The Role of DnaJ-like Proteins in Glucocorticoid Receptor·hsp90 Heterocomplex Assembly by the Reconstituted hsp90·p60·hsp70 Foldosome Complex

Dittmar, Kurt D.; Banach, Maria; Galigniana, Mario D.; Pratt, William B.

doi:10.1074/jbc.273.13.7358

Cited by 154 publications

(150 citation statements)

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“…Pharmacological and antisense-RNA studies confirmed the hypothesis that MR in the hippocampus inhibits basal HPA axis activity by influencing CRH and AVP secretion from the PVN, whereas hippocampal GR acts the opposite (Dittmar et al, 1998) (Ratka et al, 1989).…”

Section: Corticosteroid Receptorssupporting

confidence: 48%

Site-specific overexpression of corticotropin-releasing hormone (CRH) in the mouse brain – modelling central CRH system hyperactivity

Steiner²,

Engelholm³

et al. 2005

Pharmacopsychiatry

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Section: Corticosteroid Receptorssupporting

confidence: 48%

Site-specific overexpression of corticotropin-releasing hormone (CRH) in the mouse brain – modelling central CRH system hyperactivity

Steiner²,

Engelholm³

et al. 2005

Pharmacopsychiatry

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“…Toward this end, Pratt and co-workers (24,25) have successfully used purified rabbit hsp70 and hsp90 plus recombinant Hop and p23 to assemble complexes of the glucocorticoid receptor. More recently, they have also shown a requirement for hsp40 (DnaJ homolog) in this system (26). Their results indicate that glucocorticoid receptor complexes capable of binding hormone can be assembled using only hsp70, hsp40, hsp90, and Hop and that p23 is not essential but it stabilizes the receptor complexes to dramatically enhance hormone binding activity.…”

mentioning

confidence: 92%

The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

Kosano¹,

Stensgard²,

Charlesworth³

et al. 1998

Journal of Biological Chemistry

231

250

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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 ...

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“…Human p23 was purified from 10 ml of bacterial lysate by chromatography on DE52 followed by hydroxyapatite chromatography as described (29). For purification of YDJ-1, bacterial sonicates were cleared by centrifugation, and YDJ-1 was purified by sequential chromatography on DE52 and hydroxyapatite as described previously (14). The bacterial expression of YDJ-1 has been described (30) as has the expression of human Hop (13).…”

Section: Assay Of Steroid Binding Capacity-immunementioning

confidence: 99%

“…Hop does not act solely as a tie rod to bring the essential chaperones together; it also influences the conformational state and function of each protein (18). The hsp90⅐Hop⅐hsp70 complexes also contain small amounts of the hsp70 co-chaperone hsp40 (14), and together they form the hsp90/hsp70-based chaperone machinery. Once the machinery has assembled the GR⅐hsp90 heterocomplex, p23 binds dynamically (19) to the ATP-dependent conformation of hsp90 (20) and stabilizes its association with the receptor.…”

mentioning

confidence: 99%

“…This machinery has been reconstituted (13), and a mixture of five purified proteins (hsp90, hsp70, 2 Hop, hsp40, and p23) is now used to achieve efficient receptor⅐hsp90 heterocomplex assembly (14,15). The chaperones hsp90 and hsp70 are both essential for opening the steroid binding cleft in the GR LBD, and hsp40, Hop (hsp70/hsp90 organizing protein), and p23 act as co-chaperones to increase the rate or extent of GR⅐hsp90 heterocomplex assembly (16).…”

mentioning

confidence: 99%

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Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

Murphy

Morishima

Chen

et al. 2003

Journal of Biological Chemistry

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A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR)⅐hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR⅐hsp70 complex that primes the receptor for subsequent ATPdependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR⅐hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size~1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR⅐hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/ 554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/ 554C, and it is the subsequent assembly step with hsp90 that is defective.Glucocorticoid receptors (GR) 1 are recovered from hormonefree cells as large multiprotein complexes containing a dimer of hsp90 (for review see Refs. 1 and 2). Hsp90 binds to the ligand binding domain (LBD) of the GR, and the steroid binding activity of the GR is absolutely hsp90-dependent (3, 4). When the GR is stripped of its associated hsp90, it immediately loses its ability to bind steroid, and steroid binding activity is regenerated when GR⅐hsp90 heterocomplexes are reformed by the hsp90/hsp70-based chaperone machinery (5, 6). The steroids bind deep in a hydrophobic cleft that appears to be collapsed in the absence of ligand, such that the LBD must change its conformation to allow entry of ligand (7). The hsp90/hsp70-dependent chaperone machinery carries out the ATP-dependent opening of the binding cleft in the GR LBD such that it can be accessed by steroid. In addition to opening the steroid binding cleft, formation of a complex with hsp90 is accompanied by conformational changes that increase the sensitivity of the GR LBD to attack by thiol-derivatizing agents and trypsin (8 -10).The multiprotein chaperone machinery that forms receptor⅐hsp90 heterocomplexes was originally identified in reticulocyte lysate (11,12). This machinery has been reconstituted (13), and a mixture of five purified proteins (hsp90, hsp70, 2 Hop, hsp40, and p23) is now used to achieve efficient receptor⅐hsp90 heterocomplex assembly (14, 15). The chaperones hsp90 and hsp70 are both essential for opening the steroid binding cleft in the GR LBD, and hsp40, Hop (hsp70/hsp90 organizing protein), and p23 act as co-chaperones to increase the rate or extent of GR⅐hsp90 heterocomplex assembly (16). Hop b...

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The Role of DnaJ-like Proteins in Glucocorticoid Receptor·hsp90 Heterocomplex Assembly by the Reconstituted hsp90·p60·hsp70 Foldosome Complex

Cited by 154 publications

References 50 publications

Site-specific overexpression of corticotropin-releasing hormone (CRH) in the mouse brain – modelling central CRH system hyperactivity

Site-specific overexpression of corticotropin-releasing hormone (CRH) in the mouse brain – modelling central CRH system hyperactivity

The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

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