Iodination of the progesterone receptor from hen oviduct spares the DNA-binding domain

The progesterone receptor from hen oviduct is isolated as a complex of two subunits, A and B. The A protein binds one molecule of progesterone and also binds to DNA with high affinity. The native A protein can be labeled with iodine with no loss of DNA binding activity. Limited Staphylococcus aureus V8 protease digestion of the labeled preparation results in a number of DNA-binding and non-DNA-binding fragments of the receptor. The progesterone-binding domain contains iodine label. However, two low-molecular-weight DNA-binding fragments do not contain iodine label, indicating a lack of susceptible tyrosine residues near the DNA-binding site of the native receptor. The labeled receptor and its fragments will facilitate studies of the isolated DNA-binding and progesterone-binding domains of the hen A protein as well as of the activity of the native receptor in the presence and absence of hormone.     

Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.     

Transformation of glucocorticoid and progesterone receptors to the DNA-binding state

This brief review explores some recent observations relating to the structure of untransformed glucocorticoid and progesterone receptors and the mechanism by which the receptors are transformed to the DNA-binding state. In their molybdatestabilized, untransformed state, progesterone and glucocorticoid receptors exist as a heteromeric 8-9S complex containing one unit of steroid binding phosphoprotein and one or two units of the 90 kD heat shock protein hsp90. When the receptors are transformed, the steroid-binding protein dissociates from hsp90. In cytosol preparations, temperature-mediated dissociation proceeds much more rapidly in the presence of hormone. The dissociated receptor binds to DNA with high affinity, regardless of whether it is in the hormone-bound or the hormone-free state. These observations raise the possibility that the primary, and perhaps the only, role for the hormone is to promote dissociation of the receptor-hsp90 complex. Molybdate, vanadate, and tungstate inhibit receptor transformation to the DNA-binding form, an effect that appears to reflect the ability of these transition metal oxyanions to stabilize the complex between the steroid receptor and hsp90. By promoting the formation of disulfide bonds, hydrogen peroxide also stabilizes the glucocorticoid receptor-hsp90 complex and prevents receptor transformation. A small, heat-stable factor present in all cytosol preparations inhibits receptor transformation, and, when the factor is removed, glucocorticoid receptors are rapidly transformed. This ubiquitous factor has the physical properties of a metal anion, and it is proposed that molybdate and vanadate affect steroid receptor complexes by interacting with a metal anion-binding site that is normally occupied by this endogenous receptor-stabilizing factor.     

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.     

Mechanisms controlling steroid receptor binding to specific DNA sequences.

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.     

Dimerization of the chicken progesterone receptor in vitro can occur in the absence of hormone and DNA

We have analyzed the dimerization of two forms of the chicken progesterone receptor (cPRA and cPRB) by nondenaturing gradient gel electrophoresis and chemical cross-linking with dimethylpimelimidate (DMP). We demonstrate by these two methods that the PRs assemble in vitro into dimers in the absence of DNA, and that dimerization does not require hormone. The cPRA homodimer binds quantitatively to its cognate DNA response element in our nondenaturing gradient gel assay. DMP cross-linking confirms that both forms of the receptor (cPRA and cPRB) assemble into dimers in solution. Finally, in a standard mobility shift assay, chemically cross-linked receptors bind to the progesterone DNA response element with high affinity. We conclude that the PR contains a dimerization motif, which can promote stable subunit-subunit contacts without the presence of hormone in vitro. The complex thus formed expresses sequence-specific DNA-binding activity indistinguishable from that observed in the presence of hormone.     

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.     

Characterization of a novel T lymphocyte protein which binds to a site related to steroid/thyroid hormone receptor response elements in the negative regulatory sequence of the human immunodeficiency virus long terminal repeat.

We have previously identified a T lymphocyte protein which binds to a site within the LTR of the human immunodeficiency virus type 1 (HIV-1) and exerts an inhibitory effect on virus gene expression. The palindromic site (site B) recognized by this protein is related to the palindromic binding sites of members of the steroid/thyroid hormone receptor family. Here we characterize the T cell protein binding to this site as a 100 kD protein which is most abundant in T cells and which binds to site B as a 200 kD complex. This protein is distinct from other members of the steroid/thyroid hormone receptor family including the COUP protein which has a closely related DNA binding specificity.     

