Reconstitution of progesterone receptor with heat shock proteins

Nonactivated chick progesterone receptor from hypotonic tissue extracts exists in a large complex containing the heat shock proteins hsp90 and hsp70 plus additional smaller proteins; activation of receptor to a DNA-binding form involves the dissociation of proteins from the complex. Whereas numerous attempts to reversibly bind components to the activated receptor have been unsuccessful, we now report conditions that promote the reassociation of hsp90 and hsp70 to progesterone receptor. Cytosolic receptor was dissociated from hsp90 and hsp70 by treatment with 0.5 M KCl and 10 mM ATP in the absence of progesterone. It was then purified by binding to immunoaffinity resins. After wash steps, the receptor-resin complex was incubated in rabbit reticulocyte lysate at 30 C, rewashed, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Saturable binding of rabbit hsp90 and hsp70 to chick receptor was found after incubation with reticulocyte lysate; hsp binding was temperature dependent, but not dependent on exogenous ATP. Incubation of dissolved receptor with oviduct cytosol, from which receptor was obtained, or with purified hsp did not result in hsp binding. Furthermore, mixing oviduct cytosol with lysate inhibited hsp reconstitution, suggesting negative factors for hsp binding in oviduct cytosol. The steroid-binding domain of the receptor was required, since no hsp binding was observed in the reconstitution system using a receptor mutant lacking this domain. When the receptor was isolated in the presence of progesterone, reconstitution with hsp90 and hsp70 did not occur. This is consistent with the in vivo effects of progesterone in promoting hsp dissociation.     

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.     

Composition, assembly and activation of the avian progesterone receptor

When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30°C), (3) presence of ATP, and (4) presence of Mg²⁺. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl₂ and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system.     

The involvement of p23, hsp90, and immunophilins in the assembly of progesterone receptor complexes

To better understand the assembly mechanism for the progesterone receptor (PR), we have developed cell-free systems for studying interactions of PR, hsp90, and other associated proteins. When PR is incubated in rabbit reticulocyte lysate, its association with hsp90, hsp70, the three immunophilins FKBP54, FKBP52 and CyP-40, and with p23 is observed. These interactions require ATP/Mg²⁺ and when ATP is limiting the PR complex is altered to one containing the proteins p60 and p48, but lacking immunophilins and p23. We have studied two pre-formed hsp90 complexes that may participate in the assembly of PR complexes. One contains hsp90 bound to hsp70 and p60 and this complex forms spontaneously in the absence of ATP. A second complex contains hsp90 bound to p23 plus the three immunophilins and some hsp70. The formation of this complex requires ATP. In further studies we have shown that purified hsp90 can bind to purified p23 and this interaction requires both ATP and molybdate. This explains, in part, the known effects of ATP and molybdate on assembly of PR complexes.     

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.     

A novel monoclonal anti-rabbit hsp90 antibody: Usefulness for studies on hsp90-steroid receptor interaction

The M_r 90,000 protein associated with steroid receptors in their non-transformed state has been identified as a heat shock protein (hsp90) but the relationship between hsp90 binding and receptor function is still poorly understood. In this work, we have obtained and characterized one monoclonal anti-rabbit hsp90 antibody (7C10), among more than 2000 wells plated. This antibody was able to complex both free and rabbit uterine progesterone receptor-associated hsp90 as demonstrated by sedimentation analysis on sucrose gradients. As assessed by ELISA, 7C10 displayed a high binding affinity for hsp90 ( 4 nM). A standardized and specific competitive binding assay was developed for accurate quantification of hsp90 in rabbit tissues including reticulocyte lysate. 7C10 also permitted immunolocalization of hsp90 in various rabbit tissues. In Western blot, the monoclonal antibody recognized a single polypeptide band of M_r 90,000 in crude or purified rabbit preparations but failed to cross-react with any other mammalian or avian hsp90. These findings suggest that hsp90, a highly conserved protein, is a weak immunogen and elicits a strict species specific immunological response. Owing to its high affinity and specificity for rabbit hsp90, the monoclonal antibody 7C10 was used for purification and total depletion of hsp90 from the reticulocyte lysate, an efficient system for in vitro receptor translation and reconstitution studies. Thus, 7C10 represents a new powerful tool to further investigate the importance of hsp90 in steroid hormone receptor function.     

Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.

To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.     

Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the hormone binding domain of the receptor to its high affinity steroid binding state. This system has been resolved into five proteins, with hsp90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperones that optimize assembly. Hop binds independently to hsp70 and hsp90 to form an hsp90.Hop.hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rejected any role for Hop in GR.hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90.Hop.hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for approximately 30% of the hsp90 and approximately 9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR.hsp90 assembly activity resides in the peak containing Hop-bound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90.hsp70 complexes with a stoichiometry of 2:1.     

