• Journal of Life Science
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• v.11 no.2
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• pp.103-107
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• 2001

The equine chorionic gonadotropin (eCG) subunits $\alpha$ and ${\beta}$ are transcribed from different genes and associate noncovalently to form the bioactive eCG heterodimer. Dimerization is rate limiting for eCG secretion, and dissociation leads to hormone inactivation. The correct conformation of the heterodimer is alto important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduction. To determine whether ${\alpha}$ and ${\beta}$ subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule by fusing the carboxyl terminus of the eCG ${\beta}$-subunit to the amino terminus of the af-subunit was construe-ted and transfected into chinese hamster ovary (CHO-Kl) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG ${\alpha}$/${\beta}$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG ${\alpha}$/${\beta}$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-K1 cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models of FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.229-236
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• 2009

The glycoprotein hormone family represents a class of heterodimers, that includes the placental hormone equine chorionic gonadotropin (eCG) and the anterior pituitary hormones- follitropin (FSH), lutropin (LH), and thyrotropin (TSH). The 4 hormones are heterodimers, with a common $\alpha$-subunit and unique $\beta$-subunits. eCG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. Recent observations using single chain glycoprotein hormone analogs in which, the $\beta$-and $\alpha$-subunits are linked, implied that heterodimeric-like quaternary configuration is not a prerequisite for receptor/signal transduction. To study the function and signal transduction of tethered rec-eCG, a single chain eCG molecule was constructed and rec-eCG protein was produced. Molecular mass of the single chain is about 45 kDa. All mice were ovulated by tethered rec-eCG treatment. The dual activity of tethered rec-eCG was determined in receptor cell lines of nonequid species; in fact, this dual activity was proven in species other than horse. Tethered rec-eCG in equids does not bind to FSH receptors, suggesting that eCG is primarily an LH-like hormone in the horse. Taken together, these data suggest that tethered rec-eCG has dual activity in nonequid species in vitro. However, it has only LH-like activity in equid species in vitro.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.221-221
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• 2004

Equine chorionic gonadotropin (eCG), which consists of highly glycosylated α- and β-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species than the horse. To determine whether α and β subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule was constructed and transfected into Chinese hamster ovary (CHO-K1) cells. (omitted)

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.210-210
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• 2004

Equinechorionic gonadotropin(eCG) is a member of the glycoprotein hormone family which includes FSH, hCG, TSH. These hormone family is characterized by a heterodimeric structure composed a common α-subunit noncovalently linked to a hormone specific β-subunit. To determine a and β-subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-molecule by fusing the carboxyl terminus of the eCG β-subunit to the amino terminus of the α-subunit was constructed and transfected into chinese hamster ovary (CHO-K1) cells. (omitted)

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.299-308
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• 2002

In horse, a single gene encodes both eCG and eLH $\beta$ subunits. The difference between eCG and eLH lies in the structure of their glycoresidues, which are both sialylated and sulfated in LH and sialylated in CG eCG consists of highly glycosyiated $\alpha$- and $\beta$-subunits and is an unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of gonadotropin structure-function relationships and the understanding of the molecular bases of the specific interactions of these hormones with their receptors. Thus, eCG is a dintinct molecule from the view points of its biological function and glycoresidue structures. The oligosaccharide at Asn 56 of the $\alpha$-subunit plays an indispensable role, whereas the carboxyl-terminal extension of the eCG $\beta$-subunit with its associated O-linked oligosaccharides is not improtant for, the in vitro LH-like activity of eCG. In contrast, both N- and O-linked oligosaccharides play important roles for FSH-like activity and increase FSH-like activity by removal of N- and O-linked oligosaccharides. Therefore, the dual LH- and FSH-like activities of eCG can be clearly separated by removal of either the N-linked oligosaccharide on the $\alpha$-subunit or CTP-associated O-linked oligosaccharides from its $\beta$-subunit. The glycoresidues seem to play crucial roles fer biological activities. The tethered-eCG was effciently secreted and showed similar LH-like activity to the dimeric eCG $\alpha$/ $\beta$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG $\alpha$/ $\beta$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-Kl cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models ot FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• Proceedings of the KSAR Conference
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• 2000.10a
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• pp.10-12
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.357-364
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.141-142
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• 2005