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—G_q-coupled P2Y₂, G_s-coupled dopamine D1 and D5, G_i-coupled dopamine D2L, and G_q/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.