Steroid-receptor Complex Research Articles

Analysis of FKBP51/FKBP52 chimeras and mutants for Hsp90 binding and association with progesterone receptor complexes.

FKBP51, FKBP52, and Cyp40 bind competitively to Hsp90 through their respective tetratricopeptide repeat (TPR) domains, and any one of the three immunophilins can be isolated in mature steroid receptor complexes. During cell-free assembly reactions, FKBP51 associates preferentially with progesterone and glucocorticoid receptors, but less preference is observed in FKBP51 association with estrogen receptor. A number of mutant FKBP forms were generated to map sequences responsible for FKBP51's preferred association with progesterone receptor. A double-point mutation in the peptidyl prolyl isomerase domain of FKBP51 that reduces enzymatic activity by greater than 90% had no observed effect on FKBP51 interactions with progesterone receptor or Hsp90. Coprecipitation of FKBP51 and FKBP52 truncation mutants with Hsp90 indicated that sequences both upstream and downstream of the TPR domain are necessary for Hsp90 binding. FKBP chimeric constructs were also generated and tested for Hsp90 binding and receptor association. The TPR domain of FKBP51 required appropriate downstream sequences for Hsp90 binding, but FKBP52's TPR domain did not. The C-terminal region of FKBP51 that functionally interacts with the TPR domain to permit Hsp90 binding also conferred preferential association with PR. In conclusion, despite the overall similarity of FKBP51 and FKBP52, these two immunophilins associate differentially with steroid receptors, and the difference relates to both the Hsp90-binding TPR domain and to poorly conserved C-terminal sequences.

Mutation of Hip's carboxy-terminal region inhibits a transitional stage of progesterone receptor assembly.

Steroid receptor complexes are assembled through an ordered, multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop, one DPEV sequence plus a partial second sequence occurs. In an effort to better understand Hip function as it relates to assembly of progesterone receptor complexes, the DPEV region of Hip was targeted for mutations. Each DPEV sequence was mutated to an APAV sequence, singly or in combination. The combined mutation, APAV2, was further combined with a deletion of Hip's tetratricopeptide repeat region that is required for Hsp70 binding or with a deletion of Hip's GGMP repeat. An additional mutant was prepared by truncation of Hip's DPEV-containing C terminus. By comparing interactions of various Hip forms with Hsp70, it was determined that mutation of the DPEV sequences created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex was not prevented from interacting with progesterone receptor, but the mutant caused a dose-dependent inhibition of receptor assembly with Hsp90. The behavior of the Hip mutant is consistent with a model in which Hip and Hop are required to facilitate the transition from an early receptor complex with Hsp70 into later complexes containing Hsp90.

Stage-specific expression of the immunophilin FKBP59 messenger ribonucleic acid and protein during differentiation of male germ cells in rabbits and rats.

FKBP59 is an immunophilin that binds the immunosuppressant drugs FK506 and rapamycin. It is a 90-kDa heat shock protein (hsp90)-binding protein that was originally discovered as a member of steroid receptor complexes. FKBP59 is ubiquitous and well conserved, and it appears to be a multifunctional protein. It has peptidylprolyl cis-trans-isomerase activity and therefore may be involved in protein folding as a molecular chaperon. FKBP59 also includes in its structure a tetratricopeptide repeat (TPR) motif and could have a function in the cell division process. In situ hybridization experiments revealed an overexpression of FKBP59 mRNA in rabbit and rat testes in comparison with other organs. This high level of expression was restricted to germ cells of the seminiferous epithelium. Increasing levels of FKBP59 mRNA became obvious from the midpachytene stage, and the strongest signal was observed in the late pachytene, diplotene, and diakinesis primary spermatocytes. The expression then declined progressively in postmeiotic early spermatids. High expression of FKBP59 mRNA did not occur in earlier and later germ cell stages. During prepubertal development, Northern blot and in situ hybridization of rat testes examined at various postnatal ages revealed that FKBP59 mRNA was not expressed at over a basal level until the pachytene stage. High expression of the FKBP59 protein was demonstrated in the rabbit testis by Western blot and was localized by immunohistochemistry from late pachytene spermatocytes to round spermatids. The cell type-specific and developmental stage-specific expression of FKBP59 at a restricted period of male germ cell differentiation suggests that FKBP59 is involved in a specific function during the cell division process.

