( This application note describ...)Gq-coupled Receptor Assays Using FLEXstation

This application note describes the use of FLEXstation, a 96-well microplate fluorescence reader with fluid transfer capabilities, to measure the activity of the canine P2Y₁₁ (cP2Y₁₁) G_q-coupled receptor (1). The P2 superfamily of proteins includes the P2X family of ligand-gated ion channels and the P2Y family of G-coupled receptors (2). The P2Y receptors selectively respond to nucleotides and their analogs. To date, six subtypes have been cloned in various organisms, including human (3). All P2Y subtypes couple to phosphoinositide-Ca²⁺ pathway (4). Interestingly, P2Y₁₁ mediates signaling through both phosphoinositide-Ca²⁺ and cAMP pathways (3, 4, 5, Fig. 1). This receptor plays a putative role in differentiation of immunocytes and renal disease (3, 6, 7, 8).

Here, we demonstrate that FLEXstation is an accurate, efficient, and rapid alternative to IP₃ measurement in generating EC₅₀ values for cP2Y₁₁ agonists.

BACKGROUND
Cells. The canine P2Y₁₁ receptor was cloned by rapid amplification of cDNA ends (RACE) from the highly differentiated Madin-Darby canine kidney epithelial cell line MDCK-D1 (1, 9). The cP2Y₁₁ gene was cloned into the retroviral expression vector pLXSN, which was used to transfect PA317 retroviral packing cells. The resulting virus was used to infect the canine thymocyte cell line CF2Th. Infected cells were exposed to 0.5 mg/mL G418 for at least 2 weeks. G418-resistant cells were used in subsequent experiments (1).

Characterization of cP2Y₁₁ by phosphoinositide hydrolysis assay. The receptor was previously characterized, and its agonist selectivity profile is shown below:

Adenosine 5'-(2-O-thio) diphosphate (ADPβS) = 2-methylthioadenosine 5' diphosphate (2MT-ADP)>>ADP>ATP (1). The EC<
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