Human Glycoprotein Hormone Alpha-subunit Research Articles

The Antigenic Structure of the Human Glycoprotein Hormone α-Subunit: III. Solution- and Solid-Phase Mapping Using Synthetic Peptides*

Twenty-seven synthetic peptides, representing the entire structure of the human glycoprotein hormone alpha-subunit were used to map the antigenic structure of the alpha-subunit. Solution phase and solid phase assays were performed with these peptides and a panel of eight monoclonal antibodies (MAb). Two dominant regions were localized between residues 22-37 and 70-87. All eight antibodies recognized these regions, but differed somewhat with respect to whether they saw the more N-terminal, middle, or C-terminal portions of these regions. The sequence of residues 13-22 was recognized by three MAbs. The C-terminal region from residues 84-92 was recognized by three MAbs. All MAbs recognized conformational epitopes in that they reacted with two or more regions. Three MAbs (two against free alpha and one against human CG) have linear amino acid sequences as part of their conformational epitope.

A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP.

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.

Amplification of the Transcriptional Signal Mediated by the Tandem cAMP Response Elements of the Glycoprotein Hormone α-Subunit Gene Occurs through Several Distinct Mechanisms

cAMP stimulates transcription of the human glycoprotein hormone alpha-subunit gene in choriocarcinoma cells. Combined treatment with phorbol esters potentiates this effect. Tandem cAMP response elements (CREs) in the proximal 5'-flanking sequence mediate the effect of cAMP. In this report, we show that the CREs can also mediate the synergistic effect of phorbol esters. In addition to serving as an inducible cis-acting element, the two CREs act synergistically to increase basal transcription. We now provide direct evidence via equilibrium binding studies that tandem CREs bind their trans-acting factors cooperatively. However, the level of cooperativity is insufficient to explain the high degree of transcriptional synergism, suggesting that another element in the alpha-subunit promoter may be required for complete synergism. In support of this hypothesis, we show that synergism is drastically reduced when the CREs are removed from the context of their native promoter and linked to a heterologous promoter. Thus, amplification of the transcriptional signal mediated by the tandem CREs occurs through at least three distinct mechanisms. First, at the level of signal transduction by convergence of the A- and C-kinase pathways; second, through homotropic interactions of trans-acting factors binding to tandem CREs; and finally through heterotropic interaction of the two CREs with another, as yet, undefined cis-acting element(s) in the human alpha-subunit promoter.

Cell Lines of the Pituitary Gonadotrope Lineage Derived by Targeted Oncogenesis in Transgenic Mice

Study of the molecular and cellular biology of the gonadotropin hormones would be greatly facilitated by the availability of immortalized anterior pituitary gonadotrope cell lines. We directed expression of the simian virus-40 (SV40) T-antigen (Tag) oncogene to specific cells in the anterior pituitary of transgenic mice using the promoter/enhancer region from the human glycoprotein hormone alpha-subunit gene. Transgenic mice carrying this fusion gene developed anterior pituitary tumors. Clonal cell lines established from these tumors express the endogenous mouse alpha-subunit gene and synthesize and secrete alpha-subunit protein. However, they do not express beta-subunit genes. Alpha-subunit mRNA is induced by GnRH in a dose- and time-dependent manner, but is not regulated by TRH. Thus, we have targeted tumorigenesis in transgenic mice to anterior pituitary cells of the gonadotrope lineage to immortalize this specific endocrine cell while maintaining several highly differentiated functions unique to gonadotropes.

Regulation of the Transfected Human Glycoprotein Hormone α-Subunit Gene by Dexamethasone and Thyroid Hormone

A chimeric plasmid, (-1,500)h alpha CAT, containing approximately 1,500 bp of 5'-flanking DNA of the human glycoprotein hormone alpha-subunit gene directing the expression of the bacterial chloramphenicol acetyl transferase (CAT) gene, was transfected transiently into rat pituitary-derived GH3 cells. (-1,500)h alpha CAT expression was stimulated 5- to 20-fold by dexamethasone and 3- to 5-fold by 8-bromo-cAMP (8-Br-cAMP), and was inhibited by 50% by L-triiodothyronine (T3). Thus, suppression by T3 in this system was similar to that seen in pituitary thyrotropes. Induction of (-1,500)h alpha CAT expression by dexamethasone was antagonized by T3 but was unaffected by 8-Br-cAMP. However, T3 augmented the stimulation of (-1,500)h alpha CAT activity by 8-Br-cAMP. Deletants containing less than 346 bp of 5'-flanking alpha DNA showed a stepwise decrease in induction by dexamethasone, suggesting that multiple sequence elements located in this region are required for full induction of h alpha CAT activity. Deletion analysis also indicated that a thyroid hormone response element is located between 207 and 172 bp of the alpha gene transcriptional start site. Our finding of induction of alpha expression by dexamethasone in pituitary cells contrasts with the inhibition of alpha gene activity by glucocorticoids which has previously been shown in placental cells. Therefore, these data indicate that cell-type-specific factors play an important role in the modulation of alpha gene transcription.

CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation.

