Only a small portion of the cytoplasmic progesterone receptor is associated with Hsp90 in vivo.

In cell extracts all of the nonliganded steroid receptor molecules are found as an oligomeric complex with Hsp90 and other proteins. In previous studies we have shown that Wild-type Hsp90 and progesterone receptor (PR) are located in different cell compartments (Tuohimaa et al. [1993] Proc. Natl. Acad. Sci. USA 90:5848-5852). In the present work we studied whether PR and Hsp90 can efficiently associate provided they are present in the same cell compartment. The association of Hsp90 with PR in vivo was studied by nuclear cotranslocation and immunohistochemistry with an antibody (alphaD) which can distinguish between the oligomeric and dissociated form. Upon expression of a cytoplasmic mutant of PR with Wild-type (cytoplasmic) Hsp90 and Wild-type (nuclear) PR with NLS-Hsp90 (a Hsp90 with a nuclear localization signal), we noted that the epitope of alphaD in PR was exposed in both cases. Also, in vivo crosslinking and treatment of cells with substances which stabilize the oligomeric complex in vitro were inefficient in demonstrating or inducing a similar oligomeric receptor form detectable in vitro in cell homogenates. However, when the cytoplasmic PR mutant (DeltaPR) was coexpressed with a nuclear form of Hsp90 (NLS-Hsp90), a portion of PR was cotranslocated into the nucleus. This would indicate that steroid receptors are indeed associated with Hsp90 in intact cells, but the Hsp90-associated receptor pool represents only a small portion of the receptors. This suggests that the majority of oligomeric complexes seen in cell extracts are formed during cell fractionation.     

Nuclear origin of progesterone receptor of the chick oviduct cytosol

Summary Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.