DNA binding of glucocorticoid receptor protein a fusion proteins expressed in E. coli

Bonifer, Constanze; Dahlman, Karin; Strömstedt, Per-Erik; Flock, Jan Ingmar; Gustafsson, Jan Åke

doi:10.1016/0022-4731(89)90006-x

Cited by 9 publications

(4 citation statements)

References 24 publications

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“…However, in addition to the association with hsp90, at this time we cannot rule out the possibility of other receptor modifications induced by lysate treatment, e.g., phosphorylation. Our findings can also explain, at least in part, why a purified GR-fusion construct generated in bacteria (Bonifer et al, 1989), bearing the complete DNAand steroid-binding domains, does not bind steroid. To more strictly correlate the functional requirements of the MR versus GR, we have recently used our bacterial system for the production of a GST-GR fusion receptor (unpublished results).…”

Section: Discussionmentioning

confidence: 74%

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics

Caamaño

Morano

Patel³

et al. 1993

Biochemistry

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A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 74%

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics

Caamaño

Morano

Patel³

et al. 1993

Biochemistry

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“…For GR415, several proteolytic fragments were also detected. Most of the expressed proteins were recovered in an insoluble fraction as reported previously (Bonifer et al, 1989). However, specific [3H]TA binding was found in a soluble fraction.…”

Section: Resultsmentioning

confidence: 99%

“…E. coli (MZ1) cells were transformed with the plasmid, and colonies producing the protein A fused steroid-binding domain were selected. The plasmid containing the sequence of DNAand steroid-binding domains (amino acids 415-777) was constructed as described previously (Bonifer et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

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The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

Nemoto

Ohara‐Nemoto²,

Denis

et al. 1990

Biochemistry

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High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, we decided to investigate the possible relationship between these two phenomena. Steroid-free GR was converted from a 9S to a 4S form by exposure to 0.4 M NaCl. The binding of [3H]triamcinolone acetonide [( 3H]TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas [3H]TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR exposed to 0.4 M NaCl in the presence of 10 mM molybdate showed the presence of two types of binding sites with apparent dissociation constants of 0.52 +/- 0.07 and 64.1 +/- 16.2 nM, respectively. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low [3H]TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. Immunoblot analysis using anti-GR monoclonal antibody revealed no difference in molecular size (Mr 94000) between the high- and low-affinity entities. These results indicate that the transformed GR has a reduced [3H]TA-binding affinity as compared to the nontransformed GR.(ABSTRACT TRUNCATED AT 250 WORDS)

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Functional Analysis of the Glucocorticoid Receptor

Strömstedt¹,

Carlstedt‐Duke²,

Gustafsson³

1995

Signalling Mechanisms — From Transcription Factors to Oxidative Stress

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DNA binding of glucocorticoid receptor protein a fusion proteins expressed in E. coli

Cited by 9 publications

References 24 publications

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

Functional Analysis of the Glucocorticoid Receptor

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