The glucocorticoid receptor (GR) is a steroid hormone-activated transcription factor involved in the processes of inflammation, glucose homeostasis, bone cell turnover, cell differentiation, and lung maturation (Reichardt et al., 2000). It belongs to the extensive superfamily of nuclear receptors, which includes mineralcorticoid, estrogen, progestin, androgen, peroxisome proliferator, vitamin D and thyroid hormone receptors. GR is modular, made up of an N-terminal activation function-1 domain (AF-1), a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD) (Bledsoe et al, 2002). GR ligands are corticosteroid analogs, including dexamethasone and prednisolone. When not bound to a ligand, chaperone proteins such as hsp90 and p23 retain GR in the cytoplasm. Once a hormone binds, the chaperone proteins are released and dimerization occurs, along with nuclear translocation of the entire receptor. Once inside the nucleus, GR can bind to specific DNA promoter elements or 'cross-talk' with specific transcription factors to repress gene activation.
Therapeutically, GR is of great interest for two reasons. First, mutations in GR play a role in Cushing’s syndrome (an endocrine disorder caused by excessive levels of cortisol, a corticosteroid), autoimmune diseases, and some cancers. Second, GR ligands are already used to treat a variety of medical conditions, such as asthma, rheumatoid arthritis, and leukemia (Barnes et al., 1998). The use of these ligands as therapy, however, is limited due to negative side effects, such as bone loss, growth retardation, and hypothalamic-pituitary-adrenal axis suppression. A better understanding of the glucocorticoid receptor would aid in the hunt for a GR ligand and possibly result in a treatment possessing all of the anti-inflammatory benefits without the disabling side-effects.
In the absence of a ligand, the glucocorticoid receptor is retained in the cytoplasm by chaperone proteins that bind in place of a ligand in the C-terminal ligand binding domain (LBD) <> (Pratt and Toft, 1997). This interaction occurs in a hydrophobic pocket embedded within a canonical three-layer helical sandwhich, regulated by the C- terminal activation function 2 (AF-2) helix <>. When the LBD is in the apo-state it is bound to a chaperone protein - hsp90 or p23. In this deactivated state the AF-2 helix is destabilized and the LBD is able to interact with corepressors (Chen and Evans, 1995). When the appropriate ligand binds, in this case dexamethasone <>, a stabilizing conformational change in the AF-2 helix occurs. This change switches the receptor into an active conformation, enabling it to interact with coactivator proteins, such as transcriptional intermediary factor 2 <>. (TIF2; Onate et al., 1996; Voegel et al., 1996) The AF-2 partially participates in a charge clamp that stabilizes the helical ends of a conserved LLXXLL (L = Leu, X = any residue) two-turn alpha helix in the coactivator (Darimonet et al., 1998). The LBD also aids in formation of the glucocorticoid receptor homodimer, however, the arrangement of the AF-2 in that structure has yet to be defined.
Dimer formation:
The two LBD monomers <> are symmetrically arranged in the dimer. The dimer interface is maintained by hydrophobic and hydrogen bond interactions. The main hydrophobic interface consists of reciprocal interactions between Pro 625 <> and Ile 628 <> residues in the beta turns of strands 3 and 4. Hydrogen bonds between residue 547 of each monomer in helices 1 and 3 (likewise for each residue 548-551 of each monomer) <> and Gln 615 of helix 5 <> surround this hydrophobic interface and may aid in stabilizing the dimer configuration.
Recognition of TIF2:
TIF2 <>, along with other coactivators of the GR-LBD, have three conserved LLXXLL motifs. One of which, the Leu-Leu-Arg-Tyr-Leu-Leu <> sequence, forms a two-turn alpha helix. The hydrophobic leucine side chains <> fit into a groove formed by the AF-2 helix and residues from helices 3 and 4 <>. Glu 755 of the AF-2 helix and Lys 579 of helix 3 <> clamp down the N- and C-terminal ends of the coactivator helix. A second charge clamp is formed by the GR residues Asp 590 and Arg 585 <>, which interact with another LLXXLL motif of the coactivator. These charge clamps contribute to the selective binding to GR.
Recognition of Dexamethasone:
The bottom half of the LBD completely encloses dexamethasone <> in a ligand binding pocket composed of residues from the surrounding helices (3-7,10), the AF-2 helix, and residues from beta strands 1 and 2 <>. Van der Waals forces, along with extensive hydrophobic and hydrophilic interactions, are key factors in ligand binding. Almost every atom in the steroid core of dexamethasone is in contact with one or more hydrophobic residues within the LBD. The hydrophilic groups of the ligand form hydrogen bonds with the LBD. For example, the aromatic ring carbonyl of dexamethasone forms hydrogen bonds with Arg 611 and Gln 570 <> residues. In addition, Leu 753 of the AF-2 helix and Ile 747 and Phe 749 of the peptide loop preceding the AF-2 helix <> interact directly with dexamethasone. These interactions have a stabilizing effect of the AF-2 helix, aiding its active form, and may serve as a molecular basis for the ligand-dependent activation of the glucocorticoid receptor.
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Once ligand binding domain the entire receptor protein is transported into the cell's nucleus and the DNA binding domain becomes active. The DNA binding region is illustrated here as two identical monomers, each of which can be divided into two submodules. Each monomer (monomer A and monomer B) is composed of three α helices <
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Once inside the nucleus, the glucocorticoid receptor binds DNA to regulate transcription. Each of the DNA-binding domains binds a specific DNA sequence by inserting the amino- and carboxy-terminal fingers into adjacent major grooves of a DNA molecule. The DNA binding site possesses dyad symmetry, and a three base-pair non-specific ‘spacer’ region between the two half sites. In order to fully understand the nature of the protein-DNA interaction, however, a DNA oligo with a 4 base-pair spacer was used to increase crystal resolution, resulting in a specific protein-DNA in only one of the monomers.
Each DNA half site is six base-pairs long with the conserved sequence 5’-AGAACA-tcga-TGTTCT-3’ <
Each monomer also makes several phosphate <
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