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

Abstract

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.

Heat shock protein 90 and the nuclear transport of progesterone receptor

“…Our previous immunohistochemical studies, performed with antibodies that distinguish the oligomeric and dissociated forms of the receptors, are also in concert with this interpretation (Passinen et al 1999). It has been demonstrated that recombinant Hsp90, artificially targeted to the nucleus by a heterologous NLS, cotransported a fraction of cytoplasmic steroid receptor mutants into the nucleus (Kang et al 1994;Passinen et al 1999). Kang et al (1994) reasoned that if NLS-Hsp90 could transport the cytoplasmic receptors into the nucleus, intact steroid receptors should reciprocally cotransport cytoplasmic Hsp90 to the nucleus.…”