Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader

Abstract

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca2+ appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca2+ signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca2+ fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested - Gq-coupled P2Y2, Gs-coupled dopamine D1 and D5, Gi-coupled dopamine D2L, and Gq/11-coupled muscarinic acetylcholine M1 - yielded a significant rise in intracellular free [Ca2+] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y2 receptors (EC50 = 1 μM), SKF38393 stimulation of hD1 and hD5 (EC50 = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC50 = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca2+ response. Furthermore, the Ca2+ assay served to screen suramin analogs for antagonistic activity at P2Y2 receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca2+ fluxes.