How are molecular chaperones able to assist correct folding of a plethora of structurally divergent proteins? In general, the various chaperone factors protect non-native protein chains from misfolding and aggregation, but do not contribute conformational information to the folding process 
. They interact with features of non-native protein folds that are common to many proteins, such as hydrophobic stretches and unstructured backbone regions, and provide nano-compartments to shield proteins during their folding process from other proteins. Hsp90 regulates mainly a wide range of signal transduction molecules, and thus belongs to the more specialised, but still very versatile chaperones 
. Our study provides a better understanding of this versatility through combinatorial compositions of the Hsp90-client heterocomplexes.
Of the six steroid receptors, the closely homologous GR, MR and PR exhibited the strongest reaction to changes in the TPR-protein make-up of the cell ( and ). AR, and the ERs in particular, were less affected by co-expressing any of the co-chaperones. This may be explained by a diminished Hsp90-dependency of ER, at least in our cellular set-up, corroborated by the ineffectiveness of GA towards ER. Others have also provided evidence that ER may operate independently of Hsp90 
, which contrasts reports on lower ER activity where Hsp90 function was compromised 
. It should be noted, though, that high doses of GA of 0.2–1 µg/ml have been used in these reports. We used a 20–100 fold lower concentration of GA, which efficiently reduced GR activity, like we also have observed previously 
. We cannot exclude the possibility that ER activity could be impaired also in our cellular system at very high concentrations of GA, which however, would raise the question of non-specific effects of GA. We propose that the Hsp90-dependency of ER is cell-type dependent, and possibly affected by the presence or absence of additional, yet to be revealed factors. In addition, high doses of GA have been reported to induce reactive oxygen species in cells 
, which might contribute to differences in the effects of GA on ER at different concentrations.
Our study also documents numerous differences in the efficacies of the TPR proteins' influence on SR. Cyp40 exhibited only a minor effect on AR and PR, and no effect on GR, MR and the ERs, which concurs with its small binding affinity to Hsp90 and steroid receptor heterocomplexes in comparison to other TPR proteins (–). Work in yeast, which expresses the two Cyp40 homologues Cpr6 and Cpr7, revealed an involvement of Cpr7, but not Cpr6 in the hormone-dependent activity of GR 
. In addition, Cpr6 did not influence Hsp90 activity 
. In mammalian cells, the effect of Cyp40 on steroid receptors has not been directly assessed. However, cyclosporine A, which is known to target Cyp40 as well as Cyp18, somewhat diminished AR function in LNCaP cells 
CHIP efficiently inhibited the transactivational activity of GR, MR, PR, and moderately affected AR. It has been reported that CHIP induces degradation of GR, AR and ERα 
and reduces hormone binding of GR 
. With respect to steroid receptor degradation, we observed a tendency towards lower receptor amounts, but no consistently significant effect. Since saturating concentrations of hormone greatly attenuated the inhibitory effect of CHIP on all steroid receptors ( for GR and data not shown for MR, PR, AR), mechanisms in addition to protein degradation must be responsible for the observed inhibition, most likely reduction of hormone binding. Moreover, the interaction of CHIP with Hsp70 may lead to an influence on SR at early stages of the folding cycle, similarly to TPR2 
. AR may be a special case, as CHIP interacts not only via Hsp90 with the LBD of this receptor, but also via its C-terminus with a conserved motif at the N-terminus of the receptor 
Increasing or reducing the levels of TPR2 has been shown to reduce the activity of GR and PR 
, while other steroid receptors had not been analyzed before. In our experiments, increased levels of TPR2 resulted in a strong reduction of the activity of all SR, in contrast to the other investigated TPR proteins, which exhibited at least some selectivity in their action on SR. Our finding of strong interaction of TPR2 with Hsp70, but only moderate interaction with Hsp90 in comparison with other TPR proteins, supports the hypothesis that TPR2 acts by interference at early stages of the SR folding cycle 
. Furthermore, TPR2 still displayed considerable inhibitory activity at saturating conditions of hormone. Thus, TPR2 most likely operates through mechanisms in addition to reducing hormone binding affinity 
For XAP2, a moderate interaction with Hsp90 has been found before 
, but there were no reports on incorporation into SR heterocomplexes. We reveal here the potential of XAP2 to interact with SR. This leads to a differential impact on the transcriptional activity of the receptors, with the strongest effects observed for GR and PR, while MR displayed little reaction to the presence of XAP2. XAP2 also interacts with other receptors, such as AhR 
, peroxisome proliferator-activated receptor α 
and thyroid hormone receptor β1 
. These interactions go along with an inhibition of the transcriptional activity of PPARα, and a stimulation of AhR and THRβ1. XAP2 also affects nuclear translocation of AhR 
and GR 
FKBP51 and FKBP52 are the most intensely investigated TPR cofactors of steroid receptors. In particular for GR, important insight was gained from experiments in yeast, that characterised FKBP52 as stimulatory GR cofactor, while FKBP51 had no effect 
. Studies in mammalian cells reported a strong inhibitory action of FKBP51 on GR, while over-expression of FKBP52 had no effect 
. In at least some mammalian cells, a positive effect of FKBP52 on AR- and GR-signaling has been observed 
. Gene knock-out studies in mice revealed an essential influence of FKBP52 on AR- and PR-related physiological processes, while ablation of the FKBP51 gene did not result in an overt phenotype 
. Very recently, a stimulatory effect of FKBP51 on AR has been reported in prostate cancer cells 
. We have obtained preliminary evidence that this may be a cell-type-specific effect (data not shown).
