Glucocorticoid receptors (GRs) must heterocomplex with hsp90 to have an open steroid binding cleft that can be accessed by steroid. We reported that a seven-amino acid sequence (547-553 of rat GR) overlapping the amino-terminal end of the ligand binding domain is required for hsp90 binding to GR. We have now conducted saturation mutagenesis of this sequence, which appears to be part of the surface where the ligand binding cleft merges with the surface of the ligand binding domain. No single

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... t mutation causes significant changes in any of a variety of biochemical and biological properties in addition to hsp90 binding. A triple mutation (P548A/ T549A/V551A) increases by >100-fold the steroid concentration required for half-maximal induction without affecting the level of maximal induction or coactivator response. Interestingly, this triple mutant displays reduced binding of steroid and hsp90 in whole cells, but it possesses wild type affinity for steroid and normal hsp90 binding capacity under cell-free conditions. This phenotype of a dramatic shift in the dose response for transactivation would be expected from an increase in the rate of disassembly of the triple mutant GR⅐hsp90 heterocomplex in the cell. Mutation of the entire sevenamino acid region to CAAAAAC maintains the presence of a critical ␣-helical structure and heterocomplex formation with hsp90 but eliminates steroid binding and transcriptional activation, thus disconnecting hsp90 binding from opening of the ligand binding cleft and steroid binding.