Association of the 90-kDa heat shock protein does not affect the ligand-binding ability of androgen receptor

An N-terminal truncated androgen receptor with putative DNA- and ligand- binding domains (AR438) and that with a ligand-binding domain (AR612) were expressed under control of the T7 promoter in E. coli or translated in vitro with rabbit reticulocyte lysate, and their ligand-binding properties and the interaction with HSP90 were investigated. Bacterially expressed AR438 and AR612 bound a synthetic androgen, [³H]R1881, with apparent dissociation constant of 2.6 ± 0.2 and 3.1 ± 0.7 nM, respectively, values which are comparable to those of androgen receptor in target tissues. The recombinant androgen receptors sedimented at the 4–5 S region irrespective of the presence of 10 mM tungstate, indicating that the receptor exists free from HtpG, which is the bacterial homolog of eukaryotic HSP90. The apparent dissociation constant of truncated androgen receptors translated in vitro was 0.1 nM for AR438 and 0.2 nM for AR612. Sedimentation coefficients of in vitro translated molecules were converted from 7–8 S in the presence of tungstate to 3 S in the absence of tungstate. Both AR438 and AR612 translated in vitro were retained by anti-rat HSP90 antibody-protein A Sepharose. Exposure to 0.3 M NaCl in the presence of ligand caused dissociation of AR438 and AR612 from HSP90, and concomitantly, the DNA-cellulose binding ability of AR438 was enhanced. Thus, we conclude that the androgen receptor associates with HSP90 through the ligand-binding domain and that this association prevents the interaction of the androgen receptor with DNA. However, HSP90 seems to have little effect on the ligand-binding characteristics of the androgen receptor.     

A chemiluminescence-based assessment of androgen-binding activity in a large pedigree affected with androgen insensitivity syndrome.

We report a novel chemiluminescence (CL)-based method for assaying the ligand-binding activity of the androgen receptor. The central parts of this method are the utilization of the steroid CL marker as the replacement of the radioactive label in the conventional ligand-binding assay and the determination of the binding activity by the light measurement of the bound CL-label under an H(2)O(2)-microperoxidase system. The properties and reliability of this assay were investigated and verified using genital skin fibroblasts (GSF) from seven normal males. The method is precise (CV < 7% for both B(max) and K(d)) with high correlation coefficients (r > 0.93) in each Scatchard linear regression analysis. This assay can determine the androgen binding properties using only a quarter of the cells (approximately 40 000 cells/data point) of that required by the radiolabelling approach. The utility of the method was illustrated by binding experiment on the GSFs of several patients from a large Chinese family affected with androgen insensitivity syndrome. The familial distinct feature is that all patients shared an identical Arg840Cys substitution in the androgen receptor but displayed high phenotypic variation in disorders of male sexual development. The patients selected for the present study represent a wide spectrum of this phenotypic variation. This study thus provides insights on the pleiotropic effects of the mutation. In conclusion, the CL-based method can serve as an effective, precise and reliable replacement for the radiolabelling approach and has the advantages of simplicity, cost-effectiveness and health and environmental safety over the counterpart.     

A 58-kDa resident protein of the cis Golgi cisterna is not terminally glycosylated

