Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences

A combination of the gel retardation assay and interference by hydroxyl radical modification (missing nucleoside technique) was used to analyze the interaction of the glucocorticoid receptor (GR) with various glucocorticoid responsive elements (GRE). Short oligonucleotides containing the 15-bp GRE and 1 to 3 flanking base pairs on each side, are bound with very low affinity. The same GREs, when positioned in the center of a large DNA fragment (40-50 bp), show high affinity for the receptor. However, when the GRE is positioned at the border of a 54-bp fragment, the affinity of the GR for the GRE decreases markedly. The DNA binding affinity increases linearly with each added flanking base pair and optimal binding is observed with 8-10 flanking bp. Thus, the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. Using larger DNA fragments (greater than 100 bp) and a smaller form of the receptor (40 kD), two retarded complexes are found that correspond to monomeric and homodimeric receptor DNA complexes. The DNA-binding domain of the GR (20 kD), expressed in bacteria, binds to the GRE as a monomer as well as a dimer and can form heterodimers with the native 94-kD GR. Insertion or deletion of one single base pair between the two halves of the GRE reduces the affinity for the homodimeric form of the native GR, and inhibits the function of the GRE in gene transfer experiments, suggesting that a dimer of the GR is the functional entity that binds to the GRE.     

The effects of ras gene expression on glucocorticoid receptors in mouse fibroblasts

Analysis of induction of glutamine synthetase activity by dexamethasone showed a 2-fold increase in NIH3T3 but no change in NIH3T3 ras (EJ-ras) cells. The observed increase could be abolished by the antagonist RU486. The lack of response in ras transformed cells might reflect oncoprotein effects on the glucocorticoid receptor (GR). Several GR parameters were studied in order to clarify this point. Total GR level was the same for both cells; cytoplasmic receptor level however, was 3 times lower in NIH3T3 ras than in NIH3T3 cells. Hormone-receptor binding affinity, specificity, thermostability, sedimentation coefficient, molecular weight as well as the cytoplasmic GR transformation ratio were similar for the two cell lines. On the other hand, the fraction of the total receptor pool involved with the recycling process was approximately 20% lower in NIH3T3 ras than in NIH3T3 cells. After 24 h of dexamethasone treatment, no GR down regulation was observed in NIH3T3 ras cells, whereas normal NIH3T3 cells exhibited a decrease of GR binding capacity around 80%. Further studies are necessary to define the mechanisms underlying the association between glucocorticoid insensitivity, and modifications in the GR nuclear/cytoplasmic ratio, in the recycling GR fraction and in the down-regulation process observed in ras transformed cells.     

Antibodies raised against the extracellular tail of the CCKB/gastrin receptor inhibit gastrin-stimulated signalling

INTRODUCTION: Gastrin acts to stimulate gastric acid secretion and is an acknowledged growth factor for human gastrointestinal (GI) cancer. The identity of the exact receptor type mediating the growth promoting effects of gastrin in tumours is uncertain. However, the best-characterised gastrin receptor is the CCK receptor type B (CCKB)/gastrin receptor. The anti-GRE1 antibody is a polyclonal, affinity-purified antibody raised against GRE1, a synthetic 21 amino acid peptide homologous to part of the extracellular, N-terminal tail of the CCKB receptor. We have recently proven that GRE1 antiserum specifically localises CCKB receptors on CCKB receptor transfected NIH3T3 cells and human gastrointestinal tumour cells by Western blotting and immunocytochemistry. GRE1 antiserum also inhibits liver invasion in the C170HM2 colorectal liver-metastasis model. AIM: To relate the ability of GRE1 antiserum to displace G17 from CCKB receptors with its impact on cellular transduction effects. METHODS: Radioligand binding studies were performed with 125IG17 and Calcium mobilisation studies by use of the fluorescent dye Fura 2-am. RESULTS: GRE1 antiserum competitively displaced 50% radiolabelled gastrin-17 from whole cell NIH3T3 CCKB transfectants at a protein concentration of 250 microg x ml(-1). GRE1 antiserum did not stimulate calcium ion influx in the transfectant NIH3T3 cells when used at a range of protein concentrations. Pre-incubation with GRE1 antiserum was required to inhibit gastrin-stimulated calcium ion influx. This was found to be concentration-dependent, with inhibition shown at 30 and 5 microg x ml(-1) but not at 500 ng x ml(-1) or below. CONCLUSION: The GRE1 antiserum is specific for the CCKB receptor and may act to inhibit gastrin-stimulated signalling in tumour cells.     

Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element

Gel retardation analysis with full- and half-palindromic sequences using partially purified glucocorticoid receptor (GR) resulted in GR-glucocorticoid response element (GRE) species of identical mobilities, suggesting that formation of the dimeric GR protein complex is not catalyzed by DNA binding. These results are in contrast to the behavior of the isolated DNA binding domain of the glucocorticoid receptor where dimerization occurred on the GRE. Density gradient centrifugation of cytosolic GR resulted in two forms, a 4 S peak characteristic of the monomeric GR and a fraction which sediments at 6 S which is consistent with the observed size of the dimeric GR. These two forms were found to differ in their ability to bind to specific DNA sequences with the 6 S species having a higher affinity for a GRE. Taken together our results are consistent with a two-step model for hormone-induced transformation of GR: dissociation of the multimeric untransformed complex and dimerization of the GR to yield a high affinity DNA binding species.     

Differential regulation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene by human mineralocorticoid hormone-receptor complexes.

The brain tissues of the rat and mouse express two types of corticosteroid binding proteins, the glucocorticoid (GR) and aldosterone (MR) receptors. Unlike the type II (GR) receptor, type I receptor has a high affinity for aldosterone (ALDO) and corticosterone and is structurally similar to the kidney mineralocorticoid receptor (MR). The results reported in this study provide direct evidence for the interaction of dexamethasone (DEX), triamcinolone acetonide (TA), dexamethasone-21-mesylate (DXM) and 11-deoxycorticosterone (DOC) with human MR expressed in cells by transient co-transfection of a hMR expression vector. The interactions of hMR with DEX, TA, DXM, DOC, promegestone (R5020) and methyltrienelone (R1881) were measured by trans-activation of mouse mammary tumor virus long terminal repeat fused to bacterial chloramphenicol acetyltransferase (MMTV-tk-CAT) in gene co-transfection experiments and by cell free hormone binding assay. The incubation of various steroid hormones in the presence of [3H]ALDO in a competition assay with extracts prepared from HeLa cells co-transfected with hMR expression vector, showed that hMR expressed under these conditions has a high relative affinity for DEX which is similar to ALDO, TA and DOC. Incubation with DXM under these conditions showed very little competition, as was observed with R1881 and R5020. Incubation of the co-transfected cells with DEX, ALDO, DOC, R5020, TA, R1881 and DXM demonstrated that the level of trans-activation did not reflect the previously observed order of binding affinity for the hMR. The level of transactivation was always higher with DEX and TA compared to ALDO and DOC. Analysis of the binding of labeled glucocorticoid regulatory element (GRE) and hMR incubated with DEX, ALDO and DXM by gel shift analysis demonstrated that the trans-activation of MMTV-tk-CAT by hMR is a result of the interaction of hMR with GRE in the MMTV-LTR.     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.     

Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells.

Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types.     

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.     

Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton.

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.     

UV cross-linking/immunoprecipitation assay: glucocorticoid receptor-adrenergic receptor gene sequence interaction

The rat beta 1-adrenergic receptor (beta 1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither beta 1-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat beta 1-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the beta 1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immunoprecipitation assay in the detection of mammalian GR interaction with beta 1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonucleotide probes containing the beta 1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rate beta 1-AR gene sequences.