which was identified by screening an expression library 1 Corresponding author using as probe the glucocorticoid receptor overexpressed in the baculovirus system. It also interacts with other A ubiquitously expressed nuclear receptor-associating members of the nuclear receptor superfamily (Zeiner and protein of~46 kDa (RAP46) was identified recently.Gehring, 1995). Murine BAG-1 was obtained by use of a Interaction experiments with in vitro-translated prosimilar screening procedure in which baculovirusteins and proteins contained in cell extracts revealed expressed Bcl-2 or the hepatocyte growth factor receptor that a great variety of cellular regulators associate were employed to identify interacting proteins (Takayama with RAP46. However, in direct interaction tests Bardelli et al., 1996). Subsequent studies the far-Western technique, only 70 kDa proteins showed showed (see below) that the interaction of RAP46 is not up and were identified as members of the 70 kDa heat restricted to nuclear receptors but that various unrelated shock protein (hsp70) family. Interaction is specific regulatory proteins, including Bcl-2, also react with RAP46 since not all members of the hsp70 family bind to in pull-down experiments. This then raised the question RAP46; interaction occurs through their ATP-binding of whether such interactions might be mediated by some domain. RAP46 forms complexes with hsp70 in mamcommon interacting protein(s). In the present study, we malian cells and interacts with hsp70 in the yeast twoidentified members of the hsp70 family as direct and hybrid system. Consistent with the fact that hsp70 can specific interaction partners. Through binding to members bind a multitude of proteins, we identified heteromeric of the hsp70 family, a multitude of proteins might associate complexes of RAP46-hsp70 with some selected prowith RAP46. Indeed, we detected heteromeric complexes teins, most notably c-Jun. Complex formation is in which hsp70 serves as adaptor. The interaction of increased significantly by pre-treatment with alkaline RAP46 with hsp70s occurs via their ATP-binding domain. phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally Results denatured luciferase. Moreover, ATP-dependent bind- Proteins interacting with RAP46 ing of misfolded proteins to hsp70 was greatly inhibitedOriginally, RAP46 was identified by binding the activated by RAP46. These data suggest that RAP46 functions glucocorticoid receptor (Zeiner and Gehring, 1995). In as a regulator of hsp70 in higher eukaryotes.pull-down assays with RAP46 fused to glutathione-SKeywords: c-Jun/dephosphorylation/hsp70 heat shock transferase (GST) and bound to glutathione (GSH)-proteins/nuclear receptors/protein refolding Sepharose, we obtained similar associations with other mammalian steroid hormone receptors (Zeiner and Gehring, 1995) and with ecdysteroid, thyroid and retinoic acid receptors (data not shown). Using this technique, we

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver Agtll expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence Nuclear hormone receptors are a large family of eukaryotic transcriptional activators which depend on extracellular hormonal signals for affecting the expression of specific genes. Steroid hormone receptors are an important subgroup of this family (for reviews, see refs. 1-3). They preexist within target cells in a nonactivated state in association with heat shock proteins (for reviews, see refs. 4-6). After binding of the hormonal ligand, these receptor-associated proteins dissociate under physiological conditions (5, 6). This results in receptor activation to molecular forms which are then able to interact with DNA as homodimers. DNA binding occurs to specific hormone response elements of characteristic nucleotide sequences (1-3) which on the linear DNA may be quite remote from the promoter regions of the particular genes they control. This brings up the question of how DNA-bound receptors communicate with the transcriptional apparatus of the cell. It appears likely that coactivator proteins are involved in this and that they function as bridging molecules between receptors and the basal transcription factor complex TFIID (1, 3, 7). Recently, several proteins have been described (8-13) which may act as mediators between steroid and thyroid hormone receptors and the transcription initiation complex.We set out to use very general approaches to identify such receptor-associating factors. First, we incubated isolated HeLa cell nuclei with 35S-labeled glucocorticoid receptor in its activated form and applied chemical crosslinkers in order to stabilize complexes formed between the receptor and nuclear proteins. SDS/PAGE indeed showed that the receptor wasThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.completely converted into somewhat heterogeneous protein material of roughly 300 kDa as well as some material that did not enter the gel (unpublished data). As this suggests that the receptor indeed interacts with a range of nuclear proteins, we decided to search for such partner proteins by use of an interaction screening approach (14). We used a liver cDNA expression library and screened with the activated glucocorticoid receptor. We identified one clone which codes for a protein that interacts with activated steroid hormone receptors. We have characterized this receptor-associating protein (RAP) and present its primary structure.* MATERIALS AND METHODSPlasmids. cDNAs for the wild-type human estrogen receptor (HEGO) and a deletion mutant (HE19c; aa 1-180 missing) were in the pSG5 vector (15). Plasmid pcDNAI/Amp-hTRI3-1 contains the cDNA for human thyroid receptor ,B1.Cell Culture and Cell Extracts. WEHI-7 mouse thymoma cells, MCF-7 human mammary ca...

