Solution structure of the alpha-subunit of human chorionic gonadotropin. (1/266)

The three-dimensional solution structure of the alpha-subunit in the alpha, beta heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo-beta-N-acetylglucosaminidase-B (dg-alpha hCG), was determined using 2D homonuclear and 2D heteronuclear 1H, 13C NMR spectroscopy at natural abundance in conjunction with the program package XPLOR. The distance geometry/simulated annealing protocol was modified to allow for the efficient modelling of the cystine knot motif present in alpha hCG. The protein structure was modelled with 620 interproton distance restraints and the GlcNAc residue linked to Asn78 was modelled with 30 protein-carbohydrate and 3 intraresidual NOEs. The solution structure of dg-alpha hCG is represented by an ensemble of 27 structures. In comparison to the crystal structure of the dimer, the solution structure of free dg-alpha hCG exhibits: (a) an increased structural disorder (residues 33-57); (b) a different backbone conformation near Val76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the beta-subunit. Consequently, in free dg-alpha hCG, compared to the intact dimer, the two hairpin loops 20-23 and 70-74 are arranged differently with respect to each other. The beta-GlcNAc(78) is tightly associated with the hydrophobic protein-core in between the beta-hairpins. This conclusion is based on the NOEs from the axial H1, H3, H5 atoms and the N-acetyl protons of beta-GlcNAc(78) to the protein-core. The hydrophobic protein-core between the beta-hairpins is thereby shielded from the solvent.  (+info)

An upstream regulator of the glycoprotein hormone alpha-subunit gene mediates pituitary cell type activation and repression by different mechanisms. (2/266)

Targeting of alpha-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived alpha-TSH and alpha-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in alpha-TSH and alpha-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal alpha-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in alpha-TSH and alpha-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.  (+info)

Cyclic AMP- and differentiation-dependent regulation of the proximal alphaHCG gene promoter in term villous trophoblasts. (3/266)

Although the regulatory mechanisms controlling alpha and beta human chorionic gonadotrophin (HCG) expression have been investigated in choriocarcinoma cell model systems, little is known about the regulation of HCG subunit synthesis in non-tumourigenic trophoblasts. We therefore investigated alphaHCG mRNA transcription in villous cytotrophoblasts isolated from term placentae and have shown for the first time that the proximal alphaHCG gene promoter is functional in these cells. By establishing conditions which allow efficient transient transfection of immunopurified cells, we have demonstrated that a 363 bp sequence in the proximal 5' flanking region of the alphaHCG gene is sufficient to direct trophoblast-specific expression of a luciferase reporter. After 12-60 h cultivation, an increase in endogenous alphaHCG mRNA expression could be detected, indicating that aggregated villous trophoblasts undergo biochemical differentiation. Concomitantly, we observed induction of alphaHCG promoter-driven luciferase activity, suggesting that the 363 bp sequence of the proximal 5' flanking region is sufficient to direct differentiation-dependent increase of alphaHCG mRNA. Continuous luciferase expression required functional cAMP-response elements (CREs), since deletion of both recognition sequences eliminated differentiation-dependent transcription of the reporter. Elevation of cAMP values increased transcription of the wild-type construct; however, it did not affect promoter activity of the mutant plasmid. Moreover, we have demonstrated that during in-vitro differentiation, CREs interacted with increasing amounts of phosphorylated activating transcription factor/cyclic AMP response element-binding protein (ATF-1/CREB-1) suggesting that these cAMP-dependent DNA-binding factors are major determinants in regulating alphaHCG gene expression in villous trophoblasts.  (+info)

Cloning, sequencing and functional expression of zebra (Equus burchelli) LH. (4/266)

Although donkey luteinizing hormone exhibits a very high degree of amino acid sequence identity with horse LH, its FSH activity in non-equine species is tenfold lower. The coding regions of the common zebra (Equus burchelli) glycoprotein hormone alpha-subunit and LH beta-subunit transcripts were cloned by reverse transcription-PCR from pituitary gland RNA to investigate more precisely the structure-function relationships of this gonadotrophin family. Zebra LH was then expressed in COS-7 cells and its LH and FSH activities were assessed in a rat Leydig cell bioassay (for LH) and in a cell line stably expressing the human FSH receptor bioassay (for FSH). The recombinant zebra LH, although displaying LH activity similar to that of recombinant donkey and horse LH, had no detectable FSH activity. The LH amino acid sequences of these three species are very similar, leaving only very few amino acids as potential candidates to explain the difference in their FSH activities. Moreover, according to the difference in FSH bioactivity and to the percentage identity between the sequences, the common zebra is phylogenetically closer to the donkey than it is to the horse.  (+info)

