Effect of immunosuppressants FK506 and rapamycin on the function of progesterone receptor: protein "p59-HBI", intersection between immunology and endocrinology?]

In the absence of hormonal ligand, inactive, heterooligomeric, 8-10S steroid receptor complexes include a p59 protein (apparent M(r) approximately 59 kDa) bound to th heat shock protein hsp90 (apparent M(r) approximately 90 kDa), which itself binds to the ligand binding domain LBD of the receptor molecule, p59 is thus an hsp binding immunophilin HBI, which, through its interaction with a chaperone, may intervene in several cellular functions. We report that, in cell-free experiments at 0 degrees C, FK506 and rapamycin do not release p59 nor hsp90 from the 9.5S rabbit uterus progesterone receptor, suggesting that the binding of p59 to hsp90 does not interfere with the rotamase site of HBI. There is no "transformation/activation" of the receptor, but an up to 2 fold increase in progesterone agonist and antagonist binding to the receptor is observed. It is suggested that a functional interaction between HBI and receptor activity may be mediated by hsp90.     

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.     

The role of chaperone proteins in the aryl hydrocarbon receptor core complex

The aryl hydrocarbon receptor (AhR) exists in the absence of a ligand as a tetrameric complex composed of a 95-105 kDa ligand binding subunit, a dimer of hsp90, and the immunophilin-like X-associated protein 2 (XAP2). XAP2 has a highly conserved carboxy terminal tetratricopeptide repeat domain that is required for both hsp90 and AhR binding. Hsp 90 appears to be involved in the initial folding of newly synthesized AhR, stabilization of ligand binding conformation of the receptor, and inhibition of constitutive dimerization with ARNT. XAP2 is capable of stabilizing the AhR, as well as enhancing cytoplasmic localization of the receptor. XAP2 binds to both the AhR and hsp90 in the receptor complex, and is capable of independently binding to both hsp90 and the AhR. However, the exact functional role for XAP2 in the AhR complex remains to be fully established.     

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.     

Different immunoelectron microscopic locations of progesterone receptor and HSP90 in chick oviduct epithelial cells.

The intracellular locations of the components of the heterooligomeric progesterone receptor (PR), heat-shock protein (hsp90), and the ligand-binding component were studied by immunoelectron microscopy in the chick oviduct, using immunogold double labeling and peroxidase techniques with monoclonal antibodies (MAb) against hsp90 (7D alpha and 4F3) and against PR (PR6 and PR13). PR was located in the nuclei of epithelial cells independently of the presence or absence of ligand. Cells with apically located nuclei were often PR negative. Ten minutes after progesterone administration no apparent change was seen in PR immunoreactivity, but chromatin underwent extensive rearrangement and PR was seen at the margins of the hetero- and euchromatin. The nucleoli did not contain PR. Hsp90 was located in the cytoplasm as aggregates, often inside small vesicles. In the apical part of the cell, hsp90 was located at the Golgi complex. The nuclei contained no detectable amounts of hsp90 except for that in the nucleoli. Ten minutes after progesterone administration the location or immunoreactivity of hsp90 did not alter. Thus, there seems to be a clear difference in the intracellular distribution of PR and hsp90. The epithelium also exhibited some cells with high levels of hsp90 and no or low levels of PR. These results raise the question of whether PR is associated with hsp90 in intact cells.     

Progesterone receptor and hsp90 are not complexed in intact nuclei

In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.     

Novel antiprogestins Org 31806 and 31710: interaction with mammalian progesterone receptor and DNA binding of antisteroid receptor complexes.

We have examined steroid binding parameters and transformation of calf uterine progesterone receptor (PR) liganded with progestins (progesterone and R5020) and the newly synthesized antiprogestins (Org 31806 and 31710). Species specificity analysis indicated that [3H]R5020 binding in the chicken oviduct cytosol could be eliminated in the presence of 100-fold excess radioinert progesterone and R5020 but not Org 31806 and 31710. In the calf uterine cytosol, the progestins and the antiprogestins appeared to interact with the same PR as revealed by the displacement of [3H]R5020 by all of the above steroids. When the extent of [3H]R5020 binding was examined in the presence of different concentrations of radioinert steroids, the relative affinity with which these compounds interacted with the uterine PR was found to be comparable. A 23 degrees C incubation of cytosol transformed the progestin-bound PR complexes increasing their binding to DNA-cellulose from 5 (0 degrees C, nontransformed) to 35%. Under these conditions, 20% Org 31710- and RU486-occupied PR complexes bound to DNA-cellulose whereas only 10% Org 31806-receptor complexes were retained by the resin. Transformation (23 degrees C) of cytosol receptor caused a loss of the larger 8 S form and an increase in the smaller 4 S form. In its unliganded state or when it was complexed with R5020 or the antiprogestins, incubation of PR at 23 degrees C led to dissociation of the receptor-associated 90 kDa heat-shock protein (hsp90). The PR-hsp90 association was stabilized in the presence of 10 mM iodoacetamide when the ligand binding site was occupied by Org 31806 and 31710. The R5020-receptor complexes, however, allowed release of hsp90 under the above transforming conditions. Our results indicate that although Org 31806 and 31710 show no affinity for the avian PR, these steroids interact with the mammalian PR. We propose that the reported antiprogestational effects of Org 31806 and 31710 are mediated via their interaction with PR which appears similar to one that exists between PR and RU486.     

The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.     

Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell.

We have used bifunctional reagents to examine the subunit composition of the non-DNA-binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross-linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90-kDa heat-shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid-binding subunit. Cross-linking of the receptor complex in the cytosol completely prevented salt-induced dissociation of the subunits. The cross-linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53-kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross-linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.     

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.     

Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts

A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.     

Characterization and localization of estrogen and progesterone receptors of human fallopian tube

The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively high concentration of immunoreactive PR whereas it was almost undetectable in menopausal tissues. These results suggests that ER and PR are regulated by the changing ovarian steroid hormones.     

Structure of the progesterone receptor of chick oviduct. The 110 kDa B protein which binds the hormone has affinity for DNA

Purified "B" protein (MW approximately 110 kDa) that binds progesterone, and more than 90% of the activated receptor labelled with tritiated hormone in the oviduct cytosol of chicks pretreated with estrogen have the same chromatography behavior on DNA-cellulose. Conversely, neither the nonactivated "8S" receptor, that includes the heat-shock protein hsp90, nor the later bind to DNA. With other biochemical and immunological arguments, these results indicate that the hormone binding B unit of the progesterone receptor binds DNA as do all steroid hormone receptors.     

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.     

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.     

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.