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 approximately 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.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.     

hsp70 interacting protein Hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of BAG-1

Reticulocyte lysate contains a chaperone system that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes. Using purified proteins, we have prepared a five-protein heterocomplex assembly system consisting of two proteins essential for heterocomplex assembly-hsp90 and hsp70-and three proteins that act as co-chaperones to enhance assembly-Hop, hsp40, p23 [Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900]. The hsp70 co-chaperone Hip has been recovered in receptor.hsp90 heterocomplexes at an intermediate stage of assembly in reticulocyte lysate, and Hip is also thought to be an intrinsic component of the assembly machinery. Here we show that immunodepletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR.hsp90 heterocomplex assembly or the activation of steroid binding activity that occurs with assembly. Despite the fact that Hip does not affect assembly, it is recovered in GR.hsp90 heterocomplexes assembled by both systems. In the five-protein system, Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1, and cotransfection of Hip with BAG-1 opposes BAG-1 reduction of steroid binding activity in COS cells. We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in opposition to BAG-1.     

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.     

Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates.

Inhibition of protein synthesis in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation is due to the activation of a heme-regulated protein kinase (HRI) which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. In this report, immunoadsorption with monoclonal antibodies (mAbs) and Western blot analysis were used to investigate the interaction of HRI, the 90-kDa heat shock protein (hsp 90), hsp 70, and the EC1 antigen in rabbit reticulocyte lysates under protein synthesizing conditions. The data indicate that hsp 90, hsp 70, and the EC1 antigen interact with HRI in rabbit reticulocyte lysate. The EC1 antigen is a protein that has been demonstrated to be associated with several steroid hormone receptor-hsp 90 complexes and reacts with the KN 382/EC1 mAb (EC1). The association of HRI with hsp 90 and the EC1 antigen in the reticulocyte lysate was found to be dependent on the presence of hemin at a concentration of 5 microM or higher; little HRI was coadsorbed by the 8D3 anti-hsp 90 mAb or the EC1 mAb in the absence of hemin. Hsp 70 remains associated with HRI in the absence of hemin, suggesting that hsp 90 and 70 may bind to HRI at different sites. The immunological properties of the hsp 70 associated with HRI indicate that it may be the constitutively express heat shock cognate protein (hsc 73). The results suggest that the association of HRI with hsp 90 and the EC1 antigen may be in a dynamic equilibrium, in which complex formation is either facilitated or stabilized by the presence of hemin, and supports the notion that these proteins in conjunction with hsp 70 may play a role in regulating HRI activity or activation in situ.     

Monovalent cation selectivity for ATP-dependent association of the glucocorticoid receptor with hsp70 and hsp90.

We have reported previously that incubation of the immunopurified transformed hormone-free glucocorticoid receptor with rabbit reticulocyte lysate reconstitutes the receptor complex with hsp90 and that reconstitution is accompanied by concomitant repression of DNA binding activity and regeneration of the steroid binding conformation (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). In this work we further characterize this system by defining the small M(r) components of reticulocyte lysate required for both structural and functional reconstitution of the receptor-hsp90 complex. Reconstitution is ATP-dependent and there is a direct relationship between the extent of hsp90 binding to the receptor and the number of specific steroid binding sites that are generated. Dialysis of reticulocyte lysate inactivates its reconstituting activity. Addition of an ATP-regenerating system or readdition of small M(r) lysate components (in the form of a Centricon C30 filtrate) has little effect, but the presence of both restores full reconstituting activity to dialyzed lysate, as assayed by steroid binding activity and by the binding of hsp90 and hsp70 to the receptor. The small M(r) activity is heat-stable, and it can be completely replaced by NH+4, K+, and Rb+, with K+ producing a maximal effect at the concentration normally present in undialyzed lysate. Na+ and Li+ have no reconstituting activity. This ion selectivity demonstrates that a monovalent cation binding site is involved in receptor heterocomplex reconstitution. It is intriguing that the protein unfoldase (e.g. clathrin uncoating ATPase) activity of hsp70 is known to have a similar monovalent cation dependence, and that under all conditions where hsp90 becomes bound to the receptor, we find that hsp70 is also bound.     

Direct evidence that the glucocorticoid receptor binds to hsp90 at or near the termination of receptor translation in vitro

We have translated the rat glucocorticoid receptor in both reticulocyte lysate and in wheat germ extract. Receptor synthesized in the reticulocyte lysate is immunoadsorbed by the 8D3 monoclonal antibody directed against the 90-kDa heat shock protein (hsp90) and it has a normal ability to bind glucocorticoid in a high affinity manner. Although the wheat germ extract synthesizes the full length receptor, the receptor is not immunoadsorbed by 8D3 and we cannot demonstrate high affinity steroid binding. Receptor synthesized by the reticulocyte lysate can be immunoadsorbed by antibody directed against hsp90 as soon as the translation product is full length, suggesting that the receptor becomes associated with hsp90 late during translation or immediately at the termination of translation. When newly synthesized receptor is bound with steroid and incubated at 25 degrees C, it is converted to a form that binds to DNA. This study provides direct evidence that association of hsp90 with the glucocorticoid receptor is a very early event and that the newly formed heteromeric receptor-hsp90 complex is fully competent to undergo transformation.     

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60^src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60^src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accomodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.     

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket

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 GR.hsp70 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.     

The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.