TY - JOUR
T1 - The steroid-binding properties of recombinant glucocorticoid receptor
T2 - A putative role for heat shock protein hsp90
AU - Ohara-Nemoto, Yuko
AU - Strömstedt, Per Erik
AU - Dahlman-Wright, Karin
AU - Nemoto, Takayuki
AU - Gustafsson, Jan Åke
AU - Carlstedt-Duke, Jan
PY - 1990/11/30
Y1 - 1990/11/30
N2 - The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide ∼ 70 nM). Glycerol gradient analysis of the E. coli lysate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lysate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2 nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.
AB - The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide ∼ 70 nM). Glycerol gradient analysis of the E. coli lysate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lysate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2 nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.
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U2 - 10.1016/0960-0760(90)90391-W
DO - 10.1016/0960-0760(90)90391-W
M3 - Article
C2 - 2278831
AN - SCOPUS:0025598281
SN - 0960-0760
VL - 37
SP - 481
EP - 490
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
IS - 4
ER -