Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Summary

An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Abstract

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways ^1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process.

The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid ³. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored ⁴ . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [³H]steroid.

Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 ⁵. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source ⁶.

In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K⁺, which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity ⁷. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding ^8-11.

Protocol

1. Preparation of cell cytosol containing functional GR

1. Technical note: All buffers should be refrigerated and each step of this protocol, including centrifugations and incubation, should be performed on ice or at 4 °C, unless otherwise noted. The low temperature is essential to prevent degradation of the GR and protein complexes.
2. Using a refrigerated centrifuge, obtain a ˜1-5 ml pellet of cells that expresses a high concentration of functional GR. Examples of cell sources rich in GR include mouse fibroblast L929 cells, which express a high concentration of endogenous GR, and Sf9 cells that have been infected with recombinant GR baculovirus (e.g.....

Discussion

The assay described above can be adapted to test a myriad of conditions affecting the chaperone actions of the hsp90/hsp70-based chaperone machinery as well as GR steroid binding. It is a modification of previously reported methods ^4,8,9,17 and is designed to take advantage of recent advances in electrophoresis technology and be accessible to a broader research community. Additional cofactors or proteins of interest may be added to the GR•hsp90 heterocomplex reconstitution mixture, described in Step 4 of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sf9 insect cells	Invitrogen		for baculovirus expression of recombinant mouse GR
L929 cells	ATCC	CRL-2173	for endogenous mouse GR cytosol preparation (alternative to baculovirus expression)
FiGR hybridoma cells	ATCC	CRL-2173	for preparing ascites containing anti-GR monoclonal antibody (alternative to purified BuGR2 antibody)
Complete-Mini protease inhibitor, EDTA-free	Roche Group	11836170001
protein A-Sepharose	Sigma-Aldrich	P3391
rabbit reticulocyte lysate	Green Hectares (Oregon, WI)		rich source of endogenous hsp90/hsp70 chaperone machinery
creatine phosphokinase	Sigma-Aldrich	C3755	for ATP-regenerating system
phosphocreatine	Sigma-Aldrich	P7936	for ATP-regenerating system
anti-GR mouse monoclonal antibody (BuGR2)	Pierce, Thermo Scientific	MA1-510	used for GR immunoadsorption and as primary antibody in western blotting
anti-hsp90 mouse monoclonal antibody (AC88)	Enzo Life Sciences	ADI-SPA-830	used as primary antibody in western blotting
anti-hsp70 mouse monoclonal antibody (N27F3-4)	Enzo Life Sciences	ADI-SPA-820	used as primary antibody in western blotting
IgG from mouse serum	Sigma-Aldrich	038K7690	used as nonimmune antibody for negative control of immunoadsorption
anti-mouse IgG-peroxidase conjugate	Sigma-Aldrich	A4416	used as secondary antibody in western blotting
Experion microfluidic electrophoresis system	Bio-Rad	7007001	alternative to conventional SDS-PAGE
[1,2,4,6,7-3H] dexamethasone	PerkinElmer, Inc.	NET1192001MC	follow safety precautions