Fourth-generation model for corticosteroid pharmacodynamics: a model for methylprednisolone effects on receptor/gene-mediated glucocorticoid receptor down-regulation and tyrosine aminotransferase induction in rat liver.

A fourth-generation pharmacokinetic/pharmacodynamic (PK/PD) model for receptor/genemediated effects of corticosteroids was developed. Male adrenalectomized Wistar rats received a 50 mg/kg i.v. bolus dose of methylprednisolone (MPL). Plasma concentrations of MPL, hepatic glucocorticoid receptor (GR) messenger RNA (mRNA) and GR density, tyrosine amino-transferase (TAT) mRNA, and TAT activity in liver were determined at various time points up to 72 hr after MPL dosing. Down-regulation of GR mRNA and GR density were observed: GR mRNA level declined to 45-50% of the baseline in 8-10 hr, and slowly returned to predose level in about 3 days; GR density fell to 0 soon after dosing and returned to the baseline in two phases. The first phase, occurring in the first 10 hr, entailed recovery from 0 to 30%. The second phase was parallel to the GR mRNA recovery phase. Two indirect response models were applied for GR mRNA dynamics regulated by activated steroid-receptor complex. A full PK/PD model for GR mRNA/GR down-regulation was proposed, including GR recycling theory. TAT mRNA began to increase at about 1.5 hr, reached the maximum at about 5.5 hr, and declined to the baseline at about 14 hr after MPL dosing. TAT induction followed a similar pattern with a delay of about 1-2 hr. A transcription compartment was applied as one of the cascade events leading to TAT mRNA and TAT induction. Pharmacodynamic parameters were obtained by fitting seven differential equations piecewise using the maximum likelihood method in the ADAPT II program. This model can describe GR down-regulation and the precursor/product relationship between TAT mRNA and TAT in receptor/gene-mediated corticosteroid effects.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: regulation of HSP90-binding activity of FKBP52.

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

Expression and function of calcineurin in the mammalian nephron: Physiological roles, receptor signaling, and ion transport

Protein phosphorylation is central to the regulation of sodium transport and other cellular processes in the nephron. Complex interactions between protein kinases and phosphatases catalyze the reversible phosphorylation of ion transporting proteins on the apical and basolateral surfaces of renal epithelia. Although the role of protein kinases in regulating sodium transport has been extensively studied, the function of phosphatases in the nephron is less well understood. Calcineurin is a serine-threonine phosphatase that was shown to be the target of cyclosporin A (CsA) and FK-506 in lymphocytes. Calcineurin exists in the cytosol as a heterotrimeric protein composed of an α-catalytic subunit, β-regulatory subunit, and calmodulin; its activity depends on calcium and calmodulin. Three isoforms of the α-subunit (α-1, α-2, α-3) and two isoforms of the β-subunit (β-1 and β-2) of calcineurin have been identified. In proximal tubules, α-1 isoforms are predominant and exceed α-2 expression by fourfold. In the CCD, α-1 and α-2 expression are approximately equal, whereas α-2 subunit expression is greatest in medullary thick ascending limbs (mTAL). α-3 was not detected in any nephron segment. Calcineurin phosphatase activity in the proximal tubule is approximately 10-fold higher than in the connecting tubules (CNT), cortical collecting ducts (CCD), or the mTAL. Protein phosphatases 1 and 2a are also expressed in CCD, and only protein phosphatase 1 can be detected in the proximal tubule. Calcineurin influences basal and stimulated Na/K-ATPase activity in the proximal and distal nephron. In the CCD, CsA or FK-506 decrease Na/K-ATPase activity by 35% and 85%, respectively; Na/K-ATPase activity in mTAL is decreased by 53% and 56%. Activation of membrane receptors, including adrenergic, dopamanergic, and angiotensin I receptors, also regulates Na/K-ATPase activity through processes that involve calcineurin. Lastly, steroid hormones including glucocorticoids and mineralocorticoids appear to activate calcineurin phosphatase activity. The mechanism is independent of transcription and appears to involve mechanisms involving heat shock proteins associated with the steroid receptor complex.

