Kurt D. Dittmar

Folding of the Glucocorticoid Receptor by the Heat Shock Protein (hsp) 90-based Chaperone Machinery THE ROLE OF p23 IS TO STABILIZE RECEPTOR·hsp90 HETEROCOMPLEXES FORMED BY hsp90·p60·hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor·hsp90 heterocomplexes. We have reconstituted a minimal receptor·hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR·hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997)J. Biol. Chem. 272, 13047–13054). In this work, we show that addition of p23 to native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR·hsp90 heterocomplexes prepared with the minimal (hsp90·p60·hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR·hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90·p60·hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR·hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90·p60·hsp70 forms a GR·hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR·hsp90 heterocomplex to inactivation and disassembly.

Folding of the Glucocorticoid Receptor by the Reconstituted hsp90-based Chaperone Machinery THE INITIAL hsp90·p60·hsp70-DEPENDENT STEP IS SUFFICIENT FOR CREATING THE STEROID BINDING CONFORMATION

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles steroid receptors into a complex with hsp90. The glucocorticoid receptor (GR) is bound to hsp90 via its hormone binding domain (HBD), which must be associated with hsp90 to have a steroid binding conformation. Recently, we have reconstituted a receptor·hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). In this work we show that when the GR is incubated with hsp90, hsp70, and p60, steroid binding sites are generated despite the absence of p23. In this minimal reconstituted system, the GR is incubated with the chaperones in the presence of [3H]triamcinolone acetonide ([3H]TA), which binds to the receptor as GR·hsp90 complexes are formed. When molybdate or p23 is also present during the incubation with chaperones at 30 °C, the formation of steroid binding sites can be assayed by incubating the washed GR with [3H]TA after heterocomplex assembly at 30 °C. However, in the absence of p23 or molybdate, rapid disassembly of GR·hsp90 complexes apparently occurs simultaneously with assembly, such that [3H]TA must be present during the assembly process to trap evidence of conversion of the GR HBD from a non-steroid binding to a steroid binding conformation. Mixture of purified rabbit hsp90 and hsp70 with bacterial lysate containing human p60 results in spontaneous formation of an hsp90·p60·hsp70 complex that can be adsorbed with anti-p60 antibody, and the resulting immune complex converts the GR HBD to a steroid binding state in an ATP-dependent and K+-dependent manner. When the GR is incubated with hsp90, hsp70, and p60 in the presence of the hsp90-binding antibiotic geldanamycin, GR·hsp90·p60· hsp70 complexes are formed, but they have no steroid binding activity. Our data suggest that hsp90, hsp70, and p60 work together as a chaperone complex that possesses all of the folding/unfolding activity necessary to generate the high affinity steroid binding conformation of the receptor.

The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)·Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90·Hop·Hsp70·Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR·Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358–7366). Hop has been considered essential for the formation of receptor·Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR·Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.

Studies with Purified Chaperones Advance the Understanding of the Mechanism of Glucocorticoid Receptor–hsp90 Heterocomplex Assembly

The study of the 9S, untransformed state of steroid receptors has led to the discovery of a multiprotein chaperone system that assembles heterocomplexes between hsp90 and a variety of proteins involved in signal transduction. Using the formation of glucocorticoid receptor (GR)-hsp90 heterocomplexes as a model, we have reconstituted a fully functional heterocomplex assembly system from purified components. The basic assembly system requires four proteins-hsp90, hsp70, p60/Hop and hsp40-to assemble GR-hsp90 heterocomplexes, which are then stabilized by the hsp90-interacting protein p23. The four proteins can self-assemble into an hsp90-p60/Hop-hsp70-hsp40 complex that we call a foldosome. Foldosomes isolated from reticulocyte lysate or formed from purified proteins open up a steroid-binding pocket to create a high-affinity steroid-binding state of the GR. We describe here the systematic reconstitution of the hsp90-based chaperone machinery and develop a model of the receptor-hsp90 heterocomplex assembly mechanism.

