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Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Published on: June 9, 2017
Persistent GnRH receptor activation in pituitary αT3-1 cells analyzed with a label-free technology
I Nederpelt1, R D Vergroesen1, A P IJzerman1
1Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Abstract:
The gonadotropin-releasing hormone (GnRH) receptor is a drug target for certain hormone-dependent diseases such as prostate cancer. In this study, we examined the activation profiles of the endogenous ligand, GnRH and a well-known marketed analog, buserelin using a label-free assay in pituitary αT3-1 cells with endogenous GnRH receptor expression. This whole cell impedance-based technology allows for the real-time measurement of morphological cellular changes. Both agonists dose-dependently decreased the impedance as a result of GnRH receptor activation with potencies of 9.3 ± 0.1 (pEC50 value, buserelin) and 7.8 ± 0.06 (pEC50 value, GnRH). Subsequently, GnRH receptor activation was completely abolished with a selective Gαq inhibitor, thereby confirming the Gαq-coupling of the GnRH receptor in pituitary αT3-1 cells. Additionally, we observed continued responses after agonist stimulation of αT3-1 cells indicating long-lasting cellular effects. Wash-out experiments demonstrated that the long-lasting effects induced by GnRH were most likely caused by rebinding since over 70% of the original response was abolished after wash-out. In contrast, a long receptor residence time was responsible for the prolonged effects caused by buserelin, with over 70% of the original response remaining after wash-out. In summary, we validated that impedance-based label-free technology is suited for studying receptor-mediated activation in cell lines endogenously expressing the target of interest. Moreover, this real-time monitoring allows the examination of binding kinetics and its influence on receptor activation at a cellular level.
Insights
This study validates label-free impedance technology for real-time GnRH receptor activation analysis. It reveals distinct cellular responses and binding kinetics for GnRH and buserelin, aiding drug development for hormone-dependent diseases.
Area of Science:
- Pharmacology and Drug Discovery
- Cellular and Molecular Biology
- Endocrinology
Background:
- The GnRH receptor is a key target for hormone-dependent diseases like prostate cancer.
- Understanding GnRH receptor activation dynamics is crucial for developing effective therapeutics.
- Existing methods may not fully capture real-time cellular responses to GnRH receptor agonists.
Purpose of the Study:
- To evaluate label-free impedance-based technology for studying GnRH receptor activation.
- To compare the activation profiles and cellular effects of GnRH and buserelin.
- To elucidate the mechanisms underlying long-lasting GnRH receptor activation.
Main Methods:
- Utilized whole-cell impedance-based assays in pituitary αT3-1 cells with endogenous GnRH receptor expression.
- Measured real-time morphological cellular changes in response to GnRH and buserelin.
- Employed a Gαq inhibitor to confirm receptor signaling pathways and conducted wash-out experiments to assess binding kinetics.
Main Results:
- Both GnRH and buserelin dose-dependently decreased impedance, indicating GnRH receptor activation.
- GnRH receptor activation was confirmed to involve Gαq-coupling in these cells.
- GnRH-induced responses were primarily due to rebinding, while buserelin effects stemmed from long receptor residence time.
Conclusions:
- Impedance-based label-free technology is suitable for studying receptor-mediated activation in cells with endogenous target expression.
- Real-time monitoring effectively reveals differences in binding kinetics and their impact on cellular activation.
- This approach provides valuable insights into GnRH receptor pharmacology for drug development.
