Effects of adenine nucleotides on choriogonadotropin alpha and beta subunit synthesis

The alpha subunit of human chorionic gonadotropin (CG) contains a discrete cAMP response element in the 5' flanking region of the gene. Since cAMP also stimulates the synthesis of the CG beta subunit the presence of a cAMP cis element in the CG beta gene was examined. Deletion mutants bearing various lengths of CG beta 5' region in front of the chloramphenicol acetyl transferase (CAT) gene were transfected in placental tumor cells. No discrete cAMP response element could be identified. Unexpectedly we also observed that AMP and adenosine not only stimulated CAT activity driven by CG beta promoter sequences but also enhanced synthesis of CG alpha and beta subunits in cultured choriocarcinoma cells. GMP, CMP, guanosine, and cytosine were inactive at comparable concentrations. These data suggest that the response of the CG alpha and beta genes to the non-cyclic adenine derivatives occurs by a mechanism that differs from cAMP.     

The gene for the beta subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5'-untranslated region

Both cDNA and genomic clones encoding the beta subunit of bovine luteinizing hormone (LH) have been isolated and characterized. The nucleotide sequence was determined for the entire gene and 776 base pairs of 5'-flanking sequence. The mRNA cap site and polyadenylation site were mapped by primer extension and S1 nuclease protection, respectively. The bovine LH beta spans less than 1.1 kilobase pairs and has three exons encoding a 550 nucleotide mRNA (excluding the poly(A) tail). Bovine LH beta is a single-copy gene, in contrast to human LH beta, which is a member of the LH/chorionic gonadotropin beta subunit multigene family. Comparison of the bovine LH beta gene with the human LH beta/chorionic gonadotropin gene family reveals a high degree of nucleotide sequence homology, both within the genes and in the 5'-flanking sequences. Despite this extensive sequence conservation, there is a major difference between the two species in the selection of a promoter site. As a result, the bovine LH beta gene produces an mRNA with an usually short 5'-untranslated region of only 6-11 nucleotides.     

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RII beta of type II cAMP-dependent protein kinase

The promoter and exon 1 of the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5'-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RII beta 5'-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp -291/-121. An enhancer element was located between bp -1426/-1018.     

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.     

Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.     

Structure and sequence analysis of the human activin beta A subunit gene.

The cloned genomic DNA containing the human activin beta A subunit gene were analyzed by restriction endonuclease mapping, Southern blotting and DNA sequencing. The activin gene is composed of two exons interrupted by the 9-kb intron. The TATA, CCAAT and CT-stretch sequences were found in the 5'-flanking region of the gene. An intronic sequence contained SV40 enhancer core element in the vicinity of the exon 1. In the 3'-flanking region, we identified eight consensus polyadenylation sequences, five ATTTA motifs, CA element consisting of (CA)14, AP-1 binding site and two SV40 enhancer core elements. A dot matrix analysis revealed the high degree of conservation between the human and rat sequences within the 3'-flanking region, suggesting a possible functional significance.