Persistent GnRH receptor activation in pituitary αT3-1 cells analyzed with a label-free technology.

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.

Biosensors & bioelectronics. 2015 Dec 21 [Epub ahead of print]

I Nederpelt, R D Vergroesen, A P IJzerman, L H Heitman

Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands. , Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands. , Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands. Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.

PubMed

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