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Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

INTRODUCTION

Many G-protein coupled receptors (GPCRs) trigger, upon binding of an agonist, a transient increase in intracellular calcium concentration. This variation acts as an internal secondary messenger and is an important modulator of many physiological mechanisms (reviewed by Rink (1990), Tsunoda (1993) and by Santella & Carafoli (1997)). Measurement of intracellular calcium concentration in cells expressing a GPCR can thus be used to monitor the efficiency of activation of a GPCR by various compounds known or suspected to be a ligand for this GPCR.

One of the methods of choice (reviewed by Mottheakis and Ohler, 2000) for such measurements is the use of cell lines expressing a GPCR and aequorin, such as described by Sheu et al (1993) or Button et al. (1993). In this system, cells are incubated with coelenterazine, which is the co-factor of aequorin. During this incubation, coelenterazine enters the cell (it is lipophylic and readily crosses the cell membrane) and conjugates with apoaequorin to form aequorin, which is the active form of the enzyme. When the cells are then exposed to an agonist of the GPCR, intracellular calcium concentration increases. This increase leads to the activation of the catalytic activity of aequorin, which oxidizes coelenterazine and yields apoaequorin, coelenteramide, CO2 and light. The intensity of light emission is proportional to the increase in intracellular calcium in the physiological range (Rizutto et al., 1995). Thus, in this system, measurement of light emission following agonist addition reflects its ability to activate the GPCR. Because light is emitted for only 20 to 30 seconds after activation of the GPCR, recording of the emitted light must be performed during the few seconds following agonist addition to the cells. This flash-type signal reflects the transient increase in calcium concentration following GPCR stimulation.


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