PUTATIVE STEROID-BINDING DOMAIN OF THE HUMAN MINERALOCORTICOID RECEPTOR, EXPRESSED IN ESCHERICHIA-COLI IN THE PRESENCE OF HEAT-SHOCK PROTEINS SHOWS TYPICAL NATIVE RECEPTOR CHARACTERISTICS

Domain E, considered as the putative hormone binding domain (HBD) of t he 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 constr ucts had no steroid binding activity per se. In fact, heat shock prote in association (hsp) is required for high affinity ligand-binding of t he MR. After incubation of purified MBP- or GST-HBD with rabbit reticu locyte lysate, known to be rich in heat shock proteins, we obtained sa turable binding of [H-3]aldosterone. The K-d value for aldosterone was 0.3 nM and the B-max = 32 pmol/mg. Hormone binding specificity was as sessed by competition studies with various steroid ligands. Sucrose gr adient assays performed with [H-3]aldosterone-MBP-HBD revealed complex sedimenting at 8.3S and 4.9S with [H-3]progesterone-MBP-HBD. Western- blot analysis of the sedimentation peak showed the concomitant presenc e of MBP-HBD by a monoclonal anti-MBP antibody, and hsp90 by a monoclo nal anti-hsp antibody. Moreover, following incubation with the anti-ra bbit hsp90 monoclonal antibody the sedimenting gradient showed a 10.4S sedimenting complex. These analyses demonstrated that the [H-3]aldost erone-MBP-HBD complex is at least associated with hsp90 in reticulocyt e lysate and that the HBD of hMR is sufficient to bind hsp90. Deletion s 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 nec essary and sufficient to bind steroid ligands, agonists or antagonists , with characteristics similar to the entire native MR. (C) 1996 Elsev ier Science Ltd.