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Biased signaling has recently emerged as an interesting mean to modulate the function of many G protein-coupled receptors (GPCRs). Previous studies reported two negative allosteric modulators (NAMs) of follicle-stimulating hormone receptor (FSHR), ADX68692 and ADX68693, with differential effects on FSHR-mediated steroidogenesis and ovulation. In this study, we attempted to pharmacologically profile these NAMs on the closely related luteinizing/chorionic gonadotropin hormone receptor (LH/CGR) with regards to its canonical Gs/cAMP pathway as well as \beta-arrestin recruitment in HEK293 cells. The NAMs effects on progesterone and testosterone production were also assessed in murine Leydig tumor cell line (mLTC-1). We found that both NAMs strongly antagonized LH/CGR signaling in both HEK293 and mLTC-1 cells. ADX68693 appeared more potent than ADX68692 to inhibit hCG-induced cAMP and \beta-arrestin 2 in HEK293 and mLTC-1 cells whereas no significant difference in their efficacy on hCG-promoted \beta-arrestin 2 recruitment. Interestingly, differential antagonism of the two NAMs on hCG-promoted steroidogenesis in mLTC-1 cells was observed with significant inhibition of testosterone but not progesterone production. These observations suggest biased effects of the two NAMs on LH/CGR-dependent pathways controlling steroidogenesis, which appeared to be different to that previously shown on FSHR. This also illustrates the complexity of signaling pathways controlling FSHR- and LH/CGR-mediated steroidogenesis, suggesting differential implication of cAMP and \beta-arrestins. Together, our data demonstrate that ADX68692 and ADX68693 are NAMs at the LH/CGR in addition to FSHR. These pharmacological characteristics are important to consider for potential contraceptive and therapeutic applications based on such compounds.

Pituitary gonadotropins play an essential and pivotal role in the control of human and animal reproduction within the hypothalamic-pituitary-gonadal (HPG) axis. The computational modeling of pituitary gonadotropin signaling encompasses phenomena of different natures such as the dynamic encoding of gonadotropin secretion, and the intracellular cascades triggered by gonadotropin binding to their cognate receptors, resulting in a variety of biological outcomes. We overview historical and ongoing issues in modeling and data analysis related to gonadotropin secretion in the field of both physiology and neuro-endocrinology. We mention the different mathematical formalisms involved, their interest and limits. We discuss open statistical questions in signal analysis associated with key endocrine issues. We also review recent advances in the modeling of the intracellular pathways activated by gonadotropins, which yields promising development for innovative approaches in drug discovery. The greatest challenge to be tackled in computational modeling of pituitary gonadotropin signaling is the embedding of gonadotropin signaling within its natural multi-scale environment, from the single cell level, to the organic and whole HPG level. The development of modeling approaches of G protein-coupled receptor signaling, together with multicellular systems biology may lead to unexampled mechanistic understanding with critical expected fallouts in the therapeutic management of reproduction., Comment: in press

Dynamic models of signaling networks allow the formulation of hypotheses on the topology and kinetic rate laws characterizing a given molecular network, in-depth exploration and confrontation with kinetic biological data. Despite its standardization, dynamic modeling of signaling networks still requires successive technical steps that need to be carefully performed. Here, we detail these steps by going through the mathematical and statistical framework. We explain how it can be applied to the understanding of \beta-arrestin-dependent signaling networks. We illustrate our methodology through the modeling of \beta-arrestin recruitment kinetics at the Follicle Stimulating Hormone (FSH) receptor supported by in-house Bioluminescence Resonance Energy Transfer (BRET) data., Comment: in press

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, \beta-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 \beta-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

Follicle-stimulating hormone (FSH) is produced in the pituitary and is essential for reproduction. It specifically binds to a membrane receptor (FSHR) expressed in somatic cells of the gonads. The FSH/FSHR system presents many peculiarities compared to classical G protein-coupled receptors (GPCRs). FSH is a large naturally heterogeneous heterodimeric glycoprotein. The FSHR is characterized by a very large NH2-terminal extracellular domain, which binds the FSH and participates to the activation/inactivation switch of the receptor. Once activated, the FSHR couples to G{\alpha}s and, in some instances, to other G{\alpha} subunits. G protein-coupled receptor kinases and \beta-arrestins are also recruited to the FSHR and account for its desensitization, the control of its trafficking and its intracellular signalling. Of note, the FSHR internalization and recycling are very fast and involve very early endosomes instead of early endosomes. All the transduction mechanisms triggered upon FSH stimulation lead to the activation of a complex signalling network that controls gene expression by acting at multiple levels. The integration of these mechanisms leads to context-adapted responses from the target gonadal cells, but also indirectly affects the fate of germ cells. Depending of the physiological/developmental stage, FSH elicits proliferation, differentiation or apoptosis in order to maintain the homeostasis of the reproductive system. Pharmacological tools targeting FSHR recently came to the fore and open promising prospects both for basic research and therapeutic applications. This paper provides an updated review of the most salient aspects and peculiarities of FSHR biology and pharmacology.