Isolation, characterization and developmental expression of the ecdysteroid-induced E75 gene of the wax moth Galleria mellonella.

Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone did not encode the N-terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75A and GmE75B, which differed in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA was induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics.

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

The human prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1 alpha,25-dihydroxyvitamin D3.

The LNCaP prostatic carcinoma cell line was examined for the presence of specific receptors for 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Whole cell binding studies identified approximately 2500 high-affinity (Kd = 1.4 x 10(-9) binding sites per cell. Competition studies revealed that these receptors are specific for the 1 alpha,25(OH)2 metabolite. Binding studies using the synthetic androgen R1881 indicate that separate androgen and vitamin D3 receptors exist in LNCaP cells. The vitamin D3 receptors sediment at approximately 3.5S on linear sucrose gradients. The sedimentation coefficient could be shifted with a monoclonal anti-vitamin D3 receptor antibody (9A7 gamma) but not with a monoclonal antibody to the androgen receptor (AN1-15). The receptor/ligand complex elutes from native DNA cellulose at 0.2 M KCl. Northern blot analysis identified an mRNA of approximately 4.6 kilobases which hybridized with a specific vitamin D3 receptor complementary DNA probe (hVDR). In the absence of androgens, 1 alpha,25(OH)2D3 stimulated growth and prostate-specific antigen production by LNCaP cells in a dose-dependent fashion. Dose-response curves indicated that at physiological concentrations (10(-9) M) 1 alpha,25(OH)2D3 was mitogenic, whereas at higher concentrations (10(-8) M) it promotes differentiation. These studies suggest that 1 alpha,25(OH)2D3 could play an important role in the natural history of and response to hormone therapy by prostatic cancer.

COUP transcription factor is a member of the steroid receptor superfamily.

The COUP (chicken ovalbumin upstream promoter) transcription factor (COUP-TF) exists in a number of different tissues and is essential for expression of the chicken ovalbumin gene. It binds to the ovalbumin promoter and, in conjunction with a second protein (S300-II), stimulates initiation of transcription in vitro. COUP-TF also binds specifically to the rat insulin promoter element, although the two binding sites share little sequence similarity. Here we report the isolation of a human complementary DNA clone encoding COUP-TF. Comparison of the amino-acid sequence of COUP-TF with known sequences reveals that it is a member of the steroid/thyroid hormone/vitamin receptor superfamily. Consequently, it is the first member of this family that has been shown to function in a cell-free transcription system. We conclude that this superfamily of gene regulators contains proteins which bind and activate distal promoter elements of eukaryotic genes.

Association of 1,25-dihydroxyvitamin D3 with cultured 3T6 mouse fibroblasts. Cellular uptake and receptor-mediated migration to the nucleus.

The uptake of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by intact cells was investigated using the cultured embryonic 3T6 mouse fibroblast as a model. Suspended cells, incubated for 60-90 min in serum-containing culture medium supplemented with 1,25-(OH)2D3 (2 nM), maximally accumulate hormone which becomes bound to a typical vitamin D 3.3 S receptor protein. Incubation of cells with varying concentrations of 1,25-(OH)2D3 reveals the presence of 21,000 receptor molecules/3T6 cell, with an apparent uptake constant of 6-8 X 10(-10) M at 37 degrees C. This value contrasts with the equilibrium dissociation constant (Kd) for 1,25-(OH)2D3 binding of 6 X 10(-11) M as determined at 2 degrees C in disrupted cell cytosol. The distribution of unoccupied (R0) receptors is predominantly (greater than 85%) cytosolic in the hormone-deprived state (1,25-(OH)2D3 less than 0.05 nM), whereas exposure to 1,25-(OH)2D3 (2 nM) leads to almost complete nuclear localization of the occupied receptor at both 2 and 37 degrees C. This phenomenon was similarly supported through reconstitution of receptor and purified 3T6 nuclei in vitro in which binding also occurs at 2 degrees C. The majority (65%) of intact cell-formed receptor-nuclear complexes can be solubilized by micrococcal nuclease treatment, suggesting the participation of DNA in the acceptor binding site for the 1,25-(OH)2D3 receptor. Consistent with these data, DNA-binding of receptor also occurred in vitro at 2 degrees C and was a characteristic of both occupied (Rs) and unoccupied receptors. However, elution of the latter occurred at reduced ionic strength, implying that the hormone does physically alter the receptor protein. This binding was also sensitive to prior ethidium bromide saturation of DNA-cellulose, but not phosphocellulose. Although the biologic effects of the 1,25-(OH)2D3 hormone in 3T6 fibroblasts are as yet unknown, the present findings support previous work with 1,25-(OH)2D3 receptors and suggest that this cell represents a good model for the study of nuclear events associated with the molecular action of 1,25-(OH)2D3.

Glucocorticoids and appearance of 1,25-dihydroxyvitamin D3 receptor in rat intestine.

The ontogenesis of the 1,25-dihydroxyvitamin D3 specific binding activity in intestine was examined in vitamin D-deficient and replete rats. The absence of binding activity in intestines during the first two postnatal weeks was not influenced by vitamin D supplementation. The concentration of binding sites peaked on day 18 in vitamin D-replete rats and preceded that in the deficient group by approximately 1 wk. The influence of glucocorticoids on 1,25-dihydroxyvitamin D3-binding protein levels was examined by sequential hydrocortisone administration and adrenalectomy. Subcutaneous hydrocortisone administration before day 14 postpartum did not induce binding activity. The concentration of binding sites was significantly increased to 369 +/- 60 fmol/mg of protein by hydrocortisone injections from days 15 to 17 postpartum when compared with an average of 182 +/- 16 fmol/mg of protein in littermate controls. Hydrocortisone administration did not further increase receptor levels in rats injected from days 19 to 21. Bilateral adrenalectomy on day 17 postpartum significantly decreased the concentration of binding sites. It is concluded that adrenal glucocorticoids play an important role in the developmental appearance of 1,25-dihydroxyvitamin D3 specific binding activity in the postnatal rat intestine.

Gonadal steroids: humoral modulators of nerve-cell function.

The gonadal steroids, estradiol, testosterone and progesterone, alter neuroendocrine function and behavior in adult mammals and do so by acting on the central nervous system and pituitary gland. Besides intracellular receptor sites, various cellular and chemical actions of gonadal steroids are being studied within the brain and pituitary. The review summarizes the present state of such research and points as well to new advances in our understanding of the cellular basis of developmental events such as brain sexual differentiation and puberty, which are influenced by gonadal steroids.

Androgen receptor in the rat brain--assays and properties.

The existence and relevance of an androgen receptor in the developing brain has been a matter of controversy. We here describe both sucrose density gradient and hydroxylapatite assays which clearly define a distinct androgen receptor in the 24-day-old rat hypothalamus, amygdala and preoptic region, but not in the cortex. This receptor has considerable affinity for estradiol-17beta, thus perhaps accounting for some uncertainty about its nature, but none for diethylstilbestrol or other estrogens, antiestrogens or glucocorticoids. Its Kd for both dihydrotestosterone and testosterone is about 1 X 10(-9) M and for estradiol about 2 X 10(-8) M. Its properties are generally consistent with those of androgen receptor reported for other tissues.