Resolution of the effects of sulfhydryl-blocking reagents on hormone-and DNA-binding activities of the chick oviduct progesterone receptor

The differential effects of sulfhydryl (SH)-blocking agents on hormone and DNA binding by the chick oviduct progesterone receptor were investigated. Previous studies have demonstrated inhibition of steroid-receptor interaction by SH-blocking agents and protection against inhibition by bound hormone. The present results indicate that the SH group required for steroid binding is within or near the hormone-binding site itself, and that a second SH group (or groups) is involved in the binding of receptor to DNA. Three findings relate to the site of action of SH-blocking agents on hormone binding. First, glycerol decreased the rate of hormone dissociation and the rate of hormone displacement by mercurial reagents by 75 to 90%. Second, mercurial reagents displaced [³H]progesterone bound to the mero-receptor, a M_r 23,000 proteolytic fragment containing the hormone-binding site, but not the site of interaction with DNA. Third, hormone displacement was still present after a 10,000-fold purification of the progesterone receptor. Mercurial reagents also inhibited binding of progesterone receptor to DNA, whereas the SH-alkylating agents N-ethylmaleimide and iodoacetamide had no effect. It is likely that distinct sulfhydryl groups are required for steroid receptor interaction with hormone and with DNA, since brief treatment with mercurial reagents blocked DNA binding, but caused only a slight displacement of bound hormone. The SH group required for hormone binding probably lies within or near the hormone-binding site, is sensitive to mercurials, alkylating agents, and 5,5′-dithiobis(2-nitrobenzoate) (DTNB), and is protected by bound hormone. The SH group required for DNA binding, in contrast, is sensitive to mercurials but not to alkylating agents, is only partially sensitive to DTNB, and is not protected by bound hormone.     

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.     

Making sense of cross-talk between steroid hormone receptors and intracellular signaling pathways: who will have the last word?

In classical models of nuclear steroid hormone receptor function, ligand binds receptor, heat shock proteins dissociate, and receptor dimers enter or are withheld in the nucleus and interact with coregulatory molecules to mediate changes in gene expression. The footnotes, "receptors become phosphorylated" and "dynamic nucleo-cytoplasmic shuttling occurs" describe well-accepted, but less well-understood aspects of receptor action. Recently, the idea that several protein kinases are activated in response to steroid hormone binding to cognate cytoplasmic or membrane-associated receptors has become fashionable. However, the precise role of steroid hormone receptor phosphorylation and our understanding of which cytoplasmic kinases are activated and their functional significance remain elusive. This review provides an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using the human progesterone receptor (PR) as a model. The functional consequences of PR phosphorylation by protein kinases classically activated in response to peptide growth factors and novel extranuclear or nongenomic functions of PR as potential independent initiators of signal transduction pathways are discussed. Intracellular protein kinases are emerging as key mediators of steroid hormone receptor action. Cross-talk between steroid receptor- and growth factor-initiated signaling events may explain how gene subsets are coordinately regulated by mitogenic stimuli in hormonally responsive normal tissues, and is suspected to play a role in their cancer biology.     

Rat antibodies as probes for the characterization of progesterone receptor A and B proteins from laying hen oviduct cytosol

The chicken oviduct contains two different hormone binding forms of the progesterone receptor, A and B. We have prepared rat antisera against both forms of the receptor partially purified from laying hen oviduct. The anti-progesterone receptor A antiserum reacts with both receptor forms on Western blots, while the anti-progesterone receptor B antiserum reacts mainly with the B form. Both antisera also react with the native progesterone receptor proteins as shown by sedimentation analysis of the antibody-receptor complexes. Receptors A and B are recognized on Western blots of total protein from dissolved tissue, indicating that both forms are likely to be physiological components. Epitope mapping experiments show that immunogenicity of both receptor molecules is restricted to structurally related protein domains of 28 kDa in receptor A and of 52 kDa in receptor B.     

Mapping the HSP90 binding region of the glucocorticoid receptor

In animal cells, unliganded steroid receptors are complexed with a 90-kDa heat shock protein, HSP90; hormone binding by the receptor leads to the release of HSP90. We found that the 795-amino acid rat glucocorticoid receptor protein formed oligomeric complexes in vitro upon synthesis in rabbit reticulocyte lysates; these oligomers also dissociated in the presence of hormone. Similar complexes formed when X795, a receptor derivative containing only the C-terminal half (amino acids 407-795) of the protein, was translated in vitro. Moreover, X795 was co-immunoadsorbed from the reticulocyte lysates together with HSP90 by three different anti-HSP90 monoclonal antibodies, indicating that the in vitro translated receptor binds HSP90 and that the interaction occurs within the C-terminal half of the receptor. To localize the HSP90 binding region in greater detail, various deletion mutants of X795 were translated in vitro and assayed for oligomer formation and for co-immunoadsorption with HSP90. The results indicated that HSP90 interacted with the receptor within a subregion of the hormone binding domain, between amino acids 568 and 616. These findings are consistent with the proposal that HSP90 may participate in the mechanism of signal transduction by steroid receptors.