Bernd Segnitz

The Function of Steroid Hormone Receptors Is Inhibited by the hsp90-specific Compound Geldanamycin

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.

Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells

Mouse lymphoma cells contain a nonactivated glucocorticoid receptor of M r∼330000 which is heteromeric in nature and is unable to bind to DNA. Following affinity labeling of the steroid‐binding subunit and subsequent cross‐linking with dimethyl suberimidate at various times either in cell extracts or in intact cells, a series of labeled bands was detected in SDS gels. From the molecular masses of completely and partially cross‐linked complexes we conclude that the large nonactivated receptor is a tetramer composed of two 90 kDa subunits, one 50 kDa polypeptide and one steroid‐binding subunit.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Dissecting SRP In the secretory pathway, inserting transmembrane and secretory proteins into and through hydrophobic cell membranes is facilitated by a highly conserved RNA and protein-containing molecular machine, the signal recognition particle (SRP). Grotwinkel et al. (p. 101) determined the x-ray crystal structures of human SRP RNA (7SL RNA) bound to the RNA-binding domain (RBD) of the protein SRP subunit SRP68, both in the presence and absence of the SRP19 subunit. The 7SL RNA is remodeled by the SRP68-RBD, which bends one domain of the RNA and remodels a loop, exposing two nucleotides, which allow direct interaction with the ribosome. The findings explain how the SRP RNA drives translation elongation arrest, which is required for membrane insertion. Structures of part of the signal recognition complex help explain how newly synthesized proteins are inserted into membranes. The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

Structural Basis for Conserved Regulation and Adaptation of the Signal Recognition Particle Targeting Complex.

The signal recognition particle (SRP) is a ribonucleoprotein complex with a key role in targeting and insertion of membrane proteins. The two SRP GTPases, SRP54 (Ffh in bacteria) and FtsY (SRα in eukaryotes), form the core of the targeting complex (TC) regulating the SRP cycle. The architecture of the TC and its stimulation by RNA has been described for the bacterial SRP system while this information is lacking for other domains of life. Here, we present the crystal structures of the GTPase heterodimers of archaeal (Sulfolobus solfataricus), eukaryotic (Homo sapiens), and chloroplast (Arabidopsis thaliana) SRP systems. The comprehensive structural comparison combined with Brownian dynamics simulations of TC formation allows for the description of the general blueprint and of specific adaptations of the quasi-symmetric heterodimer. Our work defines conserved external nucleotide-binding sites for SRP GTPase activation by RNA. Structural analyses of the GDP-bound, post-hydrolysis states reveal a conserved, magnesium-sensitive switch within the I-box. Overall, we provide a general model for SRP cycle regulation by RNA.

Structural insights into the assembly of the human and archaeal signal recognition particles.

The signal recognition particle (SRP) is a conserved ribonucleoprotein (RNP) complex that co-translationally targets membrane and secretory proteins to membranes. The assembly of the particle depends on the proper folding of the SRP RNA, which in mammalia and archaea involves an induced-fit mechanism within helices 6 and 8 in the S domain of SRP. The two helices are juxtaposed and clamped together upon binding of the SRP19 protein to their apices. In the current assembly paradigm, archaeal SRP19 causes the asymmetric loop of helix 8 to bulge out and expose the binding platform for the key player SRP54. Based on a heterologous archaeal SRP19-human SRP RNA structure, mammalian SRP19 was thought not to be able to induce this change, thus explaining the different requirements of SRP19 for SRP54 recruitment. In contrast, the crystal structures of a crenarchaeal and the all-human SRP19-SRP RNA binary complexes presented here show that the asymmetric loop is bulged out in both binary complexes. Differences in SRP assembly between mammalia and archaea are therefore independent of SRP19 and are based on differences in SRP RNA itself. A new SRP-assembly scheme is presented.

Lymphoma cell variants of decreased glucocorticoid sensitivity

Glucocorticoid-sensitive S49.1 mouse lymphoma cells were mutagenized and cloned in soft agar containing 10 nM dexamethasone. A series of clones were grown and tested for growth inhibition by dexamethasone. While most clones were completely resistant to the steroid, some were sensitive but required significantly higher glucocorticoid concentrations for the same response than wild-type cells. Two of these low-sensitivity clones were used for binding studies; they showed significantly decreased levels of glucocorticoid receptors as compared to wild-type cells. The data support the view that the level of cellular steroid hormone receptors quantitatively controls hormone responsiveness in closely related cells.

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.

Subunit Structure of the Glucocorticoid Receptor

The molecular weights of specific receptors for glucocorticoids and other steroid hormones depend heavily on the conditions of investigation. High molecular weight forms of 300 000 Da and above are detected in extracts of target cells under very mild conditions of analysis which avoid subunit dissociation and denaturation. Routinely, such receptor forms are analyzed by gel permeation chromatography and sedimentation in glycerol or sucrose gradients (Sherman and Stevens 1984). On the other hand, the hormone-binding polypeptides are most easily analyzed by affinity labeling with a radiolabeled steroid derivative and subsequent SDS gel electrophoresis (Gronemeyer 1988). These receptor polypeptides form a protein family, all members of which have a similar domain arrangement even though the sizes of the polypeptides vary considerably amongst the receptors of different hormone specificities (Gehring 1987; Evans 1988). They all contain a central DNA binding domain of less than 70 amino acid residues and a carboxy terminal region of more than 200 amino acids which is involved in hormone binding.

Subunit structure of the glucocorticoid receptor and activation to the DNA-binding state.

Glucocorticoid receptors of S49.1 mouse lymphoma cells were analyzed under a variety of conditions. The complexes with an agonist or a steroidal antagonist can be formed in cytosolic extracts, they are of high molecular weight, Mr approximately 330,000 and have a Stokes radius of 82 A. Cross-linking by several agents stabilized this structure against subunit dissociation which produces the activated receptor form of 60 A and DNA-binding ability. Careful analysis of intermediate cross-linked forms lead to the conclusion that the large receptor structure is a hetero-tetramer consisting of one hormone-bearing polypeptide of Mr approximately 94,000, two 90 kDa subunits and a protein component of Mr approximately 50,000. The 90 kDa subunits are the heat shock protein hsp90. The high molecular weight receptor form also exists in intact cells as revealed again by cross-linking. The cytosolic complex with the antagonist can become activated to the DNA-binding form upon warming but simultaneously looses the ligand. Ligand rebinding does not occur subsequent to receptor dissociation. Upon incubation of intact cells at 37 degrees C with agonist or antagonist the respective receptor-ligand complexes are formed. The agonist complex is immediately activated, however, the antagonist complex remains stable in the undissociated state. This explains the biological effect of the antagonist.