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.     

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.     

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.     

Molecular chaperones and subcellular trafficking of steroid receptors

Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl–terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.     

Purification of unactivated progesterone receptor and identification of novel receptor-associated proteins

Progesterone receptor complexes were purified from crude cytosol in a rapid, gentle, one-step procedure using anti-receptor monoclonal antibodies covalently attached to an agarose resin. Five nonreceptor proteins specifically co-purified with unactivated avian progesterone receptor; these proteins had molecular masses of approximately 90, 70, 54, 50, and 23 kDa. The 90- and 70-kDa proteins have been previously identified as the 90-kDa heat shock protein and a member of the 70-kDa heat shock protein family, respectively. The 54-, 50-, and 23-kDa proteins have not been previously described as associated with avian progesterone receptor. Two-dimensional gel electrophoresis revealed charge heterogeneities for all five proteins. Except for p70, each could be dissociated from receptor by salt, a process inhibited by sodium molybdate. However, molybdate was not required for protein association with receptor in low ionic strength. Following progesterone treatment in vivo p70 still co-purified with cytosolic receptor although the other affiliated proteins were reduced, suggesting hormone-dependent dissociation in conjunction with receptor activation. One of the proteins, p54, displayed in vitro hormone-dependent dissociation which was not prevented by molybdate.     

Direct stoichiometric evidence that the untransformed Mr 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex

We have used three methods to measure the stoichiometry of the glucocorticoid receptor and the 90-kDa heat shock protein (hsp90) in L-cell glucocorticoid receptor complexes that were purified by immunoadsorption to protein A-Sepharose with an anti-receptor monoclonal antibody, followed by a minimal washing procedure that permits retention of receptor-associated protein. In two of the methods, receptor was quantitated by radioligand binding, and receptor-specific hsp90 was quantitated against a standard curve of purified hsp90, either on Coomassie blue stained SDS gels by laser densitometry or on Western blots by quantitative immunoblotting with 125I-labeled counterantibody. The stoichiometry values obtained by densitometry and immunoblotting are 7 and 6 mol of hsp90/mol of receptor, respectively. In a third method, which detects total receptor protein rather than just steroid-bound receptor, the ratio of hsp90 to receptor was determined by immunopurifying receptor complexes from [35S]methionine-labeled L cells, and the amount of 35S incorporated into receptor and hsp90 was corrected for the established methionine content of the respective proteins. In complexes from L cells which are labeled to steady state (48 h), the ratio of hsp90 to GR is 4:1. When immunoadsorbed receptor complexes are washed extensively with 0.5 M NaCl and 0.4% Triton X-100 in the presence of molybdate, the ratio of hsp90 to GR is 2:1. In addition to hsp90, preparations of [35S]methionine-labeled untransformed receptor complex also contain a 55-kDa protein that the conclusion that the untransformed L-cell glucocorticoid receptor exists in cytosol in a much larger heteromeric complex than considered to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the atrial natriuretic peptide receptor by heat shock protein 90 complexes

Heat shock protein 90 (hsp90) is a chaperone required for the proper folding and trafficking of many proteins involved in signal transduction. We tested whether hsp90 plays a role as a chaperone for GC-A, the membrane guanylate cyclase that acts as a receptor for atrial natriuretic peptide (ANP). When cultured cells expressing recombinant GC-A were treated with geldanamycin, an inhibitor of hsp90 function, the ANP-stimulated production of cyclic GMP was inhibited. This suggested that hsp90 was required for GC-A processing and/or stability. A physical association between hsp90 and GC-A was demonstrated in coimmunoprecipitation experiments. Treatment with geldanamycin disrupted this association and led to the accumulation of complexes containing GC-A and heat shock protein 70 (hsp70). Protein folding pathways involving hsp70 and hsp90 include several pathway-specific co-chaperones. Complexes between GC-A and hsp90 contained the co-chaperone p50(cdc37), typically found associated with protein kinase.hsp90 heterocomplexes. GC-A immunoprecipitates did not contain detectable amounts of Hop, FKBP51, FKBP52, PP5, or p23, all co-chaperones found in hsp90 complexes with other signaling proteins. The association of hsp90 and p50(cdc37) with GC-A was dependent on the kinase homology domain of this receptor but not on its ANP-binding, transmembrane, or guanylate cyclase domains. The data suggest that GC-A is regulated by hsp90 complexes similar to those involved in the maturation of protein kinases.     

Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.     

Analysis of protein binding to heat shock protein 70 in pancreatic islet cells exposed to elevated temperatures or interleukin 1 beta

To elucidate a role for heat shock proteins in islet function, isolated pancreatic islets were labeled with [35S]methionine after control, heat shock, or interleukin 1 beta (IL-1 beta) treatment, extracted in the presence of detergent, and then passed over affinity columns with antibodies against heat shock protein 70 (hsp 70), hsp 70 itself, or ATP conjugated to the columns. In control or IL-1 beta-treated islets, the antibody column efficiently absorbed hsp 70 together with two other proteins of molecular masses 46 and 53 kDa. In extracts from heat-shocked cells, the binding of cellularly synthesized hsp 70 to the antibody column was inefficient but improved by the addition of unlabeled partially purified hsp 70 to the extracts. When assessing the binding of proteins in the extracts to the hsp 70 column, hsp 70 and the 46- and 53-kDa proteins among others all bound to the column. No differences in the patterns of binding to the hsp 70 column between extracts from the different islet exposures were noticed. The 46-kDa protein was identified as actin by immunoblot analysis. ATP-agarose column chromatography revealed a pattern of binding similar to that of the hsp 70 column. It is concluded that hsp 70 contains at least two functional domains, one adjacent to the epitope recognized by the antibody and active in restoring cellular function after heat shock, whereas the other has the ability to bind the 46- and 53-kDa and possibly other proteins. Furthermore, the stress induced by heat shock differs significantly from that after IL-1 beta treatment with respect to the functional behavior of hsp 70.     

Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding.

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.     

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.     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc     

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.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

Characterization of progesterone receptor binding to the 90- and 70-kDa heat shock proteins

In this study a model system for expression of the chicken progesterone receptor in cultured cells was developed using a quail fibroblast cell line, QT6. The chicken progesterone receptor form A expressed in QT6 cells was evaluated and determined to have a number of similarities to receptor isolated from chicken oviduct. These include hormone binding, sedimentation profile, phosphorylation pattern, heat shock protein (hsp) 70 and hsp90 associations and the ability to stimulate a reporter gene construct. Therefore, the receptor expressed in this system functioned adequately for further evaluation of the particular region (or regions) involved in hsp70 and hsp90 binding. Several receptor deletion mutants were tested for hsp70/hsp90 binding; only the d369-659 mutant, which has the entire steroid-binding domain deleted, was unable to bind hsp90 and hsp70. Three separate regions of the steroid-binding domain were found to partially restore hsp90 and hsp70 binding to the d369-659 mutant protein. However, hsp binding was not abolished when these or other regions of the steroid binding domain were deleted individually. These findings indicate that hsp90 and hsp70 both bind to the steroid-binding domain of the receptor through interactions at multiple locations or through some structural quality that is distributed throughout this region of the protein.     

Protein components of the nonactivated glucocorticoid receptor.

The nonactivated glucocorticoid receptor (Mr approximately 350,000) of WEHI-7 mouse lymphoma cells was investigated with respect to the stoichiometry of protein subunits. Cross-linking patterns obtained by affinity labeling and denaturing gel electrophoresis revealed a heterotetramer consisting of one receptor polypeptide in association with two 90- and one approximately 50-kDa subunits. The receptor stabilized by molybdate, disulfide bond formation, or chemical cross-linking was purified roughly 6000-fold by immunoaffinity chromatography and analyzed by gel electrophoresis and immunoblotting. The 90-kDa component was consistently detected in a 2:1 ratio with respect to the receptor polypeptide and was identified as the 90-kDa heat shock protein, hsp90. A 70-kDa heat shock protein was found in both stabilized and nonstabilized receptors and bound to the immunomatrix independent of receptor. The additional receptor subunit was unequivocally identified as the 59-kDa protein previously described (Tai, P.-K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275). This component was found only in complexes cross-linked via amino groups. It was removed from the molybdate-stabilized receptor under our purification conditions, thus leaving behind a trimer composed of the receptor polypeptide and two molecules of hsp90. In the absence of hormone, the receptor had the same subunit composition as in its presence.     

Hsp56: a novel heat shock protein associated with untransformed steroid receptor complexes

The recently-described p59 protein has been shown to be associated with untransformed steroid receptors present in rabbit uterus and rat liver cytosols (Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275; Renoir, J.-M., Radanyi, C., Faber, L. E., and Baulieu, E.-E. (1990) J. Biol. Chem. 265, 10740-10745), while a smaller version of this protein (p56) interacts with glucocorticoid receptors in human IM-9 cell cytosols (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). In addition to interacting with glucocorticoid receptors, the p56 protein of IM-9 cell cytosol is also found as part of a large heteromeric complex that contains both the 70-kDa and 90-kDa heat shock proteins (hsp70 and hsp90, respectively). Given this association of p56 with the two major stress proteins, I have speculated that p56 may itself be a heat shock protein. In this paper, the effect of heat stress on the rate of synthesis of p56 is determined. Intact IM-9 cells were exposed to 37 or 43 degrees C for 4 h, followed by pulse-labeling with [35S]methionine. Analysis of whole cytosolic extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal an increased rate of radiolabeling for hsp70, hsp90, hsp100, ad hsp110, but no heat-inducible protein of smaller relative molecular mass is detected. However, immune-purification of p56 from normal and heat-stressed cytosols with the EC1 monoclonal antibody results in the presence of a 56-kDa protein that exhibits an increased rate of synthesis in response to heat stress. The results of two-dimensional gel Western blots employing the EC1 antibody demonstrate that this heat-inducible protein is indeed the EC1-reactive p56 protein and that the induction effect is not due to unequal yields of p56 during immune-purification. Heat stress has no effect on the composition of the p56.hsp.70.hsp90 complex, except that the complex derived from heat shocked-cells contains both the constitutive and heat-inducible forms of hsp70. Induction of p56 also occurs in IM-9 cells subjected to chemical stress (sodium arsenite). It is proposed that p56 is a steroid receptor-associated heat shock protein which can now be termed hsp56. Like hsp90, hsp56 likely serves in some vital cellular role apart from any specific function it provides in steroid receptor action.