$\sp-$cells impedes the steroid-dependent translocation of the GR that normally occurs with a half-time of approximately 5 minutes. The rapid translocation of the receptor might be directed along cytoskeletal fibrils, however disruption of the three cytoskeletal systems using colcemid in combination with cytochalasin does not change the rate of translocation. To determine if a cytoskeletal pathway exists following this treatment, rat pulmonary endothelial cells were prepared for immunofluorescence to visualize proteins of the three cytoskeletal systems and the receptor-associated proteins hsp90 and p23. While microtubules and microfilaments are completely depolymerized, intermediate filaments maintain a potentially viable pathway for receptor movement and hsp90 and p23 are localized to these filaments. In summary this thesis provides evidence that both hsp56 and the cytoskeleton play a role in the nuclear translocation of the glucocorticoid receptor.