Résumé
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
K
d
value for aldosterone was 0.3 nM and the
B
max = 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 hap90 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.