title: Persistent GnRH receptor activation in pituitary αT3-1 cells analyzed with a label-free technology.
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.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: I
authors.initials: I
authors.lastname: Nederpelt
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: R D
authors.initials: RD
authors.lastname: Vergroesen
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Electronic address: ijzerman@lacdr.leidenuniv.nl.
authors.forename: A P
authors.initials: AP
authors.lastname: IJzerman
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: L H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D015374:Biosensing Techniques; D002064:Buserelin / Q000494:pharmacology; D002460:Cell Line; D017097:Electric Impedance; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011966:Receptors, LHRH / Q000819:agonists / Q000378:metabolism*
chemical_list: D007295:Inositol Phosphates; D011966:Receptors, LHRH; D002064:Buserelin
keywords: Binding kinetics; GnRH; Label-free; Receptor activation; Residence time; Wash-out