In mammals and other vertebrates, cytosine methylation in CpG sites is often negatively correlated with gene activity. Because methylation of the promoter region is most crucial for this effect, the simplest hypothesis is that CpG methylation interferes with the binding of specific transcription factors. We have examined this hypothesis with two different transcription factor-binding sites that contain a CpG dinucleotide, namely the cAMP-responsive element (CRE; 5'-TGACGTCA) and the Sp1-binding site (5'-GTGAGGCGGTGAGACT). We have reported previously that CpG methylation of the Sp1-binding site affected neither factor binding nor transcription in HeLa cells, which may be related to the fact that Sp1 is typically associated with promoters of housekeeping genes. In contrast, CREs are often associated with promoters of cell type-specific genes. A synthetic oligonucleotide containing two tandem CREs derived from the gene encoding the human glycoprotein hormone alpha-subunit was cloned upstream of a reporter gene. Transcription of this gene was dependent on the CRE sequences in both PC12 and HeLa cells. Bandshift and methylation interference assays show that similar, if not the same, factor(s) bind to the CRE in both cell lines, even though induction by cAMP was only observed in PC12 cells. CpG methylation of the CRE consensus sequences (TGACGTCA) resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types. This suggests that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.

Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.

Expression and Regulation of the Pituitary- and Placenta-Specific Human Glycoprotein Hormone Alpha-Subunit Gene Is Restricted to the Pituitary in Transgenic Mice

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.

Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer.

Although steroid hormone receptors are known to activate gene expression by binding to specific hormone-dependent enhancers, the mechanisms by which steroids inhibit the transcription of specific genes are unknown. It is shown here by gene transfer studies that the same glucocorticoid receptor that activates gene expression can negatively regulate expression of the human glycoprotein hormone alpha-subunit gene. Glucocorticoid inhibition was conferred by a 52-nucleotide region that also contains elements crucial both for adenosine 3',5'-monophosphate (cAMP) responsiveness and for placental-specific expression of this gene and was observed only under conditions in which these elements were functioning as enhancers. Purified glucocorticoid receptor was found to bind to DNA that overlap the cAMP responsive elements sites in this region. It is hypothesized that steroid receptors negatively regulate gene expression by interfering with the activity or binding of other important transcription factors.

The effects of synthetic alpha-subunit peptides on thyrotropin interaction with its receptor.

Synthetic peptides of the alpha-subunit of human glycoprotein hormones have been shown previously to inhibit binding of [125I]iodo-hCG to ovarian membranes, thus indicating the importance of the alpha-subunit in the structure-function relationships of the gonadotropic hormone. These same synthetic alpha-subunit peptides, the sequences of which are common to all human glycoprotein hormones, were found to inhibit the binding of [125I]iodo-TSH to human thyroid membrane preparations and FRTL-5 rat thyroid cells. The active portions of the subunit were represented in synthetic peptides alpha 21-35, alpha 31-45, alpha 26-46, and alpha 81-92, indicating that 2 separate sites within the alpha-subunit have binding activity for TSH. Peptides alpha 26-46 and alpha 31-45 were also found to potently inhibit the stimulation of adenylate cyclase activity by bovine TSH in TSH bioassay using FRTL-5 cells. Seven other synthetic peptides, including the remainder of the 92-amino acid sequence of the alpha-subunit, demonstrated little or no ability to inhibit binding of the tracer or inhibit the bioactivity of intact TSH. The findings were very similar to those of previous studies involving hCG binding, except that the two active sites appeared to be somewhat shifted towards the COOH-terminal end of the subunit. These studies support the concept of the importance of the alpha-subunit in receptor binding of all glycoprotein hormones and demonstrate the utility of the overlapping synthetic peptide strategy in investigations of protein structure-function relationships.

Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements.

We identified and characterized elements which confer tissue specificity and cyclic AMP (cAMP) responsiveness to the human glycoprotein alpha-subunit gene. An enhancer containing an 18-base-pair repeat conferred cAMP responsiveness in a non-tissue-specific fashion. DNase I protection assays revealed DNA-binding factors that bound to this element in both placental and nonplacental cells. It also enhanced the alpha-subunit promoter in a tissue-specific manner but had a negligible effect on a heterologous promoter. A unique element found upstream of this enhancer had no independent activity but, in combination with the cAMP-responsive enhancer, distinctly increased the tissue-specific activity of both the alpha-subunit promoter and a heterologous promoter. A factor that bound to this upstream element was found in placental but not nonplacental cells. We conclude that this novel element acts, perhaps through a specific trans-acting factor, in concert with a cAMP-responsive enhancer to confer tissue specificity to the alpha-subunit gene.

Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element.

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.

Glycoprotein hormone alpha-subunit in human stomach.

To demonstrate the immunoreactive alpha-subunit of human chorionic gonadotropin (hCG) or glycoprotein hormones in non-neoplastic gastric mucosa, and to clarify the nature and significance of alpha-subunit-immunoreactive cells, immunohistochemical studies were performed on gastric mucosa using polyclonal antibodies for hCG alpha and beta, hLH beta, hFSH beta, hTSH beta, and gastrin, and a monoclonal antibody for hCG alpha. Surgically resected stomachs were classified as follows: nearly normal (Group A); antral gastritis (Group B); fundic gastritis with pseudopyloric glands (Group C); and intestinal metaplasia (Group D). Cells immunoreactive for the alpha-subunit were present in the pyloric glands and to a lesser extent in the fundic glands (Groups A and B). Almost all alpha-subunit-immunoreactive cells were nonreactive for the beta-subunits of the four glycoprotein hormones. alpha-subunit-immunoreactive cells corresponded to gastrin-containing cells in the pyloric glands, but were unrelated to gastrin in the fundic glands. In fundic gastritis, alpha-subunit-immunoreactive cells appeared to increase (Group C), and many hyperplastic foci were observed in atrophic glands with hyperplasia of the argyrophilic cells (Groups C and D). Isolated hCG alpha or the alpha-subunit of glycoprotein hormones may be present in the endocrine cells of gastric mucosa, and alpha-subunit-immunoreactive cells in the fundic glands seem to proliferate in fundic gastritis.