We investigated the ubiquitously expressed hsp70-associating protein Hap46, which is also called RAP46 and is homologous to BAG-1, for activities independent of hsp70 interactions. We observed in vitro binding to various DNA fragments but detected no apparent sequence specificity. Deletion of the amino-terminal decapeptide, which contains two clusters of three basic amino acids each, abolished the DNA-binding ability of Hap46. Similarly, exchange of either of these positively charged clusters for three alanines resulted in loss of DNA binding. Using a fusion of Hap46 and green f luorescent protein, we found preferential accumulation in cell nuclei on heat stress as compared with unstressed cells. The repressive effect of heat shock on overall transcriptional activity in human DU145 carcinoma cells was largely prevented when Hap46 was overexpressed by transfection. Such overproduction of Hap46 also resulted in enhanced expression of specific reporter gene constructs and in increased levels of mRNAs specific for hsp70 and hsp40 after temperature stress. In vitro transcription with nuclear extracts was stimulated greatly by Hap46. Like DNA binding, transcriptional enhancement required amino-terminally located basic amino acid residues but not the carboxyl-terminal portion of Hap46 known to participate in hsp70 interaction. Our results show that Hap46 is a bifunctional protein that can interact with both hsp70s and DNA, employing different portions of the molecule. They also suggest that Hap46 is involved in temperature-sensitive regulation of transcription, acting as a general transcriptional activator.

The ansamycin antibiotic geldanamycin, which specifically interacts with the heat shock protein hsp90, was used to study the function of hsp90 in steroid hormone receptors. We observed inhibition of glucocorticoid-specific gene induction in several responsive cell systems. Hormone binding abilities of receptors for glucocorticoid, progestin, androgen, and estrogen were inhibited upon exposing intact cells to geldanamycin. Inhibition was only seen when geldanamycin was applied to cell cultures under growth conditions or was present during in vitro synthesis; presynthesized receptors in cell extracts were not affected. Upon withdrawal of geldanamycin, glucocorticoid binding ability was regained; this was partially independent of de novo protein synthesis. Geldanamycin caused decreased levels of immunoreactive glucocorticoid receptors in wild-type cells with enhanced degradation occurring through the ubiquitin-proteasome pathway. Analysis of receptors from treated cells revealed a heteromeric structure of normal size in which the receptor polypeptide is complexed with normal amounts of hsp90 and the immunophilin p59. These data support the view that hsp90 actively participates in steroid-induced signal transduction, and they suggest that geldanamycin affects receptor action without disrupting hsp90-containing heterocomplexes per se. Nevertheless, complexes synthesized and assembled in vitro in the presence of geldanamycin differ from receptors of cellular origin.Steroid hormone receptors are members of the ever growing family of nuclear receptors (for reviews, see Refs. 1 and 2). They are characterized by sequence homologies that are most prominent within the DNA binding domains. In contrast to most other members of the family, steroid hormone receptors are known to be complexed with heat shock proteins, most notably hsp90, 1 and heat shock proteins are involved in steroidal signal transduction (for reviews, see Refs. 3 and 4). The association with heat shock proteins thus prevents these receptors from interacting with specific DNA recognition sequences while they are not yet complexed with the respective steroidal ligands. On the other hand, hsp90 in association with receptor polypeptides appears to significantly contribute to the receptors' hormone binding ability, as has been established for several steroid hormone receptors (5-9).In recent years, a group of streptomyces antibiotics called benzochinone ansamycins were shown to affect the biological activity of several tyrosine-specific protein kinases (10 -17) that are known to similarly associate with hsp90. Interestingly, geldanamycin and herbimycin A, major representatives of ansamycin antibiotics, were found to directly interact with hsp90 (18). This observation prompted a series of investigations into the effects of these drugs on steroid hormone receptors. In the present study we show that geldanamycin at non-toxic concentrations inhibits hormonal inducibility in several responsive cell systems. Moreover, this drug affects the binding of glucocorticoid, pr...

We investigated several hsp70/hsc70 interacting proteins and established by two independent techniques that hsp40 and Hop/p60 specifically interact with the 257 residue carboxy-terminal domain of hsp70 while Hap-46 and Hip/p48 bind the 383 residue amino-terminal ATP binding domain. Hap-46 and Hip/p48 competed for binding to hsc70, while Hap-46 had no effect on the binding of either Hop/p60 or hsp40 to hsc70. Hap-46 inhibited the refolding of thermally denatured firefly luciferase in an hsc70 and hsp40 dependent assay, and this effect was largely compensated by Hop/p60. These interacting proteins thus appear to cooperate in affecting the chaperoning activity of hsp70/hsc70.