Hsp90 Mutants Disrupt Glucocorticoid Receptor Ligand Binding and Destabilize Aporeceptor Complexes

doi:10.1074/jbc.270.49.29433

Hsp90 Mutants Disrupt Glucocorticoid Receptor Ligand Binding and Destabilize Aporeceptor Complexes *

Sean P. Bohen(^§)

From the Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, Program in Biological Sciences (Biochemistry and Molecular Biology), University of California, San Francisco, California 94143-0448

Abstract

In order to attain competence to respond to hormone, certain steroid hormone receptors must be assembled into hetero-oligomeric aporeceptor complexes, containing Hsp90 and other proteins. Members of the Hsp90 gene family are highly conserved, strongly expressed, and required for viability in eukaryotic organisms. To elucidate the role of Hsp90 in the activity of steroid hormone receptors in vivo, four Hsp90 mutants, which cause defects in glucocorticoid receptor (GR) signaling, but support the viability of Saccharomyces cerevisiae, were previously isolated (Bohen, S. P., and Yamamoto, K. R.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11424-11428). In this study, I characterize the effects of the Hsp90 mutants on GR ligand response, ligand binding activity, and aporeceptor complex stability. The mutants fall into two classes. Three of the Hsp90 mutants cause defects in GR ligand binding in vivo and form aporeceptor complexes that are unstable in vitro, relative to those containing wild-type Hsp90. The other mutant affects GR signaling, but aporeceptor complexes with this mutant are not defective for ligand binding or stability. These findings indicate that the binding of Hsp90 to GR in the aporeceptor complex is insufficient to induce a high ligand affinity conformation, rather the high ligand affinity of GR requires a specific interaction with Hsp90, which is altered by certain Hsp90 mutants.

Footnotes

↵^§ Supported by a predoctoral fellowship from the American Heart Association, California Affiliate. Present address: Laboratory of Biochemistry, NCI, NIH, Bldg. 37, Rm. 4A01, Bethesda, Maryland 20892-4255. Tel.: 301-496-1581; Fax: 301-402-3095; bohen{at}ncifcrf.gov.
↵* This work was supported by the National Institutes of Health and a grant to K. Yamamoto from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽¹⁾ The abbreviations used are:

Hsp

heat shock protein

DR

dioxin receptor

GR

glucocorticoid receptor

PBS

phosphate-buffered saline

AEBSF

4-(2-aminoethyl)benzenesulfonyl fluoride.
↵⁽²⁾A. Kralli and K. R. Yamamoto, manuscript in preparation.
↵⁽³⁾S. Holley, S. P. Bohen, and K. Yamamoto, unpublished data.
- Received August 18, 1995.