Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor

Abstract

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

“…The present study showed that the four mutations—eLH/CGR-M398T, L457R, D564G, and D578Y—resulted in a distinctly increased cAMP response without agonist treatment, suggesting that these mutations might produce constitutively activating mutants of eLH/CGRs. Our previous observations have suggested that the same active conformations of rLHR [ 11 ], eel FSHR [ 10 ], and eFSHR [ 25 ] are involved in the stimulation of G proteins and loss of the cell-surface receptor in ligand–receptor complexes.…”

“…To construct point mutations, we introduced the cDNA, encoding the full-length eLH/CGR using an overlap extension PCR strategy to create activating and inactivating mutants, as previously described [ 10 ]. Two different sets of PCRs were performed, and the primer sequences used in these experiments are shown in Table 5 .…”

“…The gonadotropin receptors of mammals (human, rat, and equine) and fish (eel) are highly homologous, with the highest level of amino acid conservation within the transmembrane helices [ 4 , 9 , 10 ]. The agonist–receptor complex is internalized by a dynamin-dependent pathway and moved to the endosome compartment without agonist dissociation [ 11 ].…”

“…Many studies have elucidated several features of the post-endocytotic trafficking of LHR and FSHR [ 23 , 24 ]. Recently, we identified several characteristics of signal transduction of the recombinant eCG through eLH/CGR—the function of constitutively activating eFSHR mutants, eel LHR and eel FSHR—demonstrating that activating mutants produces a considerable increase in the basal cAMP response [ 9 , 10 , 25 , 26 ]. Although various agonists have been reported to induce the signal transduction of glycoprotein hormone [ 17 , 27 , 28 ], and several studies have been reported on dual biological activities and glycosylation site roles for rec-eCG, there are few reports on the characterization and function of cellular signal transduction of eLH/CGR in naturally occurring activating/inactivating mutants.…”

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

“…The CHO-K1 cells were transfected using the previously reported liposome transfection method (Byambaragchaa et al 2020 ). The CHO cells were cultured in growth medium [Ham’s F-12 media supplemented with penicillin (50 U/mL), streptomycin (50 µg/mL), glutamine (2 mM), and 10% fetal bovine serum] to 80%–90% confluency in six-well plates.…”