Abstract
The progesterone receptor can be reconstituted into hsp90-containing complexes in vitro, and the resulting complexes are needed to maintain hormone binding activity. This process requires ATP/Mg2+, K+, and several axillary proteins. We have developed a defined system for the assembly of progesterone receptor complexes using purified proteins. Five proteins are needed to form complexes that are capable of maintaining hormone binding activity. These include hsp70 and its co-chaperone, hsp40, the hsp70/hsp90-binding protein, Hop, hsp90, and the hsp90-binding protein, p23. The proteins Hip and FKBP52 were not required for this in vitro process even though they have been observed in receptor complexes. Each of the five proteins showed a characteristic concentration dependence. Similar concentrations of hsp70, hsp90, and p23 were needed for optimal assembly, but hsp40 and Hop were effective at about 1/10 the concentration of the other proteins, suggesting that these two proteins act catalytically or are needed at levels similar to the receptor concentration. ATP was required for the functioning of both hsp70 and hsp90. The binding of hsp70 to the receptor requires hsp40 and about 10 microM ATP; however, hsp90 binding appears to occur subsequent to hsp70 binding and is optimal with 1 mM ATP. A three-step model is presented to describe the assembly process.
Highlights
When extracted from tissue cytosol, receptors for progesterone (PR),1 glucocorticoid (GR), and other steroids exist in heteromeric complexes containing heat shock protein 90 and several additional proteins [1,2,3]
Identical PR samples were treated for 30 min at 30 °C in rabbit reticulocyte lysate to restore the complex with hsp90 or to make an intermediate complex by either limiting the ATP by omitting the ATP-regeneration system or by treatment with the hsp90 inhibitor, geldanamycin (GA, lane 4)
A major portion of binding activity can be maintained at 30 °C, but this is not possible when complex formation is compromised by omitting the ATP regeneration system or by treating with GA
Summary
Materials—Mouse monoclonal (IgG) antibody PR22 against the avian PR has been described previously [27]. The protein was purified from cytosol extracts to Ͼ99% purity by chromatography on columns of DEAE-cellulose, heparin-agarose, and Mono Q as described previously [29]. The soluble fraction of bacterial lysate was fractionated by DEAE-cellulose column chromatography followed by phenyl-Sepharose (hp1660) FPLC, dialyzed into 10 mM Tris-HCl, 1 mM dithiothreitol, and 1 mM EDTA, pH 7.5, and stored at Ϫ70 °C. For the reconstitution of PR complexes with purified proteins, 25 l of PR resin pellets were suspended in 200 l of incubation buffer (10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, and 2 mM dithiothreitol, pH 7.5) containing various amounts of hsp, YDJ-1 or HDJ-2, Hop, hsp, p23, and 5 mM ATP. Ten l were removed for the measurement of [3H]progesterone, and the remainder was analyzed by SDS-PAGE [34]
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