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Hsp90 is a ubiquitous ATP dependent molecular chaperone with numerous "client" proteins that depend on Hsp90 for activation. In eukaryotes, Hsp90's ability to hydrolyze ATP is essential, yet it is entirely unclear how hydrolysis is promoting client activation. One of Hsp90's clients, the Glucocorticoid Receptor (GR), is a ligand activated transcription factor that regulates many biologically important genes. Although Hsp90 is required for GR ligand binding in vivo, purified apoGR is fully capable of binding ligand with no enhancement from Hsp90. We reveal that Hsp70, known to facilitate client delivery to Hsp90, inactivates GR through partial unfolding of GR in a region important for ligand binding. Hsp90, with the cochaperones HOP and p23, reverses this inactivation, promoting ligand binding. Recovery of ligand binding requires ATP hydrolysis on Hsp90. This indicates that energy from ATP hydrolysis on Hsp90 regulates client transfer from Hsp70, likely through the coupling of the two chaperone's ATP cycles. Such coupling is embodied in novel contacts between Hsp90 and Hsp70 in the GR:Hsp70:Hsp90:HOP complex by cryoEM. While GR released from Hsp70 is aggregation prone, release from Hsp90 protects GR from aggregation and enhances its affinity for ligand. Together, this illustrates how the coordinated chaperone interactions can enhance stability, function and regulation.

The first version of a glycoprotein hormone receptor (GPHR) information resource was designed to link functional with structural GPHR information, in order to support sequence-structure-function analysis of the LH, FSH, and TSH receptors (http://ssfa-gphr.de). However, structural information on a binding- and signaling-sensitive extracellular fragment (∼100 residues), the hinge region, had been lacking. A new FSHR crystal structure of the hormone-bound extracellular domain has recently been solved. The structure comprises the leucine-rich repeat domain and most parts of the hinge region. We have not only integrated the new FSHR/FSH structure and the derived homology models of TSHR/TSH, LHCGR/CG, and LHCGR/LH into our web-based information resource, but have additionally provided novel tools to analyze the advanced structural features, with the common characteristics and distinctions between GPHRs, in a more precise manner. The hinge region with its second hormone-binding site allows us to assign functional data to the new structural features between hormone and receptor, such as binding details of a sulfated tyrosine (conserved throughout the GPHRs) extending into a pocket of the hormone. We have also implemented a protein interface analysis tool that enables the identification and visualization of extracellular contact points between interaction partners. This provides a starting point for comparing the binding patterns of GPHRs. Together with the mutagenesis data stored in the database, this will help to decipher the essential residues for ligand recognition and the molecular mechanisms of signal transduction, extending from the extracellular hormone-binding site toward the intracellular G protein-binding sites.

The SSFA-GPHR (Sequence-Structure-Function-Analysis of Glycoprotein Hormone Receptors) database provides a comprehensive set of mutation data for the glycoprotein hormone receptors (covering the lutropin, the FSH, and the TSH receptors). Moreover, it provides a platform for comparison and investigation of these homologous receptors and helps in understanding protein malfunctions associated with several diseases. Besides extending the data set (> 1100 mutations), the database has been completely redesigned and several novel features and analysis tools have been added to the web site. These tools allow the focused extraction of semiquantitative mutant data from the GPHR subtypes and different experimental approaches. Functional and structural data of the GPHRs are now linked interactively at the web interface, and new tools for data visualization (on three-dimensional protein structures) are provided. The interpretation of functional findings is supported by receptor morphings simulating intramolecular changes during the activation process, which thus help to trace the potential function of each amino acid and provide clues to the local structural environment, including potentially relocated spatial counterpart residues. Furthermore, double and triple mutations are newly included to allow the analysis of their functional effects related to their spatial interrelationship in structures or homology models. A new important feature is the search option and data visualization by interactive and user-defined snake-plots. These new tools allow fast and easy searches for specific functional data and thereby give deeper insights in the mechanisms of hormone binding, signal transduction, and signaling regulation. The web application “Sequence-Structure-Function-Analysis of GPHRs” is accessible on the internet at http://www.ssfa-gphr.de/.