A 58-kDa Golgi protein (gp58) was previously identified and found to be concentrated in cis Golgi cisternae in several cell types (Saraste, J., Palade, G.E., and Farquhar, M.G. (1987) J. Cell Biol. 105, 2021-2029). In this study the protein was partially purified from rat pancreas and mouse myeloma cells in order to characterize its oligosaccharides. It migrated on sodium dodecyl sulfate-polyacrylamide gels as a 57-58-kDa doublet under reducing conditions or as a single approximately 116-kDa band under nonreducing conditions. Pancreatic gp58 was susceptible to alpha-N-acetylgalactosaminidase digestion and it bound concanavalin A, Helix pomatia, Dolichos biflorus, soybean agglutinin, and Bauhinia purpurea lectins, but not Ricinus communis agglutinin or lectins from Griffonia simplicifolia-1, Arachis hypogaea, and Limulus polyphemus. It bound Ricinus communis agglutinin after galactosylation with GlcNAc galactosyltransferase. These data demonstrate that pancreatic p58 contains immature N-linked moieties with nonreducing terminal GlcNAc residues as well as the initiating GalNAc of O-linked glycoproteins. Myeloma gp58 was sensitive to endo-beta-N-acetylglucosaminidase H, and oligosaccharide analysis of its [3H]glucosamine-labeled glycopeptides indicated that it also contained immature N-linked glycans. Some of the latter consist of high mannose chains (high affinity for concanavalin A, endo-beta-N-acetylglucosaminidase H-sensitive), but the predominant (95%) species are neutral tri- or tetraantennary N-linked chains containing GlcNAc (no binding to concanavalin A). Glycopeptides from biosynthetically labeled myeloma cells did not contain detectable base labile oligosaccharides, indicating that unlike pancreatic p58, myeloma gp58 may not be an O-linked glycoprotein. Neither pancreatic nor myeloma gp58 contained terminally processed oligosaccharides, indicating that gp58 has not been modified by trans-Golgi glycosyltransferases. Thus, the oligosaccharide content of gp58 is consistent with the assumption that this protein is retained in the cis Golgi cisternae during biosynthesis instead of being transported across the Golgi stacks and targeted back to the cis Golgi from the trans side.     

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta

Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle.     

A 43-kDa protein of Treponema denticola is essential for dentilisin activity

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.     

The 18-kDa lectin-binding protein of Chlamydia trachomatis is different from the 18-kDa histone-like protein

Abstract Five proteins of Chlamydia trachomatis at the 18000 (18-kDa) molecular mass region were resolved by two-dimensional electrophoresis. Three proteins at 18.2 kDa, p I 6.9, 18.0 kDa, p I 6.3, and 17.9 kDa, p I 6.4 were shown to bind lectin. A fourth protein of 18.0 kDa at p I 10 was the histone-like protein. The fifth protein at 17.9 kDa, p I 7.0 was not characterized.     

Anabolic responsiveness of skeletal muscles correlates with androgen receptor protein but not mRNA

Anabolic effects of androgens on skeletal muscle are well documented, but the physiological and biochemical bases of these effects are poorly understood. Skeletal muscles that differ in their androgen responsiveness can be used to examine these mechanisms. We compared androgen receptor mRNA and protein levels of the rat levator ani, a perineal skeletal muscle that depends on androgens for its normal maintenance and function with that of the rat extensor digitorum longus, a limb muscle that does not require androgens. Western immunoblotting indicated that androgen receptor protein is significantly elevated in the levator ani relative to the extensor digitorum longus. Surprisingly, steady state androgen receptor mRNA levels were equivalent in these muscles, as determined by Northern blot analysis and quantitative RT-PCR. These results suggest that androgen responsiveness of skeletal muscles is determined by the level of androgen receptor protein in a particular muscle and that androgen receptor protein content is regulated by translational or post-translational mechanisms.     

Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor.

Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144-179] as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.     

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.     

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.     

Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein.

Platelet membrane glycoprotein IIb-IIIa has been widely studied in the last years because of its role as an activation-dependent, adhesive protein receptor. Recently we demonstrated that occupancy of glycoprotein IIb-IIIa-receptor sites by specific ligands exerts an inhibitory effect on platelet responses induced by mild stimulation, leading us to suppose that this event may interact with activation pathways. Although the mechanisms of signal transduction in human platelets are not completely elucidated, the hypothesis that GTP-binding proteins are involved is generally accepted. Our results demonstrate that platelet ConA receptors, known to be located mainly on GP IIb-IIIa, are able to bind [35S]GTP gamma S; the GTP-binding activity is specific and is due to the association with the receptors of two G-proteins, with apparent molecular masses of 25 and 21 kDa, respectively. After the purification of GP IIb-IIIa, a glycoprotein complex electrophoretically pure was obtained that was still associated with a GTP-binding activity, migrating in SDS-polyacrylamide gel electrophoresis as a narrow band of about 21 kDa.     

