Point Mutations in the 90‐kDa Heat Shock Protein Binding Region of the Glucocorticoid Receptor Affect the Functional Characteristics of the Receptor a

Heat shock proteins (hsp) are a family of highly conserved and universally expressed proteins that are induced in all organisms and cultured cells by a variety of stressors. These stress proteins are considered to act as molecular chaperones that facilitate the correct protein folding in vivo.' It is now known that steroid receptors are associated to hsps as heteromeric complexes. In the case of the glucocorticoid receptor (GR), the receptor complex is generated upon reaction of the steroid binding unit with a large cytosolic chaperone complex containing hsp90, hsp70 and hsp56.' Interestingly, the association of GR to at least hsp90 is required for the adoption of a high steroid-binding affinity state.' We previously demonstrated that the hsp90-binding site is located within the hormone-binding domain of the mouse GR, supporting a two-site model in which the critical contact site occurs in the region of amino acids 632-659. This region contains a short proline-containing hydrophobic segment and adjacent dipole-pluscysteine motif that are conserved among all of the hsp90-binding receptors in the superfamily. A second hsp90 contact site was predicted in region 574-632, which contains the only highly conserved amino acid sequence in the receptor superfamily outside the DNA-binding d ~ m a i n . ~ In order to further understand the structural requirements for the appropriate functioning of GR, we designed point mutations in the hsp90-binding region of the