Human Luteinizing Hormone and Chorionic Gonadotropin Display Biased Agonism at the LH and LH/CG Receptors

Abstract

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) have been considered biologically equivalent because of their structural similarities and their binding to the same receptor; the LH/CGR. However, accumulating evidence suggest that LH/CGR differentially responds to the two hormones triggering differential intracellular signaling and steroidogenesis. The mechanistic basis of such differential responses remains mostly unknown. Here, we compared the abilities of recombinant rhLH and rhCG to elicit cAMP, β-arrestin 2 activation, and steroidogenesis in HEK293 cells and mouse Leydig tumor cells (mLTC-1). For this, BRET and FRET technologies were used allowing quantitative analyses of hormone activities in real-time and in living cells. Our data indicate that rhLH and rhCG differentially promote cell responses mediated by LH/CGR revealing interesting divergences in their potencies, efficacies and kinetics: rhCG was more potent than rhLH in both HEK293 and mLTC-1 cells. Interestingly, partial effects of rhLH were found on β-arrestin recruitment and on progesterone production compared to rhCG. Such a link was further supported by knockdown experiments. These pharmacological differences demonstrate that rhLH and rhCG act as natural biased agonists. The discovery of novel mechanisms associated with gonadotropin-specific action may ultimately help improve and personalize assisted reproduction technologies.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

LH/CGR-mediated cAMP production. cAMP responses induced by rhCG and rhLH in HEK293 cells transiently co-expressing hLH/CGR and CAMYEL sensor (A,B) and in mLTC-1 cells transiently expressing CAMYEL sensor alone (C) or not (D). Cells were stimulated 30 minutes with increasing doses (A,C and D) or immediately with the indicated doses (B) of rhCG and rhLH, before cAMP production was measured by BRET (A,B and C) or HTRF (D). Data are means ± SEM of 3–8 independent experiments. The kinetic curves in panel B are representative of 3 experiments performed in triplicate.

Figure 2

LH/CGR-promoted β-arrestin 2 recruitment and activation in HEK293 cells. HEK293 cells transiently co-expressing either hLH/CGR-Rluc8 and yPET-β-arrestin 2 (A and B) or wildtype hLHR/CGR and Rluc8-β-arrestin 2-RGFP (C) were stimulated 30 minutes with increasing doses (A and D) or immediately with the indicated doses (B) or 0.25 µM (C) of rhCG and rhLH before BRET was measured. Data are means ± SEM of 3–5 independent experiments. The kinetic curves in panel B are representative of 3 experiments performed in triplicate.

Figure 3

LH/CGR-promoted integrated responses in HEK293 and mLTC-1 cells. (A) HEK293 transiently co-transfected with plasmids coding for LH/CGR and pSOM-Luc reporter gene were stimulated 6 hours with increasing doses of rhCG and rhLH before luciferase activity was measured (A). mLTC-1 cells transiently transfected with plasmid coding pSOM-Luc reporter gene were stimulated 6 hours with increasing doses of rhCG and rhLH before luciferase activity was measured (B). For LHR-mediated steroid production, mLTC-1 cells were stimulated 3 hours with increasing doses of rhCG and rhLH and the supernatant levels of progesterone (C) and testosterone (D) were measured by ELISA or HTRF, respectively. Data are means ± SEM of 3–5 independent experiments performed in single or duplicate.

Figure 4

Implication of β-arrestins in LH/CGR-promoted steroid production in mLTC-1 cells. mLTC-1 cells were transiently transfected with either control, β-arrestin 1 or β-arrestin 2 siRNAs. Then, cells were stimulated 3 hours with increasing doses of rhCG (A,C and E) and rhLH (B,D and F) before progesterone (A and B), testosterone (C and D) and cAMP (E and F) productions were measured. The maximal rhCG-mediated responses obtained at 10 nM with control siRNA was taken as 100%. Data are means ± SEM of 3 independent experiments performed in duplicate.