Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5.

The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

Differential recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ERalpha and ERbeta.

Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established. Further, although the two subtypes ERalpha and ERbeta appear to be similar in regard to principles of LXXLL-mediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian mediator complex, referred to as TRAP220, with ERalpha and ERbeta. In comparison to the p160 coactivator TIF2, we find that TRAP220 displays ERbeta preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend on the dominant ER subtype.

Role of important hydrophobic amino acids in the interaction between the glucocorticoid receptor tau 1-core activation domain and target factors.

In this work, we determined how altered-function mutants affecting hydrophobic residues within the tau 1-core activation domain of the human glucocorticoid receptor (GR) influence its physical interaction with different target proteins of the transcriptional machinery. Screening of putative target proteins showed that the tau 1-core can interact with the C-terminal part of the CREB-binding protein (CBP). In addition, the previously identified interactions of the tau 1-core with the TATA-binding protein (TBP) and the Ada2 adaptor protein were localized to the C- and N-terminal regions of these proteins, respectively. A panel of mutations within the tau 1-core that either decrease or increase activation potential was used to probe the interaction of the tau 1-core domain with TBP, Ada2, and CBP. We found that the pattern of effects caused by the mutations was similar for each of the interactions and that the effects on binding generally reflected effects on gene activation potential. Thus, the predominant effect of the mutations appears to influence a property of the tau 1-core that is common to all three interactions, rather than properties that are differentially required by each of the target factor interactions, individually. Such a property could be the ability of the domain to adopt a folded conformation that is generally necessary for interaction with target factors. We have also shown that TBP, Ada2, and CBP can interact with both the tau 1-core and the GR ligand-binding domain, offering a possible mechanism for synergistic interaction between the tau 1-core and other receptor activation domains. However, other target proteins (e.g., RIP140, and SRC-1), which interact with the GR C terminus, did not show significant interactions with the tau 1-core under our conditions.

Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor.

We have shown that the Ada adaptor complex is important for the gene activation capacity of the glucocorticoid receptor in yeast. The recently isolated human Ada2 protein also increases the potency of the receptor protein in mammalian cells. The Ada pathway is of key significance for the tau1 core transactivation domain (tau1c) of the receptor, which requires Ada for activity in vivo and in vitro. Ada2 can be precipitated from nuclear extracts by a glutathione S-transferase-tau1 fusion protein coupled to agarose beads, and a direct interaction between Ada2 and tau1c can be shown by using purified proteins. This interaction is strongly reduced by a mutation in tau1c that reduces transactivation activity. Mutations affecting the Ada complex do not reverse transcriptional squelching by the tau1 domain, as they do for the VP16 transactivation domain, and thus these powerful acidic activators differ in at least some important aspects of gene activation. Mutations that reduce the activity of the tau1c domain in wild-type yeast strains cause similar reductions in ada mutants that contain little or no Ada activity. Thus, gene activation mechanisms, in addition to the Ada pathway, are involved in the activity of the tau1c domain.

Role of hydrophobic amino acid clusters in the transactivation activity of the human glucocorticoid receptor.

We have performed a mutagenesis analysis of the 58-amino-acid tau1-core peptide, which represents the core transactivation activity of the tau1 transactivation domain from the glucocorticoid receptor. Mutants with altered activity were identified by phenotypic screening in the yeast Saccharomyces cerevisiae. Most mutants with reduced activity had substitutions of hydrophobic amino acids. Most single-substitution mutants with reduced activity were localized near the N terminus of the tau1-core within a segment that has been shown previously to have a propensity for alpha-helix conformation, suggesting that this helical region is of predominant importance. The particular importance of hydrophobic residues within this region was confirmed by comparing the activities of alanine substitutions of the hydrophobic residues in this and two other helical regions. The hydrophobic residues were shown to be important for the transactivation activity of both the isolated tau1-core and the intact glucocorticoid receptor in mammalian cells. Rare mutations in helical regions I and II gave rise to increased transcriptional activation activity. These mutations increase the hydrophobicity of hydrophobic patches on each of these helices, suggesting a relationship between the hydrophobicity of the patches and transactivation activity. However, certain nonhydrophobic residues are also important for activity. Interestingly, helical region I partially matches a consensus motif found in the retinoic acid receptor, VP16, and several other activator proteins.

Role of acidic and phosphorylated residues in gene activation by the glucocorticoid receptor.

To investigate the role of acidic and phosphorylated amino acids in the function of the major transactivation domain (tau 1) of the glucocorticoid receptor, we have performed a mutagenesis study. Aspartic and glutamic acid residues were neutralized in clusters of 2 to 4 amino acids throughout the tau 1 domain. The activity of the mutant proteins was determined using transactivation assays in yeast and mammalian cells. Some acidic residues in the core region of tau 1 appear to play a minor role in tau 1 activity, but, generally, individual acidic residues are not critical for activity. Mutagenesis of five serine residues that are phosphorylated in the mouse glucocorticoid receptor and which are conserved in the human receptor did not affect the transactivation activity of the tau 1 domain in yeast. As in mouse cells, these serine residues are the predominant sites of phosphorylation for ectopically expressed receptor in yeast, since the mutant protein lacking all five sites had a severely reduced phosphorylation level. Mutant proteins in which larger numbers of acidic residues are neutralized show a progressive decrease in activity indicating that acidity in general is important for tau 1 function. However, our results are not consistent with the "acid blob" theory of transactivator function that has been suggested for some other activator proteins. Other putative roles for the acidity of tau 1 are discussed.

Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor.

A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment. Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions. The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation. Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character. Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo.

The glucocorticoid receptor functions at multiple steps during transcription initiation by RNA polymerase II.

We have used a panel of monoclonal antibodies and a cell-free transcription assay to study the function of the tau 1 transactivation domain of the human glucocorticoid receptor. Three antibodies (monoclonal antibodies 250, 275, and 286) specifically inhibited tau 1-dependent transcription, but had little or no effect on either basal transcription or the activity of an unrelated yeast transcription factor. This inhibition was not due to interference of DNA binding activity, as all three antibodies super shifted tau 1-containing protein-DNA complexes. Epitopes for all three antibodies were localized to a region between amino acids 190 and 200, which lies within the recently defined 41-amino acid core region of tau 1 that is required for transactivation (Dahlman-Wright, K., Almlöf, T., McEwan, I.J., Gustafsson, J-A., and Wright, A. P.H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1619-1623). In contrast to the effect on tau 1-dependent transcription none of the antibodies tested antagonized the squelching ability of the tau 1 domain, suggesting that tau 1-mediated transactivation involves interactions in addition to those identified by the squelching assay. Consistent with this, a comparison of the kinetics of tau 1 squelching and inhibition of transactivation by monoclonal antibodies suggested a role for tau 1 mediated transcriptional induction at two or more steps during transcription initiation by RNA polymerase II.

Delineation of a small region within the major transactivation domain of the human glucocorticoid receptor that mediates transactivation of gene expression.

Previous deletion analysis localized the major transactivation function of the human glucocorticoid receptor to a 185-amino acid segment close to the N terminus of the receptor protein. This region was named tau 1 [Hollenberg, S. M. & Evans, R. M. (1988) Cell 55, 899-906]. To delineate the smallest active region within tau 1, we have systematically tested the transactivation capacity of deletion derivatives of the tau 1 domain, fused to the glucocorticoid receptor DNA-binding domain, in yeast cells. Internal scanning deletions suggested that residues near the C terminus of tau 1 are most important for activity. Deletions of N-terminal and C-terminal sequences identified a 41-amino acid "core" region near the C terminus of tau 1 that is crucial for tau 1 function. Small peptide fragments containing the tau 1 core region are competent for transactivation, while regions outside the tau 1 core are not active. We have previously demonstrated that the intact tau 1 domain squelches the activity of a minimal promoter in vivo and in vitro, suggesting involvement of interactions with a component/components of the basal transcription machinery in the mechanism of transactivation. This activity was maintained in the tau 1 core-containing segments.

Structure and function of the glucocorticoid receptor.

Glucocorticoids cause changes in the expression of target genes via interaction with an intracellular receptor protein, the glucocorticoid receptor. This signal transduction process can be divided into a number of steps, each of which represents a functional facet of the receptor protein. These steps include (i) receptor transformation to an active form resulting from specific interaction with glucocorticoid steroid hormones, (ii) homo-dimerization, (iii) DNA-binding to specific hormone response elements in the genome and (iv) modulation of the expression levels of linked genes. These aspects of glucocorticoid receptor function have been studied using a combination of tertiary structure determination, biochemical assays and a genetic approach using a yeast system to screen for mutant receptors that are altered in function. The results show that contacts involving both the DNA and steroid binding domains are involved in dimerization and high affinity DNA binding. Genetic experiments have illuminated the role of amino acids within the recognition helix of the DNA-binding domain in discriminating between cognate DNA response elements for the glucocorticoid receptor and closely related binding sites for other nuclear receptors. Squelching experiments suggest that the N-terminal transactivation domain of the receptor contacts components of the general transcriptional machinery that appear to be distinct from the TATA binding protein, TFIID, during transactivation of gene expression by the DNA-bound receptor.