Identification of Amino Acids in the  2-Region of the Mouse Glucocorticoid Receptor That Contribute to Hormone Binding and Transcriptional Activation

1997 ◽  
Vol 11 (12) ◽  
pp. 1795-1805 ◽  
Author(s):  
J. Milhon
Keyword(s):  
Amino Acids ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Hormone Binding

scholarly journals Identification of Amino Acids in the τ2-Region of the Mouse Glucocorticoid Receptor That Contribute to Hormone Binding and Transcriptional Activation

1997 ◽  
Vol 11 (12) ◽  
pp. 1795-1805 ◽  
Author(s):  
Jon Milhon ◽  
Sunyoung Lee ◽  
Kulwant Kohli ◽  
Dagang Chen ◽  
Heng Hong ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Full Length ◽  
Steroid Hormone Receptors ◽  
Single Mutation ◽  
Hormone Binding ◽  
Terminal Portion ◽  
Transactivation Activity

Abstract The τ2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding τ2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the τ2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the τ2-region of the mouse glucocorticoid receptor (mGR, amino acids 533–562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum τ2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum τ2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum τ2-fragment and the full-length GR indicated that the τ2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the τ2-region (mGR 541–547) was primarily involved in hormone binding, whereas the C-terminal portion of theτ 2-region (mGR 548–561) was primarily involved in transactivation.

scholarly journals A Novel Isoform of Human LZIP Negatively Regulates the Transactivation of the Glucocorticoid Receptor

2009 ◽  
Vol 23 (11) ◽  
pp. 1746-1757 ◽  
Author(s):  
Hyereen Kang ◽  
Yoon Suk Kim ◽  
Jesang Ko
Keyword(s):  
Amino Acids ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Transmembrane Domain ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Basic Leucine Zipper

Abstract The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as “sLZIP” contains a deleted putative transmembrane domain (amino acids 229–245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.

scholarly journals The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress.

1996 ◽  
Vol 16 (3) ◽  
pp. 839-846 ◽  
Author(s):  
E M Newton ◽  
U Knauf ◽  
M Green ◽  
R E Kingston
Keyword(s):  
Amino Acids ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Acid Activation ◽  
Regulatory Domain ◽  
Activation Domain ◽  
Control Temperature ◽  
Activation Domains ◽  
Transcriptional Activation Domains

Heat shock factor (HSF) activates transcription in response to cellular stress. Human HSF1 has a central regulatory domain which can repress the activity of its activation domains at the control temperature and render them heat shock inducible. To determine whether the regulatory domain works in tandem with specific features of the HSF1 transcriptional activation domains, we first used deletion and point mutagenesis to define these activation domains. One of the activation domains can be reduced to just 20 amino acids. A GAL4 fusion protein containing the HSF 1 regulatory domain and this 20-amino-acid activation domain is repressed at the control temperature but potently activates transcription in response to heat shock. No specific amino acids in this activation domain are required for response to the regulatory domain; in particular, none of the potentially phosphorylated serine and threonine residues are required for heat induction, implying that heat-induced phosphorylation of the transcriptional activation domains is not required for induction. The regulatory domain is able to confer heat responsiveness to an otherwise completely heterologous chimeric activator that contains a portion of the VP16 activation domain, suggesting that the regulatory domain can sense heat in the absence of other portions of HSF1.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

scholarly journals Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor

2018 ◽  
Vol 115 (46) ◽  
pp. 11688-11693 ◽  
Author(s):  
Thomas Suren ◽  
Daniel Rutz ◽  
Patrick Mößmer ◽  
Ulrich Merkel ◽  
Johannes Buchner ◽  
...  
Keyword(s):  
Free Energy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Load ◽  
Force Spectroscopy ◽  
Folding Pathway ◽  
Hormone Binding ◽  
Single Molecule Force Spectroscopy

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

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