BINDING OF HSP90 TO THE GLUCOCORTICOID RECEPTOR REQUIRES A SPECIFIC 7-AMINO ACID SEQUENCE AT THE AMINO-TERMINUS OF THE HORMONE-BINDING DOMAIN

The glucocorticoid receptor (GR) HBD must be bound to the protein chap erone hsp90 in order to acquire the high affinity steroid binding conf ormation. Despite this crucial role of hsp90, its binding site in GR r emains poorly defined. Large portions of the GR HBD have been implicat ed and no similarity has been established between steroid receptor HBD s and the catalytic domains of the protein kinases (e,g. pp60(src), Ra f) that also form stable heterocomplexes with hsp90, Thus, it has been thought that some general property of the proteins, such as exposure of hydrophobic residues in partially denatured regions, determines the assembly of stable hsp90 heterocomplexes, In this work, we have studi ed fusion proteins containing glutathione S-transferase (GST) and very short amino-terminal truncations just before and at the beginning of the rat GR HBD that are otherwise intact to the carboxyl terminus. Ove rexpression in COS cells of the chimeras GST537C and GST547C was found to yield receptors that were bound to hsp90 and had wild-type steroid binding affinity. However, removal of 7 more amino acids to form GST5 54C resulted in a fusion protein that did not bind either hsp90 or ste roid. Additional mutations revealed that the role of these 7 amino aci ds was neither to provide a spacer between protein domains nor to expo se a protein surface by introducing a bend in the conserved alpha-heli x. Instead, these observations support a model in which the sequence o f the 7 amino acids directly or indirectly affects hsp90 binding to th e GR HBD. Thus, a region of GB that has not been thought to be relevan t for hsp90 binding is now seen to be of critical importance, and thes e data argue strongly against the commonly accepted model of receptor- hsp90 heterocomplex assembly in which the chaperone initially interact s nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confir mation.