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

Munkhzaya Byambaragchaa,Hoon-Ki Seong,Dae-Jung Kim,Myung-Hwa Kang,Seung-Hee Choi,Kwan-Sik Min

doi:10.3390/ijms221910723

Abstract

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Highlights

The surface expression of equine lutropin/choriogonadotropin receptor (eLH/CGR) mutants was determined via an enzyme-linked immunosorbent assay (ELISA) in transiently transfected Chinese hamster ovary (CHO)K1 cells (Figure 2)
The present study showed that the four mutations—eLH/CGR-M398T, L457R, D564G, and D578Y—resulted in a distinctly increased cyclic adenosine monophosphate (cAMP) response without agonist treatment, suggesting that these mutations might produce constitutively activating mutants of eLH/CGRs
We suggest that the eLH/CGR-M398T mutant displays a constitutive activation of cAMP response without agonist treatment, and the basal cAMP response differs from that in the cells expressing those mutants, despite the small increase observed in this study

Summary

Introduction

The receptor of the pituitary and placental gonadotropin in species expressing placental chorionic gonadotropin (CG) during early pregnancy plays a critical role in reproductive physiology. Equine CG (eCG), a unique member of the gonadotropin family, displays both luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like activities in non-equid species [1,2]. ECG, secreted from endometrial cups during early pregnancy, exhibits only LH-like activity in equine species [3]. The β-subunits of eCG and eLH are encoded as one gene, whereas their expression differs in the placenta and pituitary gland. Rec-eCG exhibits dual activities of LH and FSH in other species [1,3]

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