A dynamic model of glucocorticoid receptor phosphorylation and cycling in intact cells

Glucocorticoid receptors have been proposed to undergo an ATP-dependent recycling process in intact cells, and a functional role for receptor phosphorylation has been suggested. To further investigate this possibility we have examined the phosphate content of the steroid-binding protein of all glucocorticoid receptor forms which have been isolated from WEHI-7 mouse thymoma cells. By labeling of intact cells with 32Pi for 18-20 h in the absence of hormone, covalent binding of [3H]dexamethasone 21-mesylate, immunopurification and SDS-PAGE analysis, the steroid binding protein was found to contain, on average, 2-3 phosphates as phosphoserine. One third of the phosphates were associated with proteolytic fragments encompassing the C-terminal steroid-binding domain. The central DNA-binding domain was not phosphorylated, leaving the other two thirds of the phosphates localized in the N-terminal domain. The phosphate content of various receptor forms from cells incubated with 32Pi and [35S]methionine was compared using 35S to normalize for quantity of protein. In ATP-depleted cells a non-steroid-binding form of the receptor (the "null" receptor) is found tightly bound to the nucleus, even without steroid. The phosphate content of null receptors was two thirds that of cytosolic receptors from normal cells, suggesting phosphorylation-dependent cycling in the absence of hormone. Addition of glucocorticoid agonists, but not antagonist, to 32P- and 35S-labeled cells increased the phosphate content of the cytosolic steroid-binding protein up to 170%, indicating an average increase in the phosphates from about 3 to 5. After 30 min of hormone treatment the phosphate content of the steroid-binding protein of cytosolic activated (DNA-binding) and nonactivated receptors, and that of nuclear receptors extractable with high salt concentrations and/or DNase I digestion, was the same. No change in the phosphate content of the 90-kDa heat shock protein associated with unliganded and nonactivated receptors was detected following association of the free protein with the receptor and following hormone binding of the receptor. Analysis of the unextractable nuclear receptors indicated that they contained less phosphate (60% of that of cytosolic receptors), similarly to null receptors, indicating that dephosphorylation is associated with the unextractable nuclear fraction. The rate of hormone-dependent phosphorylation appeared to be much faster than the rate of dephosphorylation in the presence of hormone, the latter determined by a chase of the 32P label with unlabeled phosphate. Our results show that phosphorylation and dephosphorylation are involved in the mechanism of action of glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

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.     

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.     

Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins.

As targeted proteins that move within the cell, the steroid receptors have become very useful probes for understanding the linked phenomena of protein folding and transport. From the study of steroid receptor-associated proteins it has become clear over the past two years that these receptors are bound to a multiprotein complex containing at least two heat shock proteins, hsp90 and hsp56. Attachment of receptors to this complex in a cell-free system appears to require the protein unfolding/folding activity of a third heat shock protein, hsp70. Like the oncogenic tyrosine kinase pp60src, steroid receptors bind to this complex of chaperone proteins at the time of their translation. Binding of the receptor to the hsp90 component of the system occurs through the hormone binding domain and is under strict hormonal control. The hormone binding domain of the receptor acts as a transferable regulatory unit that confers both tight hormonal control and hsp90 binding onto chimaeric proteins. The model of folding and transport being developed for steroid receptors leads to some general suggestions regarding the folding and transport of targeted proteins in the cell.     

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.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

The insulin receptor activation process involves localized conformational changes.

The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.     

Corepressor binding to progesterone and glucocorticoid receptors involves the activation function-1 domain and is inhibited by molybdate

Corepressors are known to interact via their receptor interaction domains (RIDs) with the ligand binding domain in the carboxyl terminal half of steroid/nuclear receptors. We now report that a portion of the activation function-1 domain of glucocorticoid receptors (GRs) and progesterone receptors (PRs), which is the major transactivation sequence, is necessary but not sufficient for corepressor [nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT)] RID binding to GRs and PRs in both mammalian two-hybrid and coimmunoprecipitation assays. Importantly, these two receptor sequences are functionally interchangeable in the context of GR for transactivation, corepressor binding, and corepressor modulatory activity assays. This suggests that corepressors may act in part by physically blocking portions of receptor activation function-1 domains. However, differences exist in corepressor binding to GRs and PRs. The C-terminal domain of PRs has a higher affinity for corepressor than that of GRs. The ability of some segments of the coactivator TIF2 to competitively inhibit corepressor binding to receptors is different for GRs and PRs. With each receptor, the cell-free binding of corepressors to ligand-free receptor is prevented by sodium molybdate, which is a well-known inhibitor of receptor activation to the DNA-binding state. This suggests that receptor activation precedes binding to corepressors. Collectively, these results indicate that corepressor binding to GRs and PRs involve both N- and C-terminal sequences of activated receptors but differ in ways that may contribute to the unique biological responses of each receptor in intact cells.     

Association of the heat shock protein hsp90 with steroid hormone receptors and tyrosine kinase oncogene products

Monospecific, polyclonal rabbit antibody raised against the 90-kd non-hormone binding component of molybdate-stabilized steroid hormone receptor specifically recognises the 90-kd molecular weight heat shock protein (hsp 90) in mink cell extracts. Partial proteolytic digestion experiments indicate that this protein is identical to the 90-kd phosphoprotein found in a highly stable complex with the protein products of at least three members of the tyrosine kinase family of oncogenes (src, fes, fgr).     

Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct.

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.