Specific Signal Transduction of Constitutively Activating (D576G) and Inactivating (R476H) Mutants of Agonist-Stimulated Luteinizing Hormone Receptor in Eel

Abstract

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure–function relationship of LHR–LH complexes.

Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture

“…For the expression of rec-proteins, the expression vectors were transfected into CHO-S cells using the FreeStyle TM MAX reagent, as described previously [7]. The CHO-S cells were cultured in FreeStyle CHO expression medium at 1 × 10 7 cells per 30 mL of medium for 3 days.…”

“…The family of glycoprotein hormone receptors, which includes lutropin/choriogonadotropin hormone receptor (LH/CGR) and follicle-stimulating hormone receptor (FSHR), belongs to the superfamily of G protein-coupled receptors (GPCRs) that couple extracellular agonists to intracellular effector molecules through the reactions of heterotrimeric G proteins [4][5][6][7], GPCR kinases (GRK) [8,9], β-arrestins [10][11][12], and extracellular signal-regulated kinases (ERK) [13,14].…”