The MST1 serine–threonine kinase, a component of the RASSF1-LATS tumor suppressor network, is involved in cell proliferation and apoptosis and has been implicated in cancer. However, the physiologic role of MST1 in prostate cancer (PCa) is not well understood. Here, we investigated the possibility of a biochemical and functional link between androgen receptor (AR) and MST1 signaling. We showed that MST1 forms a protein complex with AR and antagonizes AR transcriptional activity as shown by coimmunoprecipitation (co-IP), promoter reporter analysis, and molecular genetic methods. In vitro kinase and site-specific mutagenesis approaches indicate that MST1 is a potent AR kinase; however, the kinase activity of MST1 and its proapoptotic functions were shown not to be involved in inhibition of AR. MST1 was also found in AR–chromatin complexes, and enforced expression of MST1 reduced the binding of AR to a well-characterized, androgen-responsive region within the prostate-specific antigen promoter. MST1 suppressed PCa cell growth in vitro and tumor growth in mice. Because MST1 is also involved in regulating the AKT1 pathway, this kinase may be an important new link between androgenic and growth factor signaling and a novel therapeutic target in PCa. Cancer Res; 71(12); 4303–13. ©2011 AACR.

Ecdysteroids are important regulators of insect development. They perform their actions through intracellular receptors belonging to the superfamily of nuclear receptors (NRs). In the present study, the relative performance of the three Drosophila ecdysone receptor (EcR)-isoforms in terms of their affinity to the ligand Ponasterone A in mammalian cell line CHO-K1 was determined. In the absence of a heterodimerization partner hormone binding of EcR is rather weak. The presence of ecdysone response elements (EcREs) stimulates the ligand binding to different degrees depending on the EcR-isoform involved. When Ultraspiracle (Usp), the invertebrate orthologue of mammalian RXR, is used as dimerization partner, all EcR isoforms of Drosophila bind the ligand Ponasterone A with the same high affinity already in the absence of EcREs. Depending on the EcR isoform, Usp variant and EcREs, addition of DNA results in augmented ligand binding to the heterodimer EcR/Usp. In the absence of EcREs, hormone binding is not affected if the A/B domain of wild type Usp is replaced by the activation domain (AD) of herpex simplex virus (VP16) to circumvent the inhibition of transcriptional activity of the wild type Usp. By contrast, substantial differences were observed with VP16-Usp fusion proteins in the presence of EcREs. In contrast to Usp, the mammalian ortholog-RXR confers ligand affinity to the receptor complex only in the presence of an EcRE. Finally, specific mutations in the EcR ligand binding domain (LBD) showed an abolishing effect on the ligand binding function of this receptor.

Acute and chronic treatments of mice with the glutathione-depleting agent, L-buthionine-(SR)-sulfoximine (BSO), impaired the mineralocorticoid receptor (MR)-dependent biological response by inhibiting aldosterone binding. This steroid-binding inhibition was fully reversed when reducing agents were added to kidney cytosol obtained from mice treated for 5 h, but it was only partially reversed in cytosol obtained from mice treated for 10 days. Although the oligomeric structure of the MR-hsp90 heterocomplex was always unaffected, a decreased amount of MR protein was evidenced after the long term treatment. Such a deleterious effect was correlated with a post-translational modification of MR, as demonstrated by an increased level of receptor carbonylation. In addition, a failure at the elongation/termination step was also observed during the receptor translation process in a reticulocyte lysate system. Thus, a high polyribosomes/monomers ratio and both increased proteolysis and decreased ADP-ribosylatable concentration of elongation factor 2 (EF-2) were shown. Importantly, similar observations were also performed in vivo after depletion of glutathione. Notwithstanding the EF-2 functional disruption, not all renal proteins were equally affected as the MR. Interestingly, both EF-2 and MR expressed in old mice were similarly affected as in L-buthionine-(SR)-sulfoximine-treated young mice. We therefore propose that a dramatic depletion of glutathione in kidney cells mimics the cumulative effect of aging which, at the end, may lead to a renal mineralocorticoid dysfunction.

A recombinant rat thyroid hormone receptor alpha (TR alpha or c-ErbA alpha 1) was produced in E. coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TR alpha). It was found that nuclear extracts (NE) added to rec-TR alpha markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported. This T3 binding amplifying effect on rec-TR alpha occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g. ovalbumin or cytosol) only moderately enhanced T3 binding. The T3 binding amplifying nuclear factors, which are partly heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TR alpha. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR alpha D domain suggests that nuclear factors help rec-TR alpha to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXR alpha which, produced in vitro, could amplify binding of the rec-TR alpha to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.