Steroid-induced Conformational Changes at Ends of the Hormone-binding Domain in the Rat Glucocorticoid Receptor Are Independent of Agonist Versus Antagonist Activity

The underlying molecular mechanism for the expression of agonist versus antagonist activity for a given receptor-steroid complex is still not known. One attractive hypothesis, based on data from progesterone receptors, is that agonist versus antagonist binding induces unique conformations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report that the determinants of glucocorticoid receptor (GR)-antagonist complex activity are more complex. Steroid binding did cause a conformational change in the GR that was detected by partial trypsin digestion, as described previously (Simons, S. S., Jr., Sistare, F. D., and Chakraborti, P. K. (1989) J. Biol. Chem. 264, 14493-14497). However, there was no uniformity in the digestion patterns of unactivated or activated receptors bound by a series of six structurally different antagonists including the affinity labeling antiglucocorticoid dexamethasone 21-mesylate. A total of four resistant bands were observed on SDS-polyacrylamide gels in the range of 30-27 kDa. Using a series of point mutations and epitope-specific antibodies, it was determined that the 30-kDa species represented the entire C-terminal sequence of amino acids 518-795, whereas the other bands arose from additional N-terminal and/or C-terminal cleavages. Bioassays with GRs containing various point and deletion mutations failed to reveal any C-terminal alterations that could convert antagonists into biologically active agonists. Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. When compared with the current model of the ligand-binding domain, which is based on the x-ray structures of the comparable region of thyroid and retinoic acid receptors, the present results suggest that sequences outside of the model structure are relevant for the binding and biological activity of GRs.

Aldosterone and dexamethasone stimulate calcineurin activity through a transcription-independent mechanism involving steroid receptor-associated heat shock proteins.

Heat shock proteins (HSP) are components of the steroid receptor complex and are released into the cell cytosol after hormone binding. We tested whether HSPs released from steroid receptors mediate an increase in calcineurin phosphatase activity by steroid hormones. Aldosterone increased calcineurin activity in microdissected rat cortical collecting ducts (CCD) and connecting tubules, but not in proximal tubules, medullary thick ascending limb, or outer medullary collecting ducts. In contrast, 5 microM dexamethasone increased calcineurin activity in both CCD and proximal tubules. Aldosterone increased CCD calcineurin activity after 30 min and this response was blocked by spironolactone, but not by actinomycin D. An antibody recognizing HSP-56 did not change basal calcineurin activity, but completely blocked the stimulation of calcineurin by aldosterone. Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid receptor complex, also blocked the aldosterone response, whereas HSP-90 or HSP-70 increased calcineurin activity in permeabilized CCD. In summary, (a) aldosterone increases calcineurin activity in CCD through a transcription-independent process; (b) maneuvers inactivating HSP-56 or slowing HSP disassociation from the receptor complex blocks stimulation of calcineurin by steroid hormones; (c) HSP-90 and HSP-70 increase CCD calcineurin activity in the absence of steroid hormone. We conclude that HSPs released from transformed steroid receptors can stimulate calcineurin activity through a transcription-independent pathway.

Membrane binding sites and non-genomic effects of estrogen in cultured human pre-osteoclastic cells.

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17beta-estradiol (17betaE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17betaE2 may influence bone remodelling by a dual mechanism of action: to affect gene expression mediated by the nuclear activity of the steroid-receptor complex, and to initiate rapid responses triggered by a signal-generating receptor on the cell surface. Recently, we demonstrated that the human pre-osteoclastic cell line FLG 29.1 bears functional estrogen receptors. In this study we examined FLG 29.1 cells for the presence of cell surface binding sites for 17betaE2, and whether 17betaE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17betaE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 microM) doses of 17betaE2 induced a prompt and significant (P < 0.05) increase of cellular pH, as measured in single cells using an image analysis system. In addition, both cAMP and cGMP were significantly increased by 17betaE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17betaE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17betaE2 on osteoclast precursors.

Synthesis, structure and biological properties of Z-17alpha-(2-iodovinyl)-11beta-chloromethyl estradiol-17beta (Z-CMIV), a high affinity ligand for the characterization of estrogen receptor-positive tumors.

