Aequorin functional assay for characterization of G-protein-coupled receptors: Implementation with cryopreserved transiently transfected cells Bruce Jones a, * , Beverly Holskin a , Sheryl Meyer a , Thao Ung a , Vincent Dupriez b , Sandra Y. Flores b , Emmanuel Burgeon b , Mark Ator a , Emir Duzic a a Cephalon, Inc., Worldwide Discovery Research, 145 Brandywine Parkway, West Chester, PA 19380, USA b PerkinElmer Life and Analytical Sciences, 8 Imperiastraat, BE-1930 Zaventem, Belgium article info Article history: Received 27 October 2009 Received in revised form 6 January 2010 Accepted 21 January 2010 Available online 28 January 2010 Keywords: Aequorin Transient transfection Muscarinic G-protein-coupled receptor Cryopreserved cells abstract Assay technologies that measure intracellular Ca 2+ release are among the predominant methods for eval- uation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca 2+ sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor- intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M 1 receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with lit- erature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays. Ó 2010 Elsevier Inc. All rights reserved. The diversity of processes that are regulated by guanine nucle- otide binding proteins (G-proteins) via stimulation of G-protein- coupled receptors (GPCRs) 1 defines the critical importance of this class of receptors. Binding events leading to conformational changes are, in part, translated through the heterotrimeric G-proteins, result- ing in specific cellular responses [1]. Multiple techniques are avail- able to detect these responses, although functional cell-based approaches [2] are commonly employed since activation of the cel- lular pathways involves some level of amplification. These functional approaches utilize the coupling of the GPCR and an associated G-pro- tein to affect cellular responses through several second messengers. Measurement of changes in the concentration of the intracellular second messenger Ca 2+ is frequently employed as a technique for as- say of these targets. Classically, measurement of cytosolic Ca 2+ levels utilizes low molecular weight fluorescent probes whose spectral re- sponse or intensity of response changes in the presence of Ca 2+ . On chelation of Ca 2+ , these dyes emit a fluorescent response that is di- rectly proportional to the concentration of free calcium [3–7]. In recent years, recombinant photoproteins have been em- ployed as an alternative functional technique for measurement of intracellular Ca 2+ . These calcium-sensitive photoproteins emit luminescent signals in the presence of free Ca 2+ with virtually no background signal. Furthermore, by utilizing recombinant ap- proaches photoproteins can be transiently or stably expressed in a range of cellular phenotypes and directed to specific subcellular compartments, thus providing a substantial advantage over dyes to monitor Ca 2+ levels [8–10]. Aequorin (AEQ) is a calcium-sensitive photoprotein that was first isolated from the umbrella of the jellyfish Aequorea victoria [11]. The active protein (aequorin) is formed from apoaequorin (apoAEQ) and its prosthetic group, coelenterazine, which cova- lently binds to the protein in the presence of molecular oxygen [12]. Calcium binding to the holoprotein AEQ induces a conforma- tional shift that results in the oxidation of coelenterazine, yielding coelenteramide. The relaxation of the excited state of coelentera- mide bound to apoAEQ results in the flash emission of blue light at 470 nm [11]. AEQ has a low affinity for Ca 2+ [13] and large dy- namic range, making it an ideal sensor for biological Ca 2+ dynamics [14,15]. While the use of AEQ as a Ca 2+ probe to study GPCR pharmacol- ogy has several clear advantages, in practice the necessity for cell line development has been a drawback to adoption of this technique. In order to use AEQ as a Ca 2+ probe, the cell system must 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.01.028 * Corresponding author. Fax: +1 (610) 738 6305. E-mail address: bjones@cephalon.com (B. Jones). 1 Abbreviations used: Ach, acetylcholine; AEQ, aequorin; apoAEQ, apoaequorin; FBS, fetal bovine serum; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; GPCRs, G-protein-coupled receptors; hM 1 R, human M 1 muscarinic receptor; IRLU, integrated relative luminescent units; Oxo, oxotremorine; pen/strep, penicillin/ streptomycin. Analytical Biochemistry 400 (2010) 184–189 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio