Glucocorticoid Receptor Homodimer Research Articles

Multiple liver-enriched trans-acting factors interact with the glucocorticoid- (GRU) and cAMP-(CRU) responsive units within the h-IGFBP-1 promoter

In response to hormonal control, serum concentrations of insulin-growth factor-binding protein-1 (IGFBP-1) may vary as much as 10-fold, owing to strict control of its gene's expression in hepatocytes. IGFBP-1 gene transcription is increased by gluco-corticoids and cAMP and inhibited by insulin. The effect of insulin is dominant since it suppresses constitutive and both glucocorticoid- and cAMP-stimulated transcription. Close examination of the human (h) IGFBP-1 promoter sequences showed that the glucocorticoid (GRE, nt −88 to −102) and cAMP (CRE, nt −259 to −264) response elements are 5′-flanked by an A/T-rich imperfect palindrome (nt −102 to −117 and −265 to −285, respectively). These A/T-rich motifs are putative cis-elements for liver-enriched trans-acting factors. Competition experiments in electrophoretic mobility shift assay were carried out using rat liver nuclear extracts and a set of synthetic oligonucleotides designed from hIGFBP-1 Glucorticoid and cAMP Response Units (GRU and CRU), the rat transthyretin HNF3 cis-element and the ‘D-site’ of the mouse albumin promoter. The nucleotide motifs located between nt −108 and −121 of the GRU, interacted with the HNF3 family of rans-acting factors (α,β,γ), whereas those encompassing nt −181 to −104 bound DBP and/or nuclear proteins sharing similar sequence specificity (i.e. from the C/EBP family of bZIP proteins). We have also shown that the hIGFBP-1-GRE binds glucocorticoid receptor homodimers. In the case of the CRU, the cis-elements located between nt −249 and −285 bound DBP and/or nuclear proteins sharing similar sequence specificities. In addition, the nucleotide stretch lying between nt −256 and −275 was able to interact with the HNF3 family of trans-acting factors.

Novel glucocorticoid receptor complex with DNA element of the hormone-repressed POMC gene.

Previous studies defined a DNA element necessary for glucocorticoid repression of the pro-opiomelanocortin (POMC) gene. The glucocorticoid receptor (GR) binds this negative glucocorticoid response element (nGRE) with an in vitro affinity similar to that of GR for positive GREs. However, whereas GR binds GREs as homodimers, a novel GR complex which forms with nGRE appears to contain three GR molecules. Biochemical characterization of this complex as well as equilibrium binding studies suggest that it is formed by sequential binding of a GR homodimer followed by binding of a GR monomer on the opposite side of the double helix. The DNA-binding domain (DBD) of GR is sufficient for differential binding of GRE and nGRE, as bacterially-expressed DBD formed unique nGRE complexes that contain three GR polypeptides. Thus, the POMC nGRE provides the first example of an interaction between GR and DNA in which GR binds otherwise than as a homodimer. Despite its high affinity for GR, the nGRE differs significantly from GREs in that it does not activate transcription in any context. As the nGRE appears insufficient on its own to confer hormone responsiveness, other POMC promoter elements are likely to be required to mediate glucocorticoid repression.

Homodimer formation is rate-limiting for high affinity DNA binding by glucocorticoid receptor.

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor which activates transcription of specific genes by binding to a DNA sequence present in the promoters of inducible genes. These glucocorticoid response elements (GREs) have a conserved palindromic sequence. Each half-GRE palindrome binds one subunit of GR. We have assessed the relative affinity of GR monomers and homodimers for GRE and determined whether homodimer formation is rate-limiting for high affinity GRE binding. The in vitro affinity of GRE binding by GR homodimers was approximately 2 x 10(-10) M, whereas it was approximately 1 nM for GR monomers. While homodimer:GRE complexes were very stable, monomer:GRE complexes appeared less stable in vitro. At low receptor concentration, GR preferentially bound GRE as a homodimer. Prior dilution of GR (equilibrium shifted to monomers) before addition to a GRE binding reaction resulted in slower kinetics of binding by comparison to parallel reactions in which concentrated (largely homodimeric) GR was added first. Taken together, these experiments suggest that homodimer formation is rate-limiting for high affinity GRE binding. A GRE mutant which contained only a half-binding site and which was unable to bind GR homodimers was also unable to confer glucocorticoid-inducible transcription. Taken together with previous work, these experiments support the model that GR homodimers are required for hormone-dependent activation of transcription and that receptor homodimer formation is rate-limiting for GRE binding.

Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties.

We have investigated the influence of the N-terminal domain of the 94-kDa glucocorticoid receptor on the DNA:receptor interaction. An alpha-chymotrypsin-induced 39-kDa receptor fragment, containing the hormone and DNA binding domains, binds DNA with a reduced specificity compared to the intact 94-kDa receptor. Various footprinting assays did not reveal any qualitative differences when comparing the DNA contact points made by the two different receptor entities. Like the intact receptor, the 39-kDa receptor fragment binds as a dimer to DNA. Glutaraldehyde cross-linking demonstrated a difference in the protein:protein contacts of the two homodimers. Furthermore, the dimeric 94-kDa receptor did not recognize a half-DNA site, while the dissociated 94-kDa receptor dimer and the dimeric 39-kDa receptor fragment allowed binding to such a site. These results suggest that the loss of the N-terminal domain of the receptor affects the steric arrangement and/or rigidity of the two DNA binding domains of the receptor homodimer, resulting in a decreased DNA binding specificity of the 39-kDa receptor fragment.