Linkage of a 11beta-chloromethyl group to estradiol-17beta (E2) dramatically increases the binding affinity of the steroid for the estrogen receptor (ER) with the formation of a quasi-irreversible steroid-receptor complex. We have synthesized the two isomers of 11beta-chloromethyl-17alpha-iodovinyl-estradiol (E-CMIV and Z-CMIV) by a novel route. Both derivatives demonstrated high binding affinity and selectivity for ER (RBAs: ER = 820 and 1008; SHBG = 1.2 and 0.25, respectively; E2 = 100). On the basis of X-ray crystallographic data for Z-CMIV and its precursor, we have postulated that Z-CMIV might interact strongly with aromatic amino-acids within a hydrophobic groove of the ER hormone binding domain (HBD) that incorporates pockets corresponding to the 11beta and 17alpha steroid substituents. The binding properties of Z-CMIV labeled with 125I were investigated, especially its ability to detect and quantify altered ER forms with low binding affinity for E2. Sucrose density gradient analysis revealed that Z-CMIV has a higher activation potency than E2 as it converts a higher proportion of non-activated monomers in the cytosol into activated monomers with the potential to dimerize. In in vitro (MCF-7 cells) and in vivo (rat uterus) determinations of estrogenic activity, Z-CMIV was as potent as E2 in increasing progesterone receptor (PgR) concentrations and decreasing ER levels and in stimulating uterine growth. [125I]-Z-CMIV could open the way to new applications in the diagnosis and therapy of ER-positive breast cancers, especially those containing altered (variant) ERs.

The Structure and Complete Nucleotide Sequence of the Human Cyclophilin 40 (PPID) Gene

Cyclophilin 40 is a recently identified member of the cyclophilin family that is found in an unactivated steroid hormone receptor complex. Cyclophilin 40 possesses a region homologous to FKBP59, a member of the FK506-binding protein family that is also a component of the receptor complex. We report the isolation and sequencing of the entire human cyclophilin 40 (hCyP40) gene (human gene symbolPPID). The gene contains 10 exons (43 to 698 bp) and 9 introns encompassing 14.2 kb. The exon organization of the cyclophilin-like region is not similar to that of the human cyclophilin A gene (PPIA), suggesting their early divergence in evolution. Determination of the sequence of the 5′ end of the hCyP40 mRNA by an “anchor-ligation PCR” procedure showed that transcription is initiated from a cluster of sites about 80 bp upstream from the first in-frame ATG. The immediate 5′-flanking region of the gene lacks typical TATA and CAAT boxes, but is GC-rich and contains Sp1 sites, features characteristic of promoters associated with housekeeping genes. The hCyP40 gene was mapped to chromosome 4 by PCR with genomic DNA from somatic cell hybrids. As shown by “Zoo blot” analysis, the cyclophilin 40 gene appears to be highly conserved throughout evolution.

Characterization of cyclophilin 40: highly conserved protein that directly associates with Hsp90.

Cyclophilin 40 (CyP4O) is a recently identified member of the cyclophilin family that may be a component of unactivated steroid receptor complexes. It consists of an N-half portion that is highly homologous to cyclophilin A and has peptidyl prolyl isomerase (PPIase) activity, and a C-half portion that resembles the C-terminal portion of FKBP52 (FK506 binding protein 52), another component of unactivated steroid receptor complexes. To better understand the structure and functional characteristics of this new class of cyclophilin, we have raised monoclonal antibodies against the C-half portion of human CyP4O. Immunostaining with the antibodies showed its preferential localization in cytoplasm. One antibody cross-reacted with a 45 kDa protein in yeast, suggesting high conservation throughout evolution. A CyP4O-associated protein was isolated from rabbit reticulocyte lysate by means of an affinity resin, and was identified as hsp90. The C-half portion of CyP4O was necessary and sufficient for the interaction.

Binding of immunophilins to the 90 kDa heat shock protein (hsp90) via a tetratricopeptide repeat domain is a conserved protein interaction in plants.

