Abstract
Abstract: :
Purpose: There are two human glucocorticoid receptor isoforms termed GRα and GRß. GRα undergoes hormone–dependent translocation from the cytoplasm to the nucleus by associating with heat shock protein 90 (Hsp90) in a multi–protein heterocomplex. Previously we demonstrated that Hsp90 also serves as a chaperone in the nuclear import of GRß. Currently we report on the role of other components, including immunophilins FKBP51 and FKBP52, motor protein dynein, and microtubules, in the trafficking of GRß in cultured human trabecular meshwork (TM) cells. Methods: Co–immunoprecipitation and Western immunoblotting were used to investigate the multi–protein heterocomplex consisting of GRß–Hsp90–immunophilin–dynein. Confocal immunofluorescent microscopy was performed to detect the subcellular expression of FKBP51 with GRß in primary cultured normal and glaucomatous TM cell lines. Overexpression of dynamitin, a component of dynein–associated dynactin complex, to dissociate dynein from microtubules was performed by transfection of dynamitin construct pCMVH50m in transformed TM cells that are co–transfected with the GRß construct, pCMX–hGRß. Results: Immunoprecipitation and subsequent Western Blots demonstrated that both FKBP51 and FKBP52 were physically associated with GRα. Dexamethasone treatment caused translocation of the GRα–FKBP52 complex from the cytoplasm to the nucleus. In contrast, only FKBP51 complexed with GRß, which was not regulated by dexamethasone administration. Immunoprecipitation with anti–FKBP51 or anti–FKBP52 antibody also pulled down Hsp90 and dynein, and immunoprecipitation with anti–dynein, dynamitin, or ß–tubulin antibody was able to co–precipitate GRß. In the normal TM cell line, which had a high level of GRß in the nucleus, FKBP51 was also concentrated in the nuclear region, while in the glaucomatous TM cell line, both GRß and FKBP51 were present at lower concentrations in the nucleus. Results on the overexpression of dynamitin will also be presented. Conclusions: FKBP51, but not FKBP52, is associated with the transport of GRß from the cytoplasm to the nucleus. This study supports the roles of the motor protein dynein and microtubules in trafficking GRß from the cytoplasm to the nucleus.
Keywords: receptors • chaperones • cytoskeleton