title: Putative steroid binding domain of the human mineralocorticoid receptor, expressed in E. coli in the presence of heat shock proteins shows typical native receptor characteristics.
abstract: Domain E, considered as the putative hormone binding domain (HBD) of the human mineralocorticoid receptor (hMR) was expressed in Escherichia coli as a fusion protein with either maltose binding protein (MBP) or glutathione S-transferase (GST). These bacterially-produced MR constructs had no steroid binding activity per se. In fact, heat shock protein association (hsp) is required for high affinity ligand-binding of the MR. After incubation of purified MBP- or GST-HBD with rabbit reticulocyte lysate, known to be rich in heat shock proteins, we obtained saturable binding of [3H]aldosterone. The Kd value for aldosterone was 0.3 nM and the Bmax = 32 pmol/mg. Hormone binding specificity was assessed by competition studies with various steroid ligands. Sucrose gradient assays performed with [3H]aldosterone-MBP-HBD revealed complex sedimenting at 8.3S and 4.9S with [3H]progesterone-MBP-HBD. Western-blot analysis of the sedimentation peak showed the concomitant presence of MBP-HBD by a monoclonal anti-MBP antibody, and hsp90 by a monoclonal anti-hsp antibody. Moreover, following incubation with the anti-rabbit hsp90 monoclonal antibody the sedimenting gradient showed a 10.4S sedimenting complex. These analyses demonstrated that the [3H]aldosterone-MBP-HBD complex is at least associated with hsp90 in reticulocyte lysate and that the HBD of hMR is sufficient to bind hsp90. Deletions of a relatively short amino- (729-766) or carboxy-terminal (940-984) region of the HBD fragment eliminated all steroid-binding properties. Overall, these results indicate that the integrity of domain E is necessary and sufficient to bind steroid ligands, agonists or antagonists, with characteristics similar to the entire native MR.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, Montpellier, France.
authors.forename: S
authors.initials: S
authors.lastname: Jaglaguier
authors.forename: D
authors.initials: D
authors.lastname: Mesnier
authors.forename: J L
authors.initials: JL
authors.lastname: Láger
authors.forename: G
authors.initials: G
authors.lastname: Auzou
mesh_terms: D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D005591:Chemical Fractionation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D018161:Receptors, Mineralocorticoid / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000737:chemistry; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D006360:Heat-Shock Proteins; D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; D000450:Aldosterone