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

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.

Signal transduction of C-terminal phosphorylation sites for equine follicle stimulating hormone receptor (eFSHR)

“…The maximal response of eelFSHR-t614 was also drastically lower than that of eelFSHR-wt (Kim et al, 2018). We also reported that the constitutively activated eelFSHR-D540G mutant displayed remarkably increased response (13.1 to 19.6-fold) when compared to the wild type receptor (Kim et al, 2019), suggesting that cAMP production was greatly increased in the absence of eelFSH stimulation.…”

“…PathHunter CHO-K1 Parental cells are engineered to stably express the enzyme acceptor-tagged β-arrestin fusion protein. These cells were transiently transfected according to the supplier's protocol as previously described (Lee et al, 2017;Kim et al, 2018). Transfections were performed using the liposome transfection method as previously described (Byambaragchaa et al, 2018).…”

Cell-Surface Loss of Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptors

“…The cDNAs encoding wild-type and mutant eel LHR were digested with the Eco RI and Xho I restriction enzymes. The resulting fragments were then ligated into the pcDNA3.1 and pCORON1000 SP VSV-G expression vectors, as previously described (Byambaragchaa et al, 2018(Byambaragchaa et al, , 2021a. Finally, we constructed a total of five receptor genes, including WT eel LHR, mutant eel LHR-L469R, LHR-D590Y, LHR-D417N, and LHR-Y558F.…”

“…GPCR signal transduction including glycoprotein hormone receptors, has been studied in detail with respect to the loss of cell surface receptors, internalization, and recycling (Bhaskaran & Ascoli, 2005;Jacobsen et al, 2017;Foster & Bauner-Osborne, 2018). Recent research studies on eel glycoprotein hormones and receptors from our laboratory have elucidated the signal transduction of the eel LHR (Byambaragchaa et al, 2018a,b) and eel follicle-stimulating hormone receptor (FSHR; Kim et al, 2016Kim et al, , 2018Kim et al, , 2019 on deglycosylated ligands. We also first reported in fish species that the eel FSHR-D540G mutant exhibited a high increase in the basal cAMP response (Byambaragchaa et al, 2020).…”

Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/CG) Receptor

“…Therefore, eCG is a distinct molecule for biological activity and the structure of its glycoresidues. We previously reported several studies on eCG, including cDNA cloning of the eCG α-subunit and β-subunit, biological dual activities of LH-like and FSH-like in cells expressing rat LHR and rat FSHR (rLHR and rFSHR), and ovulation rate in vivo (Min et al, 1994(Min et al, , 1996(Min et al, , 2004, 2020Park et al, 2017;Kim et al, 2018;Byambaragchaa et al, 2021a,b,c;Lee et al, 2021).…”