In animal cell lysates, multiprotein complexes containing hsp90, hsp70, p60, p23, and several immunophilins can assemble steroid receptors and oncogenic protein kinases, such as v-Src and v-Raf, into heterocomplexes that contain hsp90 and either immunophilins or, in the case of protein kinases, p50. The complexes with hsp90 are required for the proper functioning of these signal transduction systems. Wheat germ lysate contains a similar protein folding activity that forms functional steroid receptor complexes with hsp90, but not all the components of this system have been identified. The plant chaperone system has conserved interactions with animal chaperones in that wheat hsp70 functions in the rabbit reticulocyte lysate heterocomplex assembly system and human p23 functions in the wheat germ lysate. Here, we ask if wheat germ lysate also contains immunophilins of the FK506-binding class (FKBPs) that bind to the hsp90 component of the chaperone complex via tetratricopeptide repeat (TPR) domains. To demonstrate the plant heterocomplex, we add purified mammalian p23, preadsorbed with the JJ3 antibody to protein A-Sepharose, to wheat germ lysate and allow ATP-dependent formation of an animal p23. plant hsp90 complex. The complex is then washed and incubated with the radiolabeled immunosuppressant drug [3H]FK506, which binds in a specific manner to a coimmunoadsorbed plant FKBP. Binding of the plant FKBP to plant hsp90 is prevented by adding to wheat germ lysate a purified fragment containing the TPR domains of human cyclophilin-40. Geldanamycin, a benzoquinone ansamycin that binds to animal hsp90s and prevents their chaperone activity, binds in a temperature-dependent manner to wheat hsp90 to block formation of the p23.hsp90.FKBP heterocomplex. These data show that immunophilin binding to hsp90 via TPR domains is conserved in the plant kingdom as well as in the animal kingdom and that geldanamycin will be an important tool for the study of hsp90-mediated protein chaperoning in plant cells.

Pharmacodynamics and pharmacokinetics of inhaled glucocorticoids

Research during the past two decades has shown that the inflammatory component of bronchial asthma has a highly complex and multifactorial pathophysiology. Inhaled corticosteroids have become first-line therapy for the treatment of moderate to severe asthma as a result of glucocorticoid action at the cellular level to attenuate the underlying disease processes. This article summarizes the findings of recent studies of the specific pharmacodynamic and pharmacokinetic mechanisms of glucocorticosteroids as a means of classifying individual drugs for use in the management of asthma and other airway diseases. In general, the glucocorticosteroid molecule is required to penetrate the cellular membrane and demonstrate affinity for the steroid-binding site on the glucocorticoid receptor, leading to activation of the receptor. Dimerization of the active steroidreceptor complex occurs, and the complex then enters the nucleus to bind to glucocorticoid responsive elements on a target gene to influence gene transcription and inhibit proinflammatory mechanisms, or to potentiate endogenous antiinflammatory mechanisms. Alternatively, a direct interaction of the glucocorticoid-receptor complex with transcription factors may also be an important determinant of steroid action and a key mechanism whereby glucocorticosteroids exert some of their antiinflammatory activities. 1

Oct-1 Protein Promotes Functional Transcription Complex Assembly on the Mouse Mammary Tumor Virus Promoter

The ubiquitous transcription factor Oct-1 stimulates basal transcription from the mouse mammary tumor virus (MMTV) promoter by binding to octamer-related sequences present in the proviral long terminal repeat. The mechanism of transcriptional activation by Oct-1 was investigated using in vitro transcription assays with a HeLa cell nuclear extract depleted of endogenous Oct-1. Oct-1-mediated transcriptional activation could be reconstituted by addition of bacterially expressed recombinant Oct-1 protein. The stimulatory effect of Oct-1 was observed only when the protein was present during formation of transcription preinitiation complexes and not when added to fully assembled complexes. Furthermore, assembled MMTV preinitiation complexes were resistant to inhibition by a competitor oligonucleotide containing MMTV octamer-related elements that could eliminate Oct-1-mediated stimulation when present during the assembly process. The time course of transcription complex assembly revealed that Oct-1 increases the number of templates on which functional transcription complexes form. Finally, experiments designed to exploit the sensitivity of discrete steps in transcription complex assembly to the anionic detergent Sarkosyl demonstrated that Oct-1 must be present during formation of an early intermediate in the assembly process.

