A conserved proline in the hsp90 binding region of the glucocorticoid receptor is required for hsp90 heterocomplex stabilization and receptor signaling.

Studies of hsp90 in yeast have supported the notion that this chaperone plays a critical role in signaling by steroid receptors. One limitation to these studies is that yeast expressing hsp90 mutants may also be deficient in fundamental cellular functions of the chaperone required for steroid-dependent induction of transcription. In this work, we have prepared mutants of the glucocorticoid receptor (GR) that permit analysis of hsp90 binding and transcriptional activity in cells with normal chaperone function. Our previous data supported a model in which hsp90 binds to the receptor steroid binding domain according to a two-site model. By amino acid mutagenesis of these two sites, we have now generated three receptor mutants and analyzed their function. Upon their translation in vitro, all three mutants interacted with hsp90 similarly to the wild-type receptor. However, one mutant, P643A (GRo), was of particular interest because, although it showed normal steroid binding and transformation to a glucocorticoid response element-specific DNA binding form, it was remarkably deficient in nuclear translocation and transcriptional function at 37 degreesC. Furthermore, GRo.hsp90 heterocomplexes formed in vivo or assembled under cell-free conditions were much less stable than wild-type GR. hsp90 heterocomplexes. Our results demonstrate that Pro-643 plays a critical role in both stabilizing the receptor.hsp90 complex and in permitting an efficient nuclear translocation and, thus, support the concept that the chaperone is an integral component of the steroid-receptor signaling pathway.