Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein.

Prapapanich, Viravan; Chen, Shiying; Nair, Satish Chandrasekhar; Rimerman, Ronald A.; Smith, David F.

doi:10.1210/mend.10.4.8721986

Cited by 70 publications

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“…Two types of proteins, Hip and the immunophilins, are missing even though these have been clearly observed in steroid receptor complexes. In rabbit reticulocyte lysate, Hip binds to PR complexes at the intermediate stages of steps 1 and 2 (17). Hip has been shown to bind to hsp70 and to stabilize the ADP-bound state of hsp70 (16).…”

Section: Resultsmentioning

confidence: 99%

“…Hop (hsp-organizing protein) is a 60-kDa stress-related protein that binds to both hsp70 and hsp90 (9,(13)(14)(15). Hip (hsp70-interacting protein) is also known to bind to hsp70 (16,17). This intermediate complex has been shown in time course studies of PR assembly, and it is the predominant form when the ATP level is suboptimal (6,12) and when further assembly is blocked by the benzoquinone ansamycin, geldanamycin (18,19), which binds to the ATP-binding site on hsp90 and blocks some of its functions (20 -23).…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

Kosano¹,

Stensgard²,

Charlesworth³

et al. 1998

Journal of Biological Chemistry

231

250

View full text Add to dashboard Cite

show abstract

“…The purified system is optimized for the amounts of proteins that produce the best overall refolding. Additional factors are most likely required for efficient refolding, and two of the most obvious candidates are the hsp70-interacting protein, Hip (46,47), and p23. An important aspect of future research will be to incorporate these, and perhaps other factors, into the chaperoning system and to define their mechanisms of cooperative action.…”

Section: Discussionmentioning

confidence: 99%

Hop Modulates hsp70/hsp90 Interactions in Protein Folding

Johnson

Schumacher

Ross

et al. 1998

Journal of Biological Chemistry

338

297

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Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90. We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase. We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate. Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system. Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90. Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1. In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90. Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70. Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23. Conversely, once p23 is bound to hsp90, Hop binding is diminished. These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.

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“…The mouse monoclonal antibody N27 (Stressgen Biotech Corp., Victoria, British Columbia, Canada) was added to the hsp7o antibody mixture for analysis of denatured samples. A mixture of mouse antibodies was used to capture p48 (2G6, 1A6, lOD1; Prapapanich et al, 1996). The P5 antibody that recognizes the y-subunit of CCT was used for nondenatured samples only (Joly et al, 1994).…”

Section: Antibodies and Immunoprecipitation Techniquesmentioning

confidence: 99%

Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

Eggers

Welch

Hansen

1997

MBoC

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Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones.We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp7O, CCT (TRiC), hsp4O, p48 (Hip), and hsp9O were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp7O was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp7O and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp7O and, perhaps, the hsp9O families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.

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Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein.

Cited by 70 publications

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The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

Hop Modulates hsp70/hsp90 Interactions in Protein Folding

Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

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