Comparison of the postsynaptic 43-kDa protein from muscle cells that differ in acetylcholine receptor clustering activity

A peripheral membrane protein of Mr = 43,000 (43-kDa protein) is closely associated with the acetylcholine receptor (AChR) in Torpedo electrocyte postsynaptic membranes and may play a role in anchoring receptors at synaptic sites. A component immunologically related to the 43-kDa protein also occurs specifically at mammalian muscle synapses and in association with receptor clusters on cultured muscle cells. We have studied this mammalian protein in two mouse muscle cell lines, C2 and BC3H1, that differ in AChR clustering activity. The 43-kDa-related protein was purified from muscle cell detergent extracts by immunoaffinity chromatography using monoclonal antibodies (mAbs) prepared against the Torpedo 43-kDa protein and identified by immunoblotting. In both C2 and BC3H1 cells, a protein of molecular mass of approximately 43,000 was recognized by two mAbs with different epitope specificity. To measure the 43-kDa protein in mammalian muscle cells, we designed a quantitative immunological assay utilizing these two mAbs. As in Torpedo electric organ, the concentration of the 43-kDa protein and receptor was approximately equimolar in C2 cells and in BC3H1 cells. Furthermore, during differentiation of both muscle cell lines, the appearance of the 43-kDa protein correlated closely with that of the receptor, raising the intriguing possibility that the expression of these two proteins is controlled by similar regulatory mechanisms. These results indicate that the inability of BC3H1 cells to form AChR clusters apparently does not result from a deficiency in the 43-kDa protein.     

A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

Protein-phosphorylation activity has been reported in chloroplast envelope membranes of several species. In spinach (Spinacia oleracea L.), we found three major phosphoproteins after incubation in vitro of envelope membranes in the presence of [-³²P]ATP. A 67-kDa phosphoprotein was associated with both inner and outer envelope membranes whereas 26- and 14-kDa proteins were observed in the inner membrane. Although the phosphorylation of the 67-kDa protein is likely to take place via its phosphoglucomutase activity (Salvucci et al., 1990, Plant Physiol. 93, 105–109), the mechanism by which ³²P is incorporated into the 26- and 14-kDa proteins remains to be elucidated. To this aim, we have compared the conditions under which phosphorylation occurs in these three proteins. The effects of Mg²⁺, Ca²⁺, pH, ATP and H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a specific inhibitor of protein-kinase C, as well as pulse-chase experiments with cold ATP, showed that the phosphorylation mechanism was identical for the 26- and 14-kDa proteins but quite different for the 67-kDa one. The protein kinase involved in the phosphorylation of the 26- and 14-kDa proteins was Ca²⁺-dependent, which was not the case of the 67-kDa protein. In addition, the use of a Triton X-114 phase-separation treatment indicated that both the 26- and 14-kDa proteins exhibited strong hydrophobic properties, in contrast to the hydrophilic character of the 67-kDa phosphoprotein. As indicated by analyses of phosphoamino acids, the three proteins were exclusively phosphorylated on serine residues. Furthermore, a treatment of envelopes by phospholipase C prior to the phosphorylation process inhibited ³²P incorporation into the three phospho-proteins to different extents (61%, 50% and 29% inhibition for the 67-, 14- and 26-kDa proteins, respectively). These results show that phosphatidylcholine and — or phosphatidylglycerol but not phosphatidylinositol were involved in this phosphorylation process.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SDS sodium dodecyl sulfate The authors are grateful to Mrs. Delphine Herrmann and Mr. Daniel Leemann for their skillful technical assistance. This study was supported by the Swiss National Science Foundation (Grant No. 31.26386.89). This work is part of a doctoral program which is carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel.     

Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown.