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

Abstract

The present study aimed to investigate the mechanism of cell surface receptor loss by two constitutively activating mutants (designated L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the luteinizing hormone receptor (LHR) in the Japanese eel Anguilla japonica, known to naturally occur in human LHR transmembrane domains. We investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in HEK 293 cells. The expression level of wild-type eel LHR was considered to be 100%, and the expression levels of L469R and D417N were 97% and 101%, respectively, whereas the expression levels of D590Y and Y558F slightly increased to approximately 110% and 106%, respectively. The constitutively activating mutants L469R and D590Y exhibited a decrease in cell surface loss in a manner similar to that of wild-type eel LHR. The rates of loss of cell surface agonist-receptor complexes were observed to be very rapid (2.6-6.2 min) in both the wild-type eel LHR and activating mutants. However, cell surface receptor loss in the cells expressing inactivating mutants D417N and Y558F was slightly observed in the cells expressing inactivating mutants D417N and Y558F, despite treatment with a high concentration of agonist. These results provide important information on LHR function in fish and the regulation of mutations of highly conserved amino acids in glycoprotein hormone receptors.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor

“…Our recent observations have suggested that activating or inactivating conformations of eel FSHR [ 3 ], equine FSHR [ 5 ], and equine LH/CGR [ 4 ] are significantly involved in the signal transduction of G proteins and the cell-surface loss of receptors by high-concentration agonist treatment. The N-linked glycosylation sites in the glycoprotein hormones are important for PKA signal transduction in the cells expressing these receptors [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

“…We have studied the GPCR signal transduction of activating and inactivating lutropin/chorionic gonadotropin receptors (LH/CG) and FSHRs, showing that the highly conserved regions in glycoprotein hormone receptors are similarly regulated in different species of both mammals and fish [ 3 , 4 , 5 ]. In recent years, we have been studying signal transduction in activating and inactivating mutants to characterize the specific function of the PKA pathway [ 17 ] and cell-surface loss of receptors [ 18 ] in eel LH/CGR. We found that the phosphorylation sites in the C-terminal region of equine LH/CGR are important for signal transduction [ 19 ].…”

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

“…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 ).…”