Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay

Élie Besserer-Offroy; Rebecca L Brouillette; Jean-Michel Longpré; Philippe Sarret

doi:10.21769/BioProtoc.3715

Assessing Gα_q/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay

Bio Protoc. 2020 Aug 20;10(16):e3715. doi: 10.21769/BioProtoc.3715.

Authors

Élie Besserer-Offroy¹, Rebecca L Brouillette^{2

3

4}, Jean-Michel Longpré^{2

3

4}, Philippe Sarret^{2

3

4}

Affiliations

¹ Dept. of Pharmacology and Therapeutics, McGill University, Montréal, QC, Canada.
² Dept. of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
³ Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada.
⁴ Centre de recherche du Centre hospitalier universitaire de Sherbrooke, CIUSSS de l'Estrie - CHUS, Sherbrooke, QC, Canada.

Abstract

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 Gα_q/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 Gα_q/15-coupled 7TMRs.

Keywords: Gα15/16 signaling Inositol phosphate; Gαq signaling; G-protein-coupled receptor; IP-One; Seven-transmembrane receptor; Signal transduction..