The steroid-binding properties of recombinant glucocorticoid receptor: A putative role for heat shock protein hsp90

Yuko Ohara-Nemoto, Per Erik Strömstedt, Karin Dahlman-Wright, Takayuki Nemoto, Jan Åke Gustafsson, Jan Carlstedt-Duke

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)481-490
Number of pages10
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume37
Issue number4
DOIs
StatePublished - Nov 30 1990

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

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