Treatment of thyrotropin-secreting pituitary adenomas with octreotide. (5/266)

Five hyperthyroid patients with TSH-secreting pituitary adenoma were treated with octreotide. Acute administration of octreotide decreased plasma TSH levels in all patients (mean decrease, 50.6 +/- 14%). Treatment with octreotide (25-300 microg/day) for 2-360 weeks resulted in reductions in plasma TSH and alpha-subunit levels in three patients, and serum free thyroxine levels were normalized with concomitant clinical improvements such as disappearance of excessive sweating, tachycardia and finger tremors. In two patients, plasma TSH and free thyroxine levels were initially decreased, but tachyphylaxis occurred 3 and 10 weeks after the initiation of therapy. Mild to marked shrinkage of the tumor was observed 2-50 weeks later in four patients. Shrinkage of the tumor seems to be reversible in one case. Frequent bowel movements and epigastric discomfort occurred in two patient. Somatostatin receptor subtype 2 (sst2) mRNAs were detected in two adenoma tissues studied by RT-PCR. Long-term treatment with octreotide is effective in controlling hyperthyroidism and tumor growth in patients with TSH-secreting pituitary adenoma.  (+info)

Mechanisms that mediate negative regulation of the thyroid-stimulating hormone alpha gene by the thyroid hormone receptor. (6/266)

A group of transcriptional cofactors for nuclear hormone receptors, referred to as corepressors (CoRs) and coactivators (CoAs), has been shown to induce transcriptional silencing and hormone-induced activation, respectively, of genes that contain positive hormone response elements. Transcriptional silencing by CoRs involves the recruitment of histone deacetylases (HDACs), whereas ligand-dependent activation is associated with the recruitment of CoAs, which possess or recruit histone acetyltransferases (HATs). In a reciprocal manner, negatively regulated genes are stimulated by nuclear receptors in the absence of ligand and are repressed in response to ligand binding to receptors. We show here that negative regulation of the thyroid-stimulating hormone alpha (TSHalpha) promoter by the thyroid hormone receptor (TR) involves a novel mechanism in which the recruitment of CoRs by TR is associated with transcriptional stimulation and histone acetylation. Expression of excess HDAC reverses the stimulation mediated by the TR.CoR complex, consistent with a pivotal role for acetylation in this event. Addition of the ligand, 3,5,3'-triiodothyronine (T3), induces transcriptional repression of the TSHalpha promoter and is associated with the loss of histone acetylation. T3-dependent repression is blocked by phosphorylation of cAMP response element binding protein, or by inhibition of HDAC, indicating that receptor action is subverted by maneuvers that stimulate histone acetylation of the target gene. We propose that negative regulation of a subset of genes by TR involves the active exchange of CoRs and CoAs with intrinsic promoter regulatory elements that normally strongly induce histone acetylation and transcriptional activation.  (+info)

Pathogenesis of ECL cell tumors in humans. (7/266)

In ECL cell tumors developed in the setting of hypergastrinemic conditions (ECL cell carcinoids type 1 and 2), hypergastrinemia is the dominant agent acting as a promoter in all steps (hyperplasia-dysplasia-neoplasia) of the tumorigenic sequence. In contrast, it apparently lacks transforming properties as shown by the absence of ECL cell carcinoids in patients exposed to hypergastrinemia alone, i.e., those with sporadic Zollinger-Ellison syndrome. The potential transforming factors include: the allelic loss of the MEN-1 suppressor gene in the genetically predisposed MEN-1 patients, an alteration that may induce ECL cell tumors even in the absence of hypergastrinemia; the still unknown factor(s) associated with atrophic corporal gastritis; agents whose role in the induction of human ECL cell tumors is still unclarified, such as basic Fibroblast Growth Factor, human Chorionic Gonadotropin-alpha and Transforming Growth Factor-alpha; and agents having a favoring role on the ECL exposure to mitogens such as BCL-2. No information is currently available on the pathogenesis of gastrin-independent, sporadic ECL cell carcinoids (type 3) or of gastric neuroendocrine carcinomas.  (+info)

Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin. (8/266)

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.  (+info)