Novel and simple two-step purification of a full-length rat glucocorticoid-receptor expressed in a baculovirus system

2001 ◽  
Vol 765 (1) ◽  
pp. 89-97 ◽  
Author(s):  
Makoto Hyodo ◽  
Kazuki Okamoto ◽  
Kiyotaka Shibata ◽  
Naoya Suematsu ◽  
Fumihide Isohashi
Keyword(s):  
Glucocorticoid Receptor ◽  
Full Length ◽  
Baculovirus System

scholarly journals N-terminal Domain Regulates Steroid Activation of Elephant Shark Glucocorticoid and Mineralocorticoid Receptors

2019 ◽  
Author(s):  
Yoshinao Katsu ◽  
Islam MD Shariful ◽  
Xiaozhi Lin ◽  
Wataru Takagi ◽  
Hiroshi Urushitani ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Mineralocorticoid Receptor ◽  
Functional Divergence ◽  
Full Length ◽  
Mineralocorticoid Receptors ◽  
Elephant Shark ◽  
Terminal Domain ◽  
Cartilaginous Fishes ◽  
Steroid Binding

AbstractOrthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD+LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.

Purification of a full-length recombinant glucocorticoid receptor

2003 ◽  
Vol 790 (1-2) ◽  
pp. 349-353 ◽  
Author(s):  
Kazuki Okamoto ◽  
Naoya Suematsu ◽  
Fumihide Isohashi
Keyword(s):  
Glucocorticoid Receptor ◽  
Full Length

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 Characterization and purification of a functional rat glucocorticoid receptor overexpressed in a baculovirus system

1991 ◽  
Vol 266 (6) ◽  
pp. 3925-3936
Author(s):  
E S Alnemri ◽  
A B Maksymowych ◽  
N M Robertson ◽  
G Litwack
Keyword(s):  
Glucocorticoid Receptor ◽  
Baculovirus System

scholarly journals N-terminal Domain Regulates Steroid Activation of Elephant Shark Glucocorticoid and Mineralocorticoid Receptors

2020 ◽  
Author(s):  
Yoshinao Katsu ◽  
Islam MD Shari ◽  
Xiaozhi Lin ◽  
Wataru Takagi ◽  
Hiroshi Urushitani ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Mineralocorticoid Receptor ◽  
Functional Divergence ◽  
Full Length ◽  
Mineralocorticoid Receptors ◽  
Elephant Shark ◽  
Terminal Domain ◽  
Cartilaginous Fishes ◽  
Steroid Binding

Abstract Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD+LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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