Differential Effects of the hsp70-binding Protein BAG-1 on Glucocorticoid Receptor Folding by the hsp90-based Chaperone Machinery

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR)·hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR·hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of ∼0.03, and its elimination by immunoadsorption did not affect GR folding and GR·hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR·hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR·hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.

Binding of hsp90 to the Glucocorticoid Receptor Requires a Specific 7-Amino Acid Sequence at the Amino Terminus of the Hormone-binding Domain

The glucocorticoid receptor (GR) HBD must be bound to the protein chaperone hsp90 in order to acquire the high affinity steroid binding conformation. Despite this crucial role of hsp90, its binding site in GR remains poorly defined. Large portions of the GR HBD have been implicated and no similarity has been established between steroid receptor HBDs and the catalytic domains of the protein kinases (e.g. pp60 src , Raf) that also form stable heterocomplexes with hsp90. Thus, it has been thought that some general property of the proteins, such as exposure of hydrophobic residues in partially denatured regions, determines the assembly of stable hsp90 heterocomplexes. In this work, we have studied fusion proteins containing glutathione S-transferase (GST) and very short amino-terminal truncations just before and at the beginning of the rat GR HBD that are otherwise intact to the carboxyl terminus. Overexpression in COS cells of the chimeras GST537C and GST547C was found to yield receptors that were bound to hsp90 and had wild-type steroid binding affinity. However, removal of 7 more amino acids to form GST554C resulted in a fusion protein that did not bind either hsp90 or steroid. Additional mutations revealed that the role of these 7 amino acids was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in the conserved α-helix. Instead, these observations support a model in which the sequence of the 7 amino acids directly or indirectly affects hsp90 binding to the GR HBD. Thus, a region of GR that has not been thought to be relevant for hsp90 binding is now seen to be of critical importance, and these data argue strongly against the commonly accepted model of receptor-hsp90 heterocomplex assembly in which the chaperone initially interacts nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confirmation.

Ability of various members of the hsp70 family of chaperones to promote assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90

To be in a conformation that binds steroid, the hormone-binding domain of the glucocorticoid receptor (GR) must be bound to the 90 kDa heat shock protein (hsp90). Rabbit reticulocyte lysate contains a protein chaperone system that assembles the receptor into a heterocomplex with hsp90 and converts it from a non-steroid-binding to a steroid-binding form. Assembly of the GR-hsp90 heterocomplex requires hsp70, and in this work we examine the activities of four members of the hsp70 protein family in GR-hsp90 heterocomplex assembly. Rabbit reticulocyte lysate was depleted of hsp70 by passing it through a column of ATP agarose, resulting in the inactivation of its GR-hsp90 heterocomplex assembly activity. Addition of purified animal (mouse) or plant (wheat germ) hsp70 to the hsp70-depleted lysate permits assembly of a GR-hsp90 heterocomplex with a high affinity steroid binding site. However, purified hsp70 homologues from bacteria (DnaK) or the endoplasmic reticulum (BiP) do not promote heterocomplex formation, despite the fact that both DnaK and BiP bind to the GR in the assay system. When added to whole (i.e. hsp70-containing) reticulocyte lysate, DnaK and BiP inhibit GR-hsp90 heterocomplex assembly. Wheat germ lysate forms a heterocomplex between mouse GR and plant hsp90, but the addition of purified rabbit hsp70 to the wheat germ lysate does not increase the amount of receptor-wheat hsp90 complex produced, despite the fact that the rabbit hsp70 binds to the GR when it is added to the wheat chaperone system. The conclusion is that binding of hsp70 to receptors does not necessarily reflect a physiologically meaningful interaction. When native receptor heterocomplexes isolated from cytosols contain hsp70, it is likely that the hsp70-bound receptors represent a minority of receptors that have not yet proceeded fully through the receptor heterocomplex assembly process, which includes the dissociation of hsp70 after the binding of hsp90.