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, 22 (2), 143-153

The C-terminal Phosphorylation Sites of Eel Follicle-Stimulating Hormone Receptor Are Important Role in the Signal Transduction

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The C-terminal Phosphorylation Sites of Eel Follicle-Stimulating Hormone Receptor Are Important Role in the Signal Transduction

Jeong-Min Kim et al. Dev Reprod.

Abstract

The large extracellular domain of glycoprotein hormone receptors is a unique feature within the G protein-coupled receptors (GPCRs) family. After interaction with the hormone, the receptor becomes coupled to Gs, which, in turn stimulates adenylyl cyclase and the production of cAMP. Potential phosphorylation sites exist in the C-terminal region of GPCRs. The experiments described herein represent attempts to determine the functions of the eel follicle-stimulating hormone receptor (eelFSHR). We constructed a mutant of eelFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 614 (eelFSHR-t614). The eelFSHR-t614 lacked all potential phosphorylation sites present in the C-terminal region of eelFSHR. In order to obtain the eelFSHR ligand, we produced recombinant follicle-stimulating hormone (rec-eelFSHβ/α) in the CHO-suspension cells. The expression level was 2-3 times higher than that of the transient expression of eelFSH in attached CHO-K1 cells. The molecular weight of the rec-eelFSHβ/α protein was identified to be approximately 34 kDa. The cells expressing eelFSHR-t614 showed an increase in agonist-induced cAMP responsiveness. The maximal cAMP responses of cells expressing eelFSHR-t614 were lower than those of cells expressing eelFSHR-wild type (eelFSHR-WT). The EC50 following C-terminal deletion in CHO-K1 cells was approximately 60.4% of that of eelFSHR-WT. The maximal response in eelFSHR-t614 cells was also drastically lower than that of eelFSHR-WT. We also found similar results in PathHunter Parental cells expressing β-arrestin. Thus, these data provide evidence that the truncation of the C-terminal cytoplasmic tail phosphorylation sites in the eelFSHR greatly decreased cAMP responsiveness and maximal response in both CHO-K1 cells and PathHunter Parental cells expressing β-arrestin.

Keywords: CHO cells; PathHunter Parental cells; cAMP; eelFSHR (eel follicle-stimulating hormone receptor); rec-eelFSHβ/α (recombinant eel follicle-stimulating hormone β/α).

Figures

Fig. 1.
Fig. 1.
Schematic diagram of rec-eelFSHβ/α. The eelFSHβ/α cDNA was amplified, constructed as a single-chain, and ligated into the pcDNA3 mammalian expression vector. rec-eelFSHβ/α, recombinant eel follicle-stimulating hormone.
Fig. 2.
Fig. 2.
Intracellular regions of the eelFSHR. The amino acid sequence of the intracellular region of the eelFSHR is shown. The 10 potential phosphorylation sites (serine and threonine residues) are S615, T629, S631, S632, T642, T645, S646, T649, T652, and S661. The location of truncation site (t614) is also shown. The amino acid sequence was amplified with ovary and testis cDNA of eel from our laboratory and sequenced according to a previoius reported method (Byambaragchaa et al., 2018). eelFSHR, eel follicle-stimulating hormone receptor.
Fig. 3.
Fig. 3.
Quantification of rec-eelFSHβ/α for transient transfection in CHO suspension cells. The media were collected and centrifuged on days 1, 3, 5, and 7 after transfection. Then, the expression of rec-eelFSHβ/α was analyzed by ELISA, as discussed in Materials and Methods. Values are expressed as mean±SEM for at least three independent experiments. rec-eelFSHβ/α, recombinant eel follicle-stimulating hormone.
Fig. 4.
Fig. 4.
Western blot of rec-eelFSHβ/α. A sample of rec-eelFSHβ/α was electrophoresed on 12.5% SDS-PAGE. The first antibody used was anti-eelFSH monoclonal antibody. The second antibody used was goat anti-mouse IgG-HRP. A band corresponding to rec-eelFSHβ/α was detected. rec-eelFSHβ/α, recombinant eel follicle-stimulating hormone; eelFSHR, eel follicle-stimulating hormone receptor.
Fig. 5.
Fig. 5.
Selection of stable cells expressing eelFSHR-C-Dels in PathHunter CHO-K1 EA Parental cells. The Delta F% value was calculated by inhibition. PathHunter CHO-K1 Parental cells were cultured in AssayCompleteTM CHO-K1 culture medium. After transfection, the cells were cultured in AssayCompleteTM medium containing G418 for 2-3 weeks to isolate cells expressing eelFSHR-C-Dels. Finally, 5 cell clone lines were isolated and cAMP accumulation was analyzed with different rec-eelFSHβ/α concentrations (0, 55, and 408 ng/mL). Each point represents the average of three independent experiments. eelFSHR, eel follicle-stimulating hormone receptor.
Fig. 6.
Fig. 6.
Dose-dependent increase in cAMP accumulation induced by rec-eelFSHβ/α in cells expressing both eelFSHR-WT and eelFSHR-t614. For the stable cells, eelFSHR-WT and eelFSHR-t614 were aliquoted at 10,000 cells per well into a 384-well plate. Standard samples were prepared to cover an average range of 0.17-712 nM. The plate was incubated for 30 min at RT after the addition of rec-eelFSHβ/α adding (0 to 408 ng/mL). cAMP d2 and anti cAMP-cryptate were added and incubated at RT for 1 hr. Inhibition of cAMP accumulation is represented by Delta F%. The cAMP nM (1×104 cells) value was calculated by GraphPad Prism. A) CHO-K1 cells, B) PathHunter Parental cells. rec-eelFSHβ/α, recombinant eel follicle-stimulating hormone; eelFSHR-WT, eel follicle-stimulating hormone receptor wild type.

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