Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay
Elie Besserer-Offroy,
Rebecca Brouillette,
Jean-Michel Longpré,
Philippe Sarret
Affiliations
Elie Besserer-Offroy
Dept. of Pharmacology and Therapeutics, McGill University, Montréal, QC, Canada
Rebecca Brouillette
Dept. of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, CanadaInstitut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada, Centre de recherche du Centre hospitalier universitaire de Sherbrooke, CIUSSS de l’Estrie – CHUS, Sherbrooke, QC, Canada
Jean-Michel Longpré
Dept. of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, CanadaInstitut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada, Centre de recherche du Centre hospitalier universitaire de Sherbrooke, CIUSSS de l’Estrie – CHUS, Sherbrooke, QC, Canada
Philippe Sarret
Dept. of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, CanadaInstitut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada, Centre de recherche du Centre hospitalier universitaire de Sherbrooke, CIUSSS de l’Estrie – CHUS, Sherbrooke, QC, Canada
Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of fluorescent probes for Ca++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [3H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express a specific 7TMR. However, when using transiently transfected mutants of a 7TMR, this assay is not ideal, as it requires a two-step protocol of cell culture. Therefore, we have optimized the IP-One assay protocol using the reverse transfection method in 384-well plates. This offers a time- and resource-efficient alternative to the two-step protocol previously used for the screening of several mutants of Gαq/15-coupled 7TMRs.