Cellular Factors Binding to a Novel cis-Acting Element Mediate Steroid Hormone Responsiveness of Mouse Mammary Tumor Virus Promoter

Steroid hormone receptors regulate mouse mammary tumor virus (MMTV) gene expression by binding to hormone response DNA elements present in the long terminal repeat. Tissue-specific expression of MMTV is unlikely to be regulated by steroid hormone-receptor complex alone, and mammary cell-specific factors might play a role in the hormone-induced transcriptional activation. In this report we have investigated the function of a novel cis-acting element designated Kil (-204 to -188) which is located adjacent to the distal glucocorticoid response element, in steroid hormone-induced transcription of MMTV. Electrophoretic mobility shift assays indicate that cellular factors bind to the Kil element, and dexamethasone stimulation results in alterations in the binding pattern of proteins in this region. By transient transfection assays using wild type and deletion mutants of the Kil element, we show that this novel cis-acting element is necessary for hormone-induced transcription of MMTV and functions in mammary tumor cells but not in NIH/3T3 cells. Mutagenesis of the Kil sequence suggests that the entire Kil element functioning as one unit is necessary for hormone-induced transcription of MMTV. When placed in the context of heterologous promoters, neither Kil element nor glucocorticoid response element is able to induce significant hormone-induced transcription of MMTV. The presence of both the DNA elements in tandem results in optimal induction of transcription in the presence of steroid hormones. Our results also indicate that the Kil element functions in human breast carcinoma cell lines such as T47D and MCF-7. These results suggest that Kil element in combination with distal glucocorticoid response element functions as a mammary cell-specific enhancer to regulate MMTV transcription.

Growth-Regulated Expression of FKBP-59 Immunophilin in Normal and Transformed Fibroblastic Cells

Expression of many primary response cellular genes is observed during the early stages of transition from a resting G0 state to DNA synthesis. To identify gene products implicated in long-term response to growth factors, we have isolated genes sequentially activated several hours after serum stimulation of fibroblastic CCL39 cells. We report here the characterization of one of them as encoding the immunophilin of 56-59 kDa (FKBP-59), a component of the steroid receptor complex. FKBP-59 is encoded in several mammalian species by a unique gene located within the transition protein 2 gene locus. FKBP-59 mRNA is ubiquitously expressed at various levels in human organs. FKBP-59 gene activation in CCL39 fibroblasts requires protein synthesis during the first 2 h of stimulation, a period of time during which proto-oncogenes such as c-fos or c-jun are expressed. FKBP-59 mRNA degrades upon serum withdrawal and accumulates in proportion to the mitogenic strength of various purified growth factors. Its expression is reduced in CCL39 mutant-transformed cells or cells able to grow in low serum-containing medium, suggesting that FKBP-59 might participate in the negative feedback control of cell proliferation.

Steroid hormone induced effects on membrane fluidity.

