Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

Byambaragchaa, Munkhzaya; Park, Ayoung; Gil, So-Jin; Lee, Hae-Won; Ko, Yun-Jeong; Choi, Seung-Hee; Kang, Myung‐Hwa; Min, Kwan-Sik

doi:10.1080/19768354.2021.1943708

Cited by 12 publications

(23 citation statements)

References 43 publications

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“…For rec-eCGβ/α production, the expression vectors were transfected into CHO-S cells using the FreeStyle™ MAX reagent, following the manufacturer’s instructions [ 41 , 42 ]. Briefly, the CHO-S cells were cultured in FreeStyle CHO expression medium at 1 × 10 7 cells per 30 mL of medium for 3 days.…”

Section: Methodsmentioning

confidence: 99%

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Byambaragchaa

Seong

Choi

et al. 2021

IJMS

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The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

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Section: Methodsmentioning

confidence: 99%

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Byambaragchaa

Seong

Choi

et al. 2021

IJMS

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“…The second messenger kinases of β ARK and GRK phosphorylate serine and/or threonine residues in the intracellular region of GPCRs following agonist-induced treatment ( Kim et al, 2018 ). We and others have shown that ligands (hCG, eCG, eFSH, eel FSH, and eel LH) of glycoprotein hormone receptors result in the accumulation of cAMP responses ( Hipkin et al, 1995a , b ; Min et al., 2004 ; Bhaskaran & Ascoli, 2005 ; Kim et al, 2018 ; Byambaragchaa et al, 2021c , d , 2022 ). Several features of the post-endocytotic trafficking of receptors (mouse, rat, and human) that demonstrate routing of most internalized receptors to the lysosomes, and degradation of hormone-receptor complexes have been elucidated ( Baratti-Elbaz et al, 1999 ; Kishi et al, 2001 ; Bhaskaran & Ascoli, 2005 ).…”

Section: Discussionmentioning

confidence: 97%

“…The supernatant was collected and concentrated using a Centricon filter. Finally, the sample was mixed 10–20 times and the quantity of rec-eCG was determined using ELISA as previously reported ( Byambaragchaa et al, 2021c , d ).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, the C-terminal region of eLH/CGR has 14 potential phosphorylation sites and a highly conserved leucine motif that is essential for trafficking ( Duvernay et al, 2004 ; Byambaragchaa et al, 2021a , b , e ). The eCG, secreted from the placenta during early pregnancy, is a unique molecule that displays both LH- and FSH-like activities in non-equid species ( Min et al., 2019 , 2020 , 2021 ; Lee et al, 2021 ) and exhibits only LH-like activity in equine species ( Lee et al, 2017 , 2021 ; Park et al, 2017 ; Min et al, 2019 , 2020 ; Byambaragchaa et al, 2021c , d ).…”

Section: Introductionmentioning

confidence: 99%

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Signal Transduction of C-Terminal Phosphorylation Regions for Equine Luteinizing Hormone/Chorionic Gonadotropin Receptor (eLH/CGR)

Byambaragchaa¹,

Joo²,

Kim³

et al. 2022

Dev. Reprod.

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This study aimed to investigate the signal transduction of phosphorylation sites at the carboxyl (C)-terminal region of equine luteinizing hormone/chorionic gonadotropin receptor (eLH/ CGR). The eLH/CGR has a large extracellular domain of glycoprotein hormone receptors within the G protein-coupled receptors. We constructed a mutant (eLH/CGR-t656) of eLH/ CGR, in which the C-terminal cytoplasmic tail was truncated at the Phe656 residue, through polymerase chain reaction. The eLH/CGR-t656 removed 14 potential phosphorylation sites in the intracellular C-terminal region. The plasmids were transfected into Chinese hamster ovary (CHO)-K1 and PathHunter Parental cells expressing β-arrestin, and agonist-induced cAMP responsiveness was analyzed. In CHO-K1 cells, those expressing eLH/CGR-t656 were lower than those expressing eLH/CGR wild-type (eLH/CGR-wt). The EC 50 of the eLH/ CGR-t656 mutant was approximately 72.2% of the expression observed in eLH/CGR-wt. The maximal response in eLH/CGR-t656 also decreased to approximately 43% of that observed in eLH/CGR-wt. However, in PathHunter Parental cells, cAMP activity and maximal response of the eLH/CGR-t656 mutant were approximately 173.5% and 100.8%, respectively, of that of eLH/CGR-wt. These results provide evidence that the signal transduction of C-terminal phosphorylation in eLH/CGR plays a pivotal role in CHO-K1 cells. The cAMP level was recovered in PathHunter Parental cells expressing β-arrestin. We suggest that the signal transduction of the C-terminal region phosphorylation sites is remarkably different depending on the cells expressing β-arrestin in CHO-K1 cells.

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“…To determine the intracellular signal transduction to cAMP via LH receptors, the amino acid residues 102 -104 of the β-subunit of sc eCG were replaced. The Rmax value of mutant eCG at amino acid 104 was the lowest, which means that this amino acid plays an essential role in the intracellular signal transduction to cAMP through LH receptors (Byambaragchaa et al, 2021b).…”

Section: Recombinant Equine Chorionic Gonadotropin (Rec-ecg)mentioning

confidence: 95%

Natural and recombinant equine chorionic gonadotropins past and future in animal reproductive technology

Duong

Bui

Hoang

et al. 2022

VIS

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Equine Chorionic Gonadotropin (eCG) previously named Pregnant Mare Serum Gonadotropin (PMSG) has been widely used since the 40s in animal reproduction control. It is extracted from the blood of pregnant mares between days 40 and 120 of gestation. Animal welfare organizations have voiced concerns against mares bleeding conditions. There is currently no effective substitute for the natural PMSG. In this review, we summarize the basic knowledge of the structure and biology of eCG, and the research on recombinant eCG production in the past five years.

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Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

Abstract: 2021): Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor, Animal Cells and Systems,

Cited by 12 publications

References 43 publications

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Signal Transduction of C-Terminal Phosphorylation Regions for Equine Luteinizing Hormone/Chorionic Gonadotropin Receptor (eLH/CGR)

Natural and recombinant equine chorionic gonadotropins past and future in animal reproductive technology

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