In the study presented here, FKBP51 and FKBP52 exhibited divergent effects on the transcriptional activities of GR, MR, PR and AR. Consistent with a previous report on GR 
, we also observed a stronger incorporation of FKBP51 in SR heterocomplexes than of FKBP52. At the same time, the interaction of both proteins with Hsp90 was comparable. Thus, the interaction with Hsp90 is not the sole determinant for the efficiency of integration into SR-heterocomplexes. Our interaction analyses further revealed a higher abundance of p23 in Hsp90 complexes with FKBP51 than in Hsp90 complexes with FKBP52. This may be explained by the possibly different interaction surfaces of Hsp90 that are engaged in binding these two immunophilins. FKBP52, in addition to the classic C-terminal MEEVD motif, recognises amino acids at the ATP binding pocket 
, which may impinge on p23 interaction. It should be noted, though, that binding of FKBP51 to this N-terminal site has not been tested yet 
. Whatever the explanation is for the differential recruitment of p23, the increased presence of p23 may be related to the inhibitory action of FKBP51 on GR 
. In addition, the recruitment of Hsp70 by FKBP51, albeit minor, could also add to the differential action of FKBP51 and FKBP52, as Hsp70 is absent in mature receptor heterocomplexes.
Our experiments revealed an inhibitory effect of PP5 that was most pronounced in the case of GR. Previous studies examining the effect of PP5 on GR produced partly inconsistent results. Expression of the TPR domain of PP5 in CV-1 cells abolished GR-dependent transcription 
, like probably the over-expression of any functional TPR-domain would do. On the other hand, over-expression of the PP5 TPR domain slightly stimulated the transactivation of ERα and ERβ 
, which reinforces the notion that the ERs differ in their TPR-protein dependency from the other SRs. Expression of full-length PP5 inhibited ERα and ERβ, probably via dephosphorylation of ER 
. Knock-down of PP5 increased GR-dependent reporter gene activity in one study 
, while another study discovered a reduction of endogenous mRNA levels of GR-dependent genes 
. In yeast, no effect of PP5 on GR was observed and PP5 was unable to compete with FKBP52 to decrease the GR-stimulation of this protein 
. The inactivity of PP5 in yeast, which is insufficiently endowed with TPR-cofactors positive for GR, is compatible with our observations of the inhibitory action of PP5 in mammalian cells. Another study in yeast reported a positive effect of the PP5 yeast homologue Ppt1 on GR function, possibly due to removal of chaperone-inhibitory phosphates on Hsp90 
. It is not known whether this seeming discrepancy can be explained by differences between PP5 and its yeast homologue Ppt1, or by differences in GR regulation between yeast and mammalian cells in general.
The effects of the TPR proteins on SR observed here were significantly attenuated by saturating concentrations of hormone. This is consistent with an effect on hormone binding affinity. Different laboratories including ours provided evidence that later steps in steroid signal transduction are also affected by TPR cofactors, for example nuclear translocation 
and dynamics of intranuclear mobility 
, which requires future experiments to clarify their relative contributions. Our results substantiate the concept that a delicate balance of TPR cofactors governs SR activity in a given cell or tissue, probably in a combinatorial fashion. The recent description of the N-terminal FKBP52 binding site on Hsp90 
opens the possibility for various combinations of two TPR proteins in the same SR heterocomplex. Alternatively, the dynamic assembly and disassembly of heterocomplexes may enable the sequential contribution of specific functions by the different TPR proteins.