The classical mechanism of steroid hormone action involves the niolecule binding to high affinity intracellular receptors. This steroid-receptor complex then travels to the nucleus where it modulates gene transcription. Some steroid hormone actions, however, are not due to genoniic changes, e.g., modification of behaviour by progesterone [ l ] and altered neuronal firing activity after gonadal and adrenal steroid hormone action 121. These actions are almost immediate or of R short latency and are insensitive to inhibitors of RNA and protein synthesis. Steroid horniones in such cases may act directly on plasma membranes. Indeed, receptors for oestradiol, testosterone and progesterone have been identified in synaptic membranes [ 3 ] . Steroid hormones may also alter the dynamic properties of membrane bilayers. The structural requirements for maxiniising the interaction of sterols with the bilayer appear to be R 3-p-hydroxy group, an isocaproyl side chain and a flat cholestene skeleton 14). Given the structural nnalogy between cholesterol and steroid hormones it is possible that such hormones both alter membrane fluidity and influence membrane protein organisation and function. Deliconstantinos [5] has observed a change in synaptic membrane ordering after exposure to steroid hormones. The influences of steroid hormones on model membrane bilayer fluidity and on natural membrane ordering was therefore investigated. Measurements of membrane fluidity were performed using the steady-state fluorescence polarisation of 1,6-diphenyl 1,3,5hexatriene (DPH) according to the method described by Shinitzky and Barenholz 161. The data obtained are expressed in terms of the emission anisotropy (r) where r = (Iw I@I + 244 . and IvH being the emission intensities observed parall8 and perpendicular to the polarisation plane of the exciting light. Lipid bilayer membranes were prepared by dispersing egg yolk phosphatidylcholine in 0.2M Tris-C1 buffer pH 7.4. Cholesterol was added to some liposomes to give a molar ratio of cholesterol to phospholipid of 0.8, similar to the ratio in synaptic membranes. Liposomal membranes containing steroid hormones were prepared by co-dissolving the hormone together with the requisite amount of phosphatidylcholine in chloroformmethanol (2: 1 v/v). The solvent was then removed under a stream of nitrogen. 0.2M Tris-CI buffer pH 7.4 was added to the dry hormone-lipid mixture and unilamellar liposomes formed by sonication. The final concentration of hormone varied between 2 and 1OpM. This method could not be used with natural membranes. In this case, the steroid hormones were incorporated into dipalmitoyl phosphatidylcholine (DPPC) liposomes. The hormone-associated DPPC WRS added in a 1:l molar ratio to the lipid in the membranes, resulting in transference of hormone from the DPPC bilayers to the membrane lipid. The suspension was then centrifuged on a 20% (w/v) sucrose layer on a 60% sucrose (w/v) cushion for 2 hours at 30,000 xg to separate the biomembrane from excess DPPC and any unassociated hormone. Progesterone was found to decrease membrane fluidity. Testosterone, however, caused no change in the ordering of the membrane whereas its metabolite 17-P-oestradiol increased membrane fluidity as did 17-a-oestradiol and oestriol. In both liposomes and natural membranes containing cholesterol the steroid hormone effect on membrane fluidity was less pronounced. It has been proposed [7] that cholesterol limits steroid hormones intercalating between lipid bilayers. They consequently bind more to the surface of the membrane, explaining the lower change in anisotropy. Thus in natural membranes the steroid hormones have more influence on sarcoplasmic reticulum membranes from rabbit white muscle with a neglible cholesterol content than in synaptic membranes. Steroid hormones have been shown to alter the dynamic properties of bilayers. Further work will show if this influences the activity of membrane-bound enzymes. Rorlesenme Oeslradiol

Effect of s-triazine compounds on testosterone metabolism in the rat prostate.

The influence of s-triazine compounds (atrazine, prometryne and deethylatrazine) on testosterone conversion and 5 alpha-dihydrotestosterone-receptor complex formation was studied in vitro and in vivo in the rat prostate. A marked in vitro influence of atrazine and prometryne (from 0.465 to 1.392 mumol) and their mixtures (in total concentration, 0.928 mumol) on 5 alpha-dihydrotestosterone formation was detected. 5 alpha-Dihydrotestosterone-specific receptor complex formation was inhibited in vitro by ca. 0.5 mumol of atrazine or deethylatrazine and only in vivo by 6 mg of atrazine 100 g-1 body wt. daily during 7 days in the prostate cytosol. The inhibition of the enzymic activities responsible for testosterone conversion and steroid hormone-receptor complex formation was non-competitive and reversible, and s-triazine compounds act as antiandrogens.

In vitro and in vivo thermal activation of steroid-receptor complexes from rats and ground squirrels ( Spermophilus citellus)

Using 3H-labelled triamcinolone acetonide ( 3HTA, synthetic steroid hormone), it was shown that the in vitro time course kinetics of thermal activation of 3HTA-receptor complexes exhibited the same temperature dependence in liver cytosols prepared from hibernating ground squirrels (Spermophilus citellus) as in cytosols from the rat. When 3HTA was injected in vivo to animals hibernating with a body temperature of 3°C, the activation and nuclear uptake of the in vivo formed steroid-receptor complexes proceeded at a slow rate, comparable to the one predicted by in vitro studies. In the hibernator, the results are not indicative of adaptive modifications at the level of thermal activation, but prove that steroid action does proceed at a temperature incompatible with hypothermic survival in the nonhibernator.