FOLDING REQUIREMENTS OF THE LIGAND-BINDING DOMAIN OF THE HUMAN MINERALOCORTICOID RECEPTOR

The effects of aldosterone are mediated by the mineralocorticoid recep tor (MR), a ligand-dependent transcription factor. We investigated the structural determinants for ligand binding to the receptor using a se ries of human MR (hMR) deletion mutants. These proteins were produced in vitro in rabbit reticulocyte lysate and analyzed for their ability to bind agonists, antagonists, and the heat shock protein hsp90, which is a prerequisite for ligand binding to hMR. Studies on N terminus-tr uncated hMRs showed that the ligand-binding domain (LBD: amino acids 7 34-984) has a lower affinity for aldosterone than the entire receptor [dissociation constant (K-d) 2.9 vs. 0.47 nM] and does not interact wi th hsp90. Addition of the five-amino acid sequence (729-733) upstream from the LED is necessary for interaction with hsp90, but a larger reg ion is needed for high aldosterone affinity. Deletions at the C-termin al end of the hMR greatly reduced both agonist and antagonist binding: deletion of the last three amino acids reduced the affinity for aldos terone to 1/20 that of the entire protein, and deletion of the last fo ur amino acids completely abolished binding, although the interaction with hsp90 was not affected. These effects can be explained by misfold ing of the receptor, since limited proteolysis assays showed that dele tions at the C-terminal end of hMR affect the accessibility of the cle avage sites within the DNA-binding domain and the N-terminal part of t he hinge region to trypsin. Thus, our results support the idea that a short sequence upstream of the LED is essential for the interaction of hMR with hsp90 and that the C terminus of hMR and hsp90 are both esse ntial for folding of the receptor in a high-affinity hormone-binding s tate.