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.

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.