Almost 30 years have passed since the original demonstration that steroid receptors, comprising a subfamily of the nuclear receptor (NR) superfamily, exist as large (6-8S) non-DNA-binding complexes in hypotonic extracts (cytosol) of target cells; later such complexes were shown to correspond to a heterooligomer composed of receptor, heat shock (Hsp), and other proteins. Subsequently, an impressive number of studies have dealt with the composition of the "nonactive" complex, its dissociation and/or reassembly in vitro, possible functions of the non-receptor components, and their subcellular compartmentalization. While there is little dispute about the chaperoning role of some Hsps in such a complex, there is still no final proof of an association in vivo of NRs and Hsps in the nuclei of target cells, which is requisite for a direct regulatory involvement of Hsps in NR function. Here we critically review the various models that have been put forward to attribute a biological function to the NR-Hsp90 interaction, evaluate the corresponding experimental data, and integrate recent concepts originating from the structural and functional analyses of NRs.
With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.Keywords: anchoring signal; C-terminal half; heat shock protein 90; hybrid molecule; progesterone receptor.Heat shock proteins (Hsp) constitute an evolutionarily highly conserved group of stress-induced proteins present in diverse species ranging from bacteria to man [1,2]. Hsp expression is increased under a wide variety of stress conditions [3]. Most of the receptor-associated proteins belong to a large family of unrelated classes of proteins commonly referred to as chaperones. Chaperones are involved in all phases of the proteins' lifetime: folding, assembly of oligomers, transport to a particular subcellular compartment, and targeting for proteolytic degradation [4]. Hsp90 is also required for the proper function of steroid receptors (SRs). Original in vitro experiments suggested that Hsp90 functions as a repressor for SRs [5 -7]. Recent genetic analysis in yeast has revealed fairly a positive effect of Hsp90 expression on the transcriptional activity of SRs. It seems that Hsp90 does not act as a bona fide transcription intermediary factor (cofactor) for SRs, but rather as a chaperone [8 -11].Nuclear localization of proteins has been shown to occur by two mechanisms, passive diffusion and active transport [12]. Proteins destined for the nucleus contain nuclear localization signals (NLS) [13]. The first signals characterized as directing nuclear import were those of SV40 large T antigen and the bipartite nucleoplasmin NLS [14][15][16][17][18][19]. Nuclear receptors have a more complex NLS comprising three nuclear localization protosignals (proto-NLS; ...
Steroid receptors exist as large oligomeric complexes in hypotonic cell extracts. In the present work, we studied the nuclear transport of the 2 major components of the oligomeric complex, the receptor itself and the heat shock protein 90 (Hsp90), by using different in vitro transport systems: digitonin permeabilized cells and purified nuclei. We demonstrate that the stabilized oligomeric complex of progesterone receptor (PR) cannot be transported into the nucleus and that unliganded PR salt dissociated from Hsp90 is transported into the nucleus. When nonstabilized PR oligomer was introduced into the nuclear transport system, the complex dissociated and the PR but not the Hsp90 was transported into the nucleus. If PR exists as an oligomeric form after synthesis, as suggested by the experiments with reticulocyte lysate, the present results suggest that the complex is short-lived and is dissociated before or during nuclear transport. Thus, the role of Hsp90 in PR action is likely to reside in the Hsp90-assisted chaperoning process of PR preceding nuclear transport of the receptor.
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.
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