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

Abstract

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.

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 ].…”

“…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 ]. The ligand glycosylation sites of eel FSH [ 8 , 9 ], eel LH [ 20 ], and equine CG [ 21 , 22 , 23 , 24 , 25 ] demonstrate that the N-linked glycosylation sites are important for signal transduction in cells expressing these receptors.…”