E. Brad Thompson

The structure of the nuclear hormone receptors.

The functions of the group of proteins known as nuclear receptors will be understood fully only when their working three-dimensional structures are known. These ligand-activated transcription factors belong to the steroid-thyroid-retinoid receptor superfamily, which include the receptors for steroids, thyroid hormone, vitamins A- and D-derived hormones, and certain fatty acids. The majority of family members are homologous proteins for which no ligand has been identified (the orphan receptors). Molecular cloning and structure/function analyses have revealed that the members of the superfamily have a common functional domain structure. This includes a variable N-terminal domain, often important for transactivation of transcription; a well conserved DNA-binding domain, crucial for recognition of specific DNA sequences and protein:protein interactions; and at the C-terminal end, a ligand-binding domain, important for hormone binding, protein: protein interactions, and additional transactivation activity. Although the structure of some independently expressed single domains of a few of these receptors have been solved, no holoreceptor structure or structure of any two domains together is yet available. Thus, the three-dimensional structure of the DNA-binding domains of the glucocorticoid, estrogen, retinoic acid-beta, and retinoid X receptors, and of the ligand-binding domains of the thyroid, retinoic acid-gamma, retinoid X, estrogen, progesterone, and peroxisome proliferator activated-gamma receptors have been solved. The secondary structure of the glucocorticoid receptor N-terminal domain, in particular the taul transcription activation region, has also been studied. The structural studies available not only provide a beginning stereochemical knowledge of these receptors, but also a basis for understanding some of the topological details of the interaction of the receptor complexes with coactivators, corepressors, and other components of the transcriptional machinery. In this review, we summarize and discuss the current information on structures of the steroid-thyroid-retinoid receptors.

Mechanism of action of glucocorticoids

Abstract The mechanism of action of glucocorticoids is reviewed from the standpoint of seeing how far current concepts of the molecular action of steroids go towards explaining the varied physiologic and metabolic effects induced. The role of plasma binding to transport proteins in modifying steroid action is considered. The interactions between glucocorticoids and other hormones, particularly those mediated by cAMP are considered along with a discussion of the mechanism of “permissive” effects. Various proposals concerning the way in which glucocorticoids induce their effects including direct interactions with enzymes, nucleic acids, lysosomes, and receptor proteins are considered in detail, and an attempt is made to consider how far each of these mechanisms can go in explaining steroid action. Those biologic effects that cannot be accounted for by currently available mechansims are mentioned. Effects of glucocorticoids on growth, differentiation, and the inflammatory response are discussed from a mechanistic point of view. Catabolic or inhibitory effects of glucocorticoids as well as enzyme induction are examined using several exemplary systems. Finally an attempt is made to summarize the current state of knowledge concerning glucocorticoid and genome interaction.

Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins

Transcription factors and other allosteric cell signaling proteins contain a disproportionate number of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases folding of these segments is coupled to binding with one or more of their interaction partners, suggesting that intrinsic disorder plays an important functional role. Despite numerous hypotheses for the role of ID domains in regulation, a mechanistic model has yet to be established that can quantitatively assess the importance of intrinsic disorder for intramolecular site-to-site communication, the hallmark property of allosteric proteins. Here, we present such a model and show that site-to-site allosteric coupling is maximized when intrinsic disorder is present in the domains or segments containing one or both of the coupled binding sites. This result not only explains the prevalence of ID domains in regulatory proteins, it also calls into question the classical mechanical view of energy propagation in proteins, which predicts that site-to-site coupling would be maximized when a well defined pathway of folded structure connects the two sites. Furthermore, in showing that the coupling mechanism conferred by intrinsic disorder is robust and independent of the network of interactions that physically link the coupled sites, unique insights are gained into the energetic ground rules that govern site-to-site communication in all proteins.

Gene regulation by the glucocorticoid receptor: Structure:function relationship

The glucocorticoid receptor (GR) belongs to the superfamily of ligand-activated transcription factors, the nuclear hormone receptors. Like other members of the family, the GR possesses a modular structure consisting of three major domains-the N-terminal (NTD), DNA binding (DBD), and ligand binding (LBD). Although the structures of independently expressed GR DBD and LBD are known, the structures of the NTD and of full-length GR are lacking. Both DBD and LBD possess overall globular structures. Not much is known about the structure of the NTD, which contains the powerful AF1/tau1/enh2 transactivation region. Several studies have shown that AF1 region is mostly unstructured and that it can acquire folded functional conformation under certain potentially physiological conditions, namely in the presence of osmolytes, when the GR DBD is bound to glucocorticoid response element (GRE), and when AF1 binds other transcription factor proteins. These conditions are discussed here. The functions of the GR will be fully understood only when its working three-dimensional structure is known. Based on the available data, we propose a model to explain data which are not adequately accounted for in the classical models of GR action. In this review, we summarize and discuss current information on the structure of the GR in the context of its functional aspects, such as protein:DNA and protein:protein interactions. Because of the close similarities in modular organization among the members of the nuclear hormone receptors, the principles discussed here for the GR should be applicable to many other receptors in the family as well.

Characterization of two forms of glucocorticoid hormone-receptor complex separated by DEAE-cellulose column chromatography.

Summary Two forms of [ 3 H]triamcinolone acetonide-receptor complex prepared from rat liver cytosol were successfully separated by DEAE-cellulose column chromatography. One form (Peak I) was eluted by 0.06 M, and the other form (Peak II) by 0.24 M, potassium phosphate buffer (pH 7.4). Exposure of extracts to 20° for up to 2 h progressively diminished the radioactivity found in Peak II while increasing that in Peak I, with conservation of total bound radioactivity. The radioactivity in Peak I could bind to either chromatin or DNA very efficiently. Little of the radioactivity in Peak II could bind to either. The properties of the glucocorticoid receptor therefore appear to differ from those described for the two forms of progesterone receptors.

Estrogen receptor status: an important variable in predicting response to endocrine therapy in metastatic breast cancer

Abstract The influence of estrogen receptor status on response rate to endocrine therapy in 85 patients with metastatic breast cancer was determined in a retrospective study. The specific purpose of this study was to assess the role of estrogen receptor determinations in the light of a host of clinical variables known or suspected to influence response rates to endocrine therapy. Thirty-four of 52 patients whose tumors contained significant amounts of estrogen receptor (> 10 fmole/mg cytoplasmic protein) had objective responses to endocrine therapy while only 333 patients whose tumors did not possess estrogen receptor ( 10 fmole/mg cytoplasmic protein) responded ( P 0.0001 ). A quantitative relationship was found between the amount of estrogen receptor and response rate. The quantity of estrogen receptor was not associated with the duration of response. The predictive value of the estrogen receptor assay was not associated with the receptor dissociation constant. Prior treatment with endocrine or chemotherapy did not diminish the ability of estrogen receptor determinations to predict response to subsequent endocrine therapy. Response rate in estrogen receptor positive tumors was not affected by extent of disease, site of involvement with metastatic tumor, or prior therapy. We conclude that these negative prognostic factors are less important in predicting response to endocrine therapy than estrogen receptor values.

Trimethylamine N-Oxide-induced Cooperative Folding of an Intrinsically Unfolded Transcription-activating Fragment of Human Glucocorticoid Receptor

A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive α-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1–500 and residues 77–262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the α-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1–500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures.

The Conformation of the Glucocorticoid Receptor AF1/tau1 Domain Induced by Osmolyte Binds Co-regulatory Proteins

The activation domains of many transcription factors appear to exist naturally in an unfolded or only partially folded state. This seems to be the case for AF1/tau1, the major transactivation domain of the human glucocorticoid receptor. We show here that in buffers containing the natural osmolyte trimethylamineN-oxide (TMAO), recombinant AF1 folds into more a compact structure, as evidenced by altered fluorescence emission, circular dichroism spectra, and ultracentrifugal analysis. This conformational transition is cooperative, a characteristic of proteins folding to natural structures. The structure resulting from incubation in TMAO causes the peptide to resist proteolysis by trypsin, chymotrypsin, endoproteinase Arg-C and endoproteinase Gluc-C. Ultracentrifugation studies indicate that AF1/tau1 exists as a monomer in aqueous solution and that the presence of TMAO does not lead to oligomerization or aggregation. It has been suggested that recombinant AF1 binds both the ubiquitous coactivator CBP and the TATA box-binding protein, TBP. Interactions with both of these are greatly enhanced in the presence of TMAO. Co-immunoadsorption experiments indicate that in TMAO each of these and the coactivator SRC-1 are found complexed with AF1. These data indicate that TMAO induces a conformation in AF1/tau1 that is important for its interaction with certain co-regulatory proteins.

Stimulation of tyrosine aminotransferase synthesis by dexamethasone phosphate in cell culture

A specific antibody, prepared by injecting rabbits with purified rat liver tyrosine aminotransferase, has been used to study the glucocorticoid induction of this enzyme in HTC cells, a tissue culture system derived from a rat hepatoma. When the enzyme activity is induced, parallel immunotitrations show that there is a corresponding increase of the antigenic activity of tyrosine aminotransferase, indicating that the induction is due to an accumulation of enzyme protein. The rates of amino acid incorporation into tyrosine aminotransferase in induced and non-induced HTC cells were compared by using the double-label isotope technique and specific immunoprecipitation. When there was maximum induction (12-fold) of enzyme activity, there was about a 20-fold increase in the rate of enzyme synthesis. These studies show that the accumulation of aminotransferase on induction is due to an increased rate of its synthesis from free amino acids and not due to a decreased rate of degradation. Since there is amino acid incorporation into tyrosine aminotransferase under both basal and fully induced conditions (when the level of the enzyme is not changing), we conclude that in both conditions a steady state is maintained by a balance between enzyme degradation and synthesis. A simplified method for the preparation of the enzyme from rat liver, which gives 40 to 50% yields of highly purified material, is also presented.

Interdomain Signaling in a Two-domain Fragment of the Human Glucocorticoid Receptor

Studies of individual domains or subdomains of the proteins making up the nuclear receptor family have stressed their modular nature. Nevertheless, these receptors function as complete proteins. Studies of specific mutations suggest that in the holoreceptors, intramolecular domain-domain interactions are important for complete function, but there is little knowledge concerning these interactions. The important transcriptional transactivation function in the N-terminal part of the glucocorticoid receptor (GR) appears to have little inherent structure. To study its interactions with the DNA binding domain (DBD) of the GR, we have expressed the complete sequence from the N-terminal through the DBD of the human GR. Circular dichroism analyses of this highly purified, multidomain protein show that it has a considerable helical content. We hypothesized that binding of its DBD to the cognate glucocorticoid response element would confer additional structure upon the N-terminal domain. Circular dichroism and fluorescence emission studies suggest that additional helicity as well as tertiary structure occur in the two-domain protein upon DNA binding. In sum, our data suggest that interdomain interactions consequent to DNA binding imparts structure to the portion of the GR that contains a major transactivation domain.