A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894 -6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR⅐hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.Unliganded steroid receptors exist in cytosols in heterocomplexes with the abundant, ubiquitous, and essential heat shock protein hsp90 1 (for review, see Ref. 1). The glucocorticoid receptor (GR) must be in heterocomplex with hsp90 for it to have steroid binding activity (2, 3). The ligand binding domain (LBD) is the region of the receptor that interacts with hsp90 (1), and biochemical data (4) coupled with data from GR mutants (5, 6) support the notion (3) that formation of a complex with hsp90 opens up a hydrophobic pocket in the LBD to access by steroid. Steroid receptor⅐hsp90 heterocomplexes are formed in an ATP-dependent process by a multiprotein chaperone system that has been studied most extensively in reticulocyte lysate (7,8) but is present in lysates of both animal and plant cells (9).The receptor⅐hsp90 heterocomplex assembly system has now been reconstituted (10 -14), and five purified proteins, including hsp90, hsp70, 2 Hop (hsp organizer protein), hsp40, and p23, are required for optimally efficient assembly (for review of heterocomplex assembly, see Refs. 15 and 16). Only two of these proteins, hsp70 and hsp90, are absolutely required for opening the steroid binding cleft in the GR LBD, and the other three proteins act as co-chaperones that increase the overall efficiency of GR⅐hsp90 heterocomplex assembly (17).Hop binds independently to hsp90 and hsp70 to form an hsp90⅐Hop⅐hsp70 complex (18). Although Hop is not required for opening of the steroid binding cleft in the GR LBD, it increases the rate of the process (17). The peptide binding activity of hsp70 is coupled to the binding of ADP versus ATP (for review...

Transition metal oxyanions, such as molybdate, tungstate and vandadate, have been shown to prevent in vitro hormone-induced activation of the glucocorticoid receptor (GR) by blocking dissociation of the GR/heat shock protein heterocomplex. In this work, we report a novel effect of vanadate: in vivo potentiation of GR-mediated gene expression. In cells stably-transfected with complex (mouse mammary tumor virus (MMTV)) or minimal GR-regulated CAT reporters, treatment with 500 M vanadate caused CAT gene expression to dramatically increase, even at saturating concentrations of dexamethasone; while no such effect was seen in response to RU486 antagonist. Similar treatment with molybdate had no effect on GR activity, suggesting that the response to vanadate was not a general property of transition metal oxyanions. Treatment with vanadate after hormone-induced nuclear translocation of the GR also caused potentiation, demonstrating that vanadate was acting on a post-transformation event, perhaps by affecting the transactivation function of DNA-bound GR. Paradoxically, vanadate caused an apparent but temporary "loss" of GR protein immediately after treatment (as measured by loss of reactivity to BuGR2 antibody and of hormone-binding capacity) that returned to normal at approximately 8 h post-treatment, suggesting that potentiation of GR transactivation function (as measured by our CAT assays) was probably occurring during the later stages (8-24 h) of this assay. However, gel shift analyses revealed that vanadate could induce binding of the hormone-free GR to glucocorticoid response element (GRE)-containing oligonucleotides immediately after treatment. Thus, the rapid vanadate-induced "loss" of GR was not due to degradation of GR protein. Yet, vanadate in the absence of hormone had no effect on CAT reporter expression, demonstrating that this form of the GR still requires agonist for its enhanced transcriptional activity. As an indication of the potential mechanism of vanadate action, vanadate was found to dramatically stimulate the mitogen-activated protein kinases, ERK-1 and ERK-2. In addition, vanadate potentiation of GR reporter gene expression was completely blocked by the tyrosine kinase inhibitor herbimycin A. Taken as a whole, our results suggest that vanadate can have dramatic and complex effects on GR structure and function, resulting in hormone-free activation of GR DNA-binding function, as well as alterations to the BuGR2 epitope and hormone-binding domains-while at the same time stimulating tyrosine phosphorylation pathways controlling GR-mediated gene transcription.