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Assessing Gonadotropin Receptor Function by Resonance Energy Transfer-Based Assays
Mohammed Akli Ayoub1, Flavie Landomiel2, Nathalie Gallay2
1Biologie et Bioinformatique des Systèmes de Signalisation (BIOS) Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements , Nouzilly , France ; CNRS, UMR7247 , Nouzilly , France ; Université François Rabelais , Tours , France ; L'Institut français du cheval et de l'équitation (IFCE) , Nouzilly , France ; LE STUDIUM ® Loire Valley Institute for Advanced Studies , Orléans , France.
New BRET and FRET assays reveal real-time dynamics of gonadotropin receptors (FSHR and LHR) in living cells. These tools uncover distinct signaling pathways and receptor trafficking, aiding reproductive research and drug discovery.
Area of Science:
- Endocrinology and Reproductive Biology
- Molecular Cell Biology
- Biophysics
Background:
- Gonadotropin receptors (FSHR and LHR) are crucial for reproductive function and are G protein-coupled receptors.
- Their signaling and trafficking are complex and tightly regulated, often not fully captured by classical assays.
- Understanding these dynamics is key for reproductive health and therapeutic development.
Purpose of the Study:
- To develop and apply bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays for real-time analysis of FSHR and LHR.
- To investigate hormone-mediated signaling pathways, including G protein activation, cAMP production, and β-arrestin 2 recruitment.
- To characterize receptor trafficking dynamics, such as internalization and recycling, in living cells.
Main Methods:
- Utilized BRET and FRET assays in human embryonic kidney 293 cells expressing transiently follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR).
- Assessed real-time dynamics of heterotrimeric G protein activation, cyclic adenosine-monophosphate (cAMP) production, and β-arrestin 2 recruitment.
- Monitored receptor internalization and recycling processes following hormone stimulation.
Main Results:
- Kinetics and dose-response analyses confirmed known properties of FSHR and LHR, with distinct EC50 values for cAMP production (picomolar) versus β-arrestin 2 recruitment/internalization (nanomolar).
- Maximal G protein activation and cAMP response occurred at <10% receptor occupancy, while >90% was needed for full β-arrestin 2 recruitment and internalization.
- Observed rapid receptor internalization followed by a recycling phase, providing mechanistic insight into the 'spare receptor' paradigm.
Conclusions:
- BRET/FRET assays offer powerful real-time insights into gonadotropin receptor pharmacology and signaling dynamics.
- Distinct receptor occupancy thresholds for cAMP and β-arrestin pathways highlight complex regulation.
- Fast receptor recycling may explain the 'spare receptor' phenomenon, with implications for drug discovery and understanding reproductive pathophysiology.
