Binding of hsp90 to the Glucocorticoid Receptor Requires a Specific 7-Amino Acid Sequence at the Amino Terminus of the Hormone-binding Domain

Min Xu,Kurt D Dittmar,Georgia Giannoukos,William B Pratt,S Stoney Simons

doi:10.1074/jbc.273.22.13918

Abstract

The glucocorticoid receptor (GR) HBD must be bound to the protein chaperone hsp90 in order to acquire the high affinity steroid binding conformation. Despite this crucial role of hsp90, its binding site in GR remains poorly defined. Large portions of the GR HBD have been implicated and no similarity has been established between steroid receptor HBDs and the catalytic domains of the protein kinases (e.g. pp60(src), Raf) 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 studied 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. Overexpression 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 GST554C resulted in a fusion protein that did not bind either hsp90 or steroid. Additional mutations revealed that the role of these 7 amino acids was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in the conserved alpha-helix. Instead, these observations support a model in which the sequence of the 7 amino acids directly or indirectly affects hsp90 binding to the GR HBD. Thus, a region of GR that has not been thought to be relevant for hsp90 binding is now seen to be of critical importance, and these data argue strongly against the commonly accepted model of receptor-hsp90 heterocomplex assembly in which the chaperone initially interacts nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confirmation.

Highlights

Ʈ To whom correspondence may be addressed: Dept. of Pharmacology, The University of Michigan Medical School, Medical Science Research Building III, Ann Arbor, MI 48109-0632
Steroid Binding Properties of glutathione S-transferase (GST)/glucocorticoid receptor (GR) hormone-binding domain (HBD) Fusion Proteins—We previously utilized a series of DHFR/GR constructs with deletions at the amino- and carboxyl-terminal ends to determine the boundaries of the HBD of the rat GR to be amino acids 550 –795 [21]
Using GST/GR HBD fusion proteins, we have identified a region at the amino terminus of the GR HBD that is required for assembly of a stable heterocomplex with hsp90

Summary

Introduction

Ʈ To whom correspondence may be addressed: Dept. of Pharmacology, The University of Michigan Medical School, Medical Science Research Building III, Ann Arbor, MI 48109-0632. Cadepond et al [14] divided the human GR HBD into three subregions of roughly equal length and showed that the fusion of each segment to GR with carboxyl-terminal truncation at amino acids 550 or 568 was sufficient to confer hsp binding. The boundaries of the steroid-binding domain were localized to 550 –795 of the rat GR [21] These results raised the question of whether the loss of steroid binding upon deletion of amino acids beyond 550 was due to the absence of sequences necessary for interaction with steroid or to the lack of residues required for hsp binding

Methods

Results

Conclusion