Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader.
Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) 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 P2Y(2), 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 [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) 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 Ca(2+) fluxes.
Kassack
Matthias U
MU
Pharmaceutical Institute, University of Bonn, Bonn, Germany. kassack@uni-bonn.de
Höfgen
Barbara
B
Lehmann
Jochen
J
Eckstein
Niels
N
Quillan
J Mark
JM
Sadée
Wolfgang
W
D002118:Calcium / Q000378:metabolism*; D002264:Carboxylic Acids; D005470:Fluorometry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D011954:Receptors, Dopamine / Q000032:analysis; D011976:Receptors, Muscarinic / Q000032:analysis
D002264:Carboxylic Acids; C436864:Oregon Green 488 carboxylic acid; D011956:Receptors, Cell Surface; D011954:Receptors, Dopamine; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D002118:Calcium