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Functional Screening of G Protein—Coupled Receptors by Measuring Intracellular Calcium with a Fluorescence Microplate Reader

Pharmaceutical Institute, University of Bonn, Bonn, Germany

Barbara Höfgen

Pharmaceutical Institute, University of Bonn, Bonn, Germany

Jochen Lehmann

Pharmaceutical Institute, University of Bonn, Bonn, Germany

Niels Eckstein

Pharmaceutical Institute, University of Bonn, Bonn, Germany

J. Mark Quillan

Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA

Wolfgang Sadée

Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA

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), SKF38393stimulation 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.

Journal of Biomolecular Screening, Vol. 7, No. 3, 233-246 (2002)
DOI: 10.1177/108705710200700307


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