The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

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/Mg 2؉ , 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 cochaperone, 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 M 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.When extracted from tissue cytosol, receptors for progesterone (PR), 1 glucocorticoid (GR), and other steroids exist in heteromeric complexes containing heat shock protein 90 (hsp90) and several additional proteins (1-3). Recent information on the assembly of these complexes has been gained mainly through the use of an in vitro system consisting of immuneisolated progesterone (4) or glucocorticoid (5) receptor incubated in rabbit reticulocyte lysate. The formation of receptor complexes in this system is dependent upon ATP hydrolysis, the ions Mg 2ϩ and K ϩ , and the participation of a number of proteins (6, 7). When assembled in vitro, the mature avian progesterone receptor complex closely resembles that obtained from oviduct cytosol and contains hsp90, any one of three immunophilins, and a 23-kDa phosphoprotein, p23. It also contains variable sub-stoichiometric amounts of hsp70. The immunophilins include the cyclosporin A-binding protein, CyP-40, and two FK506-binding proteins, FKBP51 and FKBP52. Antibody inhibitor studies or depletion and reconstitution experiments indicate that hsp70 (6, 8), Hop, an intermediate in complex assembly (9), and p23 (10, 11) are essential for the formation of hsp90 complexes, but the actual roles of these and other proteins are still unclear.An intermediate in PR complex formation has been identified that contains submaximal amounts of hsp90, substantially more hsp70 than in the mature complex, and two additional proteins, Hop (p60) and Hip (p48) (6, 12). This complex does not contain immunophilins or p23. Hop (hsp-organizing protein) is a 60-kDa stress-related protein that binds to both hsp70 and hsp90 (9, 13-15). Hip (hsp70-interacting ...

Conformational dynamics of the molecular chaperone Hsp90

“…This has been observed for both GR and PR (Dittmar & Pratt, 1997;Kosano et al, 1998). p23 may serve as a molecular timer for the activation of client proteins, and once clients are activated p23 is released, hydrolysis occurs, and client proteins dissociate.…”

“…A minimal set of necessary co-chaperones has been established for the progesterone receptor (PR), the glucocorticoid receptor (GR), and the kinase Chk1 providing information about the role of different cochaperones and about the specific requirements among client proteins. In vitro, GR requires the fewest number of co-chaperones (only Hsp70 and Hop) in addition to Hsp90 for the reconstitution of steroid binding activity (Arlander et al, 2006;Dittmar & Pratt, 1997;Kosano et al, 1998). For the in vitro reconstitution of PR, Hsp70, Hsp40, Hop and p23 are required in addition to Hsp90 (Kosano et al, 1998).…”

“…In vitro, GR requires the fewest number of co-chaperones (only Hsp70 and Hop) in addition to Hsp90 for the reconstitution of steroid binding activity (Arlander et al, 2006;Dittmar & Pratt, 1997;Kosano et al, 1998). For the in vitro reconstitution of PR, Hsp70, Hsp40, Hop and p23 are required in addition to Hsp90 (Kosano et al, 1998). The kinase Chk1 also requires Hsp90, Hsp70, Hsp40, and Hop for maximal activation, but unlike PR the kinase specific cochaperone p50 is also required and p23 is not (Arlander et al, 2006).…”

“…The steroid hormone receptors are in complex with Hsp90 and p23 at this stage. Activated client proteins are then released from the Hsp90 complex Hernandez et al, 2002;Kosano et al, 1998;Smith, 1993). The diagrams in Figure 8 detail the minimal systems required for activation, but numerous other co-chaperones have been identified and are believed to play important roles in the regulation of the chaperone cycle in vivo (Table 2).…”

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

“…As molecular chaperones, Hsp70 family members bind to exposed hydrophobic protein surfaces, inhibiting aggregation and promoting the refolding of denatured proteins (Kobayashi et al 2000;Wang et al 2005). This chaperone function is critical for the translation of proteins from ribosomes (Beckmann et al 1990), the translocation of proteins through intracellular membranes (Kang et al 1990) and is required for the remodeling of native protein structures such as clathrin-coated vesicles and steroid receptors (Chappell et al 1986;Kosano et al 1998). Hsp70 also has an important role in modulating protein homeostasis by targeting proteins for proteolytic degradation through the ubiquitin/proteasome and lysosomal systems (Dice 